Effect Of MeHg On Neuronal Cell Cycle And The Regulational Role Of Cyclin D1 And P21

Intrauterine methylmercury exposure could lead to offspring brain developmental damage and neuronal cell cycle alteration. Cyclin D1 and p21 might have important functions within this cell cycle regulation. In this study, rat experimental model was established of intrauterine methylmercury exposure. Offspring brain regions were determined cell cycle alterations at different time points and with different exposure dose. In the meantime, Cyclin D1 and p21 were also determined so as to investigate their role in the cell cycle regulation.1. Effect of intrauterine methylmercury exposure on neuronal cell cycleIntrauterine methylmercury exposure could lead to cell cycle alteration. As methylmercury exposure dose increased, in cerebrum, cell percentage in G0/G1 phase was decreased gradually, whereas it was increased in S and G2/M phase.1.5mg/kg dose group had G2/M cell cycle arrest; 3.0mg/kg dose group had S phase cell cycle arrest. In cerebellum, cell percentage in G0/G1 phase decreased gradually, and increased in S and G2/M phase.0.75mg/kg dose group had S phase cell cycle arrest. In hippocampus, cell percentage in G0/G1 and G2/M phase both decreased gradually, whereas S phase showed increase as exposure dose increased. 0.75 and 3.0mg/kg dose groups had S phase cell cycle arrest. Intrauterine methylmercury exposure showed also time effect. Cell percentage decreased at G0/G1 phase, and increased at S and G2/M phase on PND14. The trend went the countrary on PND30, where cell percentage increased at G0/G1 phase, and decreased at S and G2/M phase.2. p21 mRNA determinations by RT-PCRRT-PCR was applied to determine expression of p21Wafl/cipl mRNA. In 3.0mg/kg dose group, p2lWafl/Cipl mRNA expression in cerebrum and hippocampus decreased gradually as PND prolonged, whereas in cerebellum, p21Wafl/Cipl mRNA expression was increased.On PND 14 after intrauterine methylmercury exposure, p21Wafl/Cipl mRNA expression in cerebrum increased as methylmercury exposure dose increased where significant differences were observed in 0.75mg/kg and 1.5mg/kg dose group compared with control group. p21Wafl/Cipl mRNA in hippocampus showed lower expression in any dose group compared with control group. p21Wafl/Cipl mRNA expression in cerebellum showed the same trend as in hippocampus.3. Cyclin D1, p21, PKC and CaN determination by immunohistochemistryCyclin D1 and p21 were determined by immunohistochemistry. Results showed, on PND14, Cyclin D1 protein expression in every brain region increased gradually as exposure dose increased, where compared with control group, the medium and high dose group had significant differences compared with control group (P<0.05); of the 3.0mg/kg dose group, Cyclin D1 was determined increased as time prolonged, except that on PND30, Cyclin D1 expression in cerebellum decreased but had significant differences compared with control group (P>0.05).p21 expression showed similar situation to that of Cyclin D1. As time prolonged and exposure dose increased, p21 expression showed time and dose effect.PKC and CaN expression were identical, both of which increased in each exposure group.4. Cyclin D1 expressions by Western-BlotThe Cyclin D1 expression determined by Western-Blot was similar to that by immunohistochemistry. Cyclin D1 expression presented both time and dose effect. It increased as exposure dose increased and time prolonged.From the data above, we can see intrauterine methylmercury exposure could lead to offspring rat's brain neuronal cell cycle alterations. Cyclin D1 and p21 showed dose and time effect after methylmercury intrauterine exposure in rat. Both proteins increased as time prolonged and exposure dose increased. PKC expressioin increased in offspring rat's brain after intrauterine methylmercury exposure. This increased the capability of PKC to induce cell cycle proceeding from G0/G1 to S. The higher expression of CaN might be the reason that caused Cyclin D1 over expression. The higher expression of PKC and Cyclin D1 induced cell percentage decline at G0/G1. On the other hand, CaN can inhibit the transcription level of CDK4, which leaded to inhibition of Cyclin D1/CDK4 complex. p21 had a wide range of cyclins substrate to combine with, and posed arrest effect at any cell cycle phases. The higher expression of p21 and inhibition of CDK4 together prevented cell cycle from G0/G1 to S. The positive and negative effect balanced each other and resulted in G0/G1 cell percentage insignificantly decline. At S and G2/M phases, Cyclin D1 losed to balance the p21 effect, as Cyclin D1 mainly functioned at G0/G1 phase, which leaded to cell cycle arrest at these phases. The results also demonstrated time effect of methylmercury to cell cycle. PND1, PN14 and PND30 were chosen as the experiment time points. It showed cell cycle distribution on PND30 differred from PND1 and PND14. In fact, it was contratry to the case on PND14. Cell percentage increased at G0/G1 and decreased at S and G2/M. This indicated that G0/G1 phase cell cycle arrest on PND30. Possible reason might be that, as time prolonged, p21 showed higher expression and with higher activity as a response to more severe cell apoptosis, cell aging et al, the result of which leaded unbalanced and higher negative effect than the positive function by Cyclin D1, and finally induced cell cycle arrest at G0/G1 phase.