Effect Of Heroin On Gene Expression Of Nucleic Acid And Nucleotides Metabolism In C6 Glioma Cell

Opiates are among the most commonly abused illegal drugs all over theworld. Chronic exposure to opiates, such as morphine and heroin, can causeserious physical dependence and psychological dependence. It is still animportant task to investigate the basis of tolerance and dependence. Therelationship between opiate addiction and changes in nucleotide catabolismremains poorly understood. In this study, we choose rat C6 glioma cells anddivide into four group that exposure to heroin for 12h, 24h, 48h and 72h. Eachgroup set up with control and experiment group. MTT colorimetry methodsverify that heroin can inhibit cell multiplication. Though this essay we can alsodetermin the suitable density of heroin which is 20μg/mL. We study theinfluence of nucleic acid metabolism of heroin by using PCNA and TFⅡF. Asthe same time we clarify whether heroin affects the catabolism of purinenucleotides by regulating gene expression of ADA, XO, HGPRT and AKwhich are key enzymes of purine nucleotide metabolism to probe the substantialbasis of heroin dependence and tolerance and provide a new way for ourresearch.1. We worked over the effect of heroin on the proliferation of rat C6 gliomacells. Using MTT colorimetry methods, we investigated whether heroin hadinhibitory effects on rat C6 glioma cells which hadn't been treated with anystimulative factors before. The results showed that after exposured to heroin for24 hours, the proliferation of rat C6 glioma cells had been obviously inhibited.This inhibition is maybe caused of the adverse reaction. To depletion that possiblewe determine the cell vigor by trypan blue method. The results indicate that thereis no significant deviation between control and heroin group and cell vigor are allover 90 percent. This can confirm that the decrease of cell growth is not causedby cell death.2. We investigated the effect of heroin on nucleic acid metabolism. The geneexpression of proliferating cell neuclear antigen (PCNA), which played key rolein DNA synthesis. Using RT-PCR method, we found that heroin decreased themRNA level of PCNA. Since PCNA played important role at replicating fork inDNA synthesis, and had relationships with both the leading strand's and thelagging strand's synthesises, so we considered that it was the inhibition of PCNAgene expression that caused the proliferating inhibition of C6 cells by heroin.Using RT-PCR method, we investigated the effect of heroin on expression ofgeneral transcription factor gene TFⅡF which played important role in genetranscription (RNA synthesis). The results indicated that heroin decreased themRNA level of TFⅡF in rat C6 glioma cells. Since general transcription factorTFⅡF played key role in eukaryotic gene transcription, it indicated that heroinmight have inhibitory effects on RNA synthesis generally. Our research studiedthe effect of heroin on rat C6 glioma cells of nucleic acid metabolism, used TFⅡF and PCNA as target genes which played important roles in RNA and DNAsynthesis respectively. Our plan has innovations, we have'nt found similarresearch reports published yet.3. HGPRT,AK,ADA and XO mRNA detection of heroin dependent C6cells: heroin administration can increase the catabolism of purine nucleotide anddecrease purine salvage pathway.3.1 Effect of heroin on gene expression of enzymes catalyzing purinesalvage pathway.HGPRT transcript was decreased respectively in 12h,24h and 48h afterexposure of heroin as compared with control in corresponding time point , butHGPRT mRNA level showed little lower than corresponding control afterexposure of herion for 72h. HGPRT is the key enzyme of salvage pathway ofpurine nucleotides synthesis. The inhibition effect of heroin on HGPRT geneexpression maybe one of the mechanism of chromid heroin causing inhibition ofDNA synthesis on C6 cells. Many studies have shown that opioid and adenosinemay interact at cellular level. In this experiment there was significant AKtranscript changes in C6 cells after exposure of heroin for 24h and 48h ascompared with corresponding control, but AK mRNA level was increased after72h exposure of heroin. The heroin exposure on C6 cells causing inhibition ofAK gene expression might be one of the mechanism of increase of adenosinerelease.3.2 Effect of heroin on gene expression of enzymes catalyzing purinecatabolism in C6 glioma cell.In order to investigate the mechanism of heroin's effect on ADA and XO,RT-PCR was used to examine the gene transcripts of ADA and XO. Comparedwith that in the control group, the transcripts of ADA mRNA were significantlyhigher in cells treated with heroin. Compared with the heroin treatment groups,the effect of heroin decreased gradually in the XO mRNA levels. In summary,acute and chronic heroin administration can increase the catabolism of purinenucleotide by regulating the gene expression of key enzymes of ADA and XO.The second part of determination of effect of heroin on C6 glioma cell ADAand XO gene expression by RT-PCR in this experiment in exposure of heroin atall time point to C6 cells caused an enhancement of ADA gene expression, whileXO transcripts increased respectively in 12h ,24h, 48h and 72h after exposure ofheroin as compared with control in corresponding time point. The results indicatethat heroin can enhance the catabolism of purine nucleotide in cell level byregulating the gene expression of two key enzymes of ADA and XO.In summary, heroin administration can increase the catabolism of purinenucleotide by regulating two key enzymes ─ADA and XO in vitro. Andwithdrawal of heroin may related to salvage pathway synthesis of purinenucleotides and catabolism pathway of purine nucleotides. The key enzymessuch as HGPRT , AK, ADA and XO. This may be the basic effect of heroin andmay be one of the main causes of heroin addiction.