Maternal low-level lead exposure reduces the expression of PSA-NCAM and the activity of sialyltransferase in the hippocampi of neonatal rat pups.

Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is transiently expressed specifically in newly generated cells, and is important for cell migration and neurite outgrowth. Developmental lead (Pb) exposure has been considered to affect the expression of PSA-NCAM, which contributes to the neurotoxicity of Pb exposure. However, the effect of maternal low-level Pb exposure on the expression of PSA-NCAM in neonatal rat pups has not been reported. In the present study, female Wistar rats were exposed to vehicle or different dosages of lead chloride (0.5-4mM PbCl2) 2 weeks before and during pregnancy. This exposure protocol resulted in neonatal rat pups blood Pb levels up to 12.12+/-0.38 microg/dl, and hippocampal Pb levels up to 9.22+/-0.81 microg/g at postnatal day 1 (PND 1). Immunohistochemistry analysis and Western blot analysis revealed that the expressions of PSA-NCAM and NCAM in the hippocampi of neonatal rat pups at PND 1 were significantly reduced by the maternal low-level Pb exposures. Furthermore, the mRNA levels of NCAM and polysialyltransferases (STX and PST), measured by the fluorescent real-time quantitative RT-PCR, dosage-dependently and significantly decreased by 13.26-37.62%, 25.17-59.67%, and 10.78-47.81%, respectively. In addition, the sialyltransferase activity in neonatal rat pups was significantly reduced by 6.23-32.50% in the presence of the low-level Pb exposure, too. Taken together, these results suggest that maternal low-level Pb exposure reduces the expression of PSA-NCAM, NCAM, and the activity of sialyltransferase in the hippocampi of neonatal rat pups, which might contribute to the learning and memory impairments in the developmental pups following maternal low-level Pb exposure.

[Construction of thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene].

OBJECTIVE: To construct Thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene and to provide scientific base for deeply researching on the function of cytochrome 1A1 gene (CYP1A1) and the mechanism of carcinogenesis. METHODS: According to cDNA sequence of human CYP1A1 gene, universal primers (Pm3/Pm4) and mutant primers (Pt15/Pt16 and Pt17/Pt18) containing restriction enzyme site and mutation site were designed. The first set of primers involving Pm3/Pt16 and Pm3/Pt18 amplified a forward 1.5kb fragment from pGEM-T-CYP1A1 plasmid. The second set of primers involving Pt15/Pm4 and Pt17/Pm4 amplified a reverse 177-bp fragment from 10ng pGEM-T-CYP1A1 plasmid. The third set of primers involving Pm3/Pm4 amplified a 1.5kb fragment from the fomer PCR amplifications. The third PCR products were separated, purified and recovered from 1% agarose gel, then inserted into pMD-T vector. Subsequently the conjunct products were transformed into E. coil strain DH-5alpha., then the single clone was screened out and plasmids were extracted from such clone finally verified by restriction endonuclease analysis and sequencing. RESULTS: A 1.5kb fragment of tricycle PCR amplifications were digested by restriction endonucleases (BamHI and SailI) and sequenced bidirectionally by universal primers(T7p and SP6). The results verified that the cloned fragment including Asn461 and Val462 mutant site had 99.9% homology with the human cDNA of CYP1A1 gene in Genebank. CONCLUSION: The objective fragment containing Asn461 and Va462 mutant site with cDNA of the CYP1A1 gene has been successfully constructed in this experiment.

Aluminum-induced apoptosis in cultured cortical neurons and its effect on SAPK/JNK signal transduction pathway.

Aluminum exposure and apoptotic cell death has been implicated in several neurodegenerative diseases. The mechanisms by which aluminum interacts with the nervous system are only partly understood. In this study, we used cultured cortical neurons to investigate the ability of aluminum to induce the apoptosis of neurons and to explore the role of SAPK/JNK (stress-activated protein kinase or c-jun N-terminal kinase) signal transduction pathway on the apoptosis induced by aluminum. We found that aluminum-induced degeneration of cortical neurons involved the DNA fragmentation characteristic of apoptosis, and staining of aluminum-treated neurons with the DNA-binding fluorochrome Hoechst 33258 revealed the typical apoptotic condensation and fragmentation of chromatin. The rate of apoptosis increased significantly (from 4.9 to 13.1, 21.4, and 59.8%, P<0.01), which was measured by TdT-mediated dUTP nick end labeling. Western blot analysis showed that SAPK/JNK activities of cortical neurons varies when the exposure time of AlCl(3) were different. The phosphorylation levels were 4.2, 3.3, 1.9 and 1.1 times greater compared to control cultures for 6, 12, 24, and 48 h, respectively (P<0.01). Furthermore, a JNK pathway inhibitor, CEP-11004 (KT8138) inhibited the activation of SAPK/JNK to protect cortical neurons from apoptosis induced by aluminum chloride. Our study demonstrates that aluminum can induce the apoptosis of cortical neurons and SAPK/JNK signal transduction pathway may play an important role in the apoptosis.

[Impact of low-level lead exposure on neural cell adhesion molecule expression of primarily cultured hippocampal neurons].

OBJECTIVE: To study the impact of low-level lead exposure on neural cell adhesion molecule (NCAM) expression of primarily cultured hippocampal neurons. METHODS: Wistar rats gestated at 18th day were anaesthetized and paunched to get the pups, the hippocampi of the pups were separated and the hippocampal neurons were primarily cultured. After co-cultivated with different dosage of PbCl(2), the NCAM expression of the neurons were tested with Western blotting at different culture time. RESULTS: Normally, the expression of NCAM at the 1st culture day was very low and its integral obsorbency density was 14; the climax expression time of NCAM of the cultured hippocampal neurons was 3rd to 5th cultured day, and their integral obsorbency density were 2 542 to 2 580; henceforth, the NCAM expression declined. NCAM expression was inhibited significantly by lead during the 2nd to 4th cultured day, and dose-response relationship was observed. The inhibition of lead weakened along with the cultured time prolonged, at 5th cultured day, it disappeared, and the NCAM expression of 10(-2), 10(-3) and 10(-4) mmol/L groups even exceeded the control groups. After that, the expression of NCAM in all groups began to decline, and the dose-response relationship of lead to the NCAM expression was observed again. CONCLUSION: Low-level lead might significantly inhibit the NCAM expression of the primarily cultured Wistar rats' hippocampal neurons, and might delay the climax NCAM expression time.

Low-level lead exposure attenuates the expression of three major isoforms of neural cell adhesion molecule.

Toxic lead (Pb) exposure poses serious risks to human health, especially to children at developmental stages, even at low exposure levels. Neural cell adhesion molecule (NCAM) is considered to be a potential early target in the neurotoxicity of Pb due to its role in cell adhesion, neuronal migration, synaptic plasticity, and learning and memory. However, the effect of low-level Pb exposure on the specific expression of NCAM isoforms has not been reported. In the present study, we found that Pb could concentration-dependently (1-100 nM) inhibit the expression of three major NCAM isoforms (NCAM-180, -140, and -120) in primary cultured hippocampal neurons. Furthermore, it was verified that levels of all three major isoforms of NCAM were reduced by Pb exposure in human embryonic kidney (HEK)-293 cells transiently transfected with NCAM-120, -140, or -180 isoform cDNA constructs. In addition, low-level Pb exposure delayed the neurite outgrowth and reduced the survival rate of cultured hippocampal neurons at different time-points. Together, our results demonstrate that developmental low-level Pb exposure can attenuate the expression of all three major NCAM isoforms, which may contribute to the observed Pb-mediated neurotoxicity.