Effects Of Benzene On Mice Lymphocytes Telomerase Activity And Regulations Of Selenium In Vivo

Benzene Hydrocarbon is a kind of aromatic hydrocarbon, mainly achromatous or pale yellow transparent and evaporable oleaginous liquid, rich in aromatic odor, they including Benzene, Toluene, Xylene and so on. Benzene Hydrocarbon can lead to inhibiting of bone marrow and immune system, mainly cause hematotoxicity, can result in cytotoxicity and genotoxicity. They can give rise to many kinds of biological effects, cause hematopoietic cells apoptosis and influence hepatocytes and progenitor cells replication. They inhibit cytokine secretion, influence on T lymphocytes function and variation of its subpopulation in peripheral blood. They give rise to peripheral blood hemogram variation, cause lymphocytes and erythrocytes reduction, lead to micronuclei. They induce cells apoptosis, mutation and replication, resulting in DNA breakage, leading to lymphocytes aberration in structural and other aspects, cause chromosome damage, including aneuploidy, chromosome deletion, chromosome translocation, and so on. After benzene was absorbed in human bodies, it was transported into liver, after activated and metabolized under the mediation by cytochrome P-450 monooxygenase, their metabolin hydroquinone, Phenol, intermediates were distributed to blood, Kidney, Spleen, Thymus Gland and Bone Marrow Hematopoiesis system. They targeted at lymphocytes and myeloid cells, injured chromosome, they lead to Hematologic disease and carcinogenesis. It can cause Aplastic Anemi, Acute Myeloid Leukaemia (AML), Hemangioma, Myelodysplastic Syndrome (MDS), Thymic Lymphoma, Chronic Lymphadenoma, Chronic Myelogenous Leukemia (CML) and other Hematogenic Neoplasma. Benzene is showed to be a carcinogen cause multi-organ neoplasma. Benzene and its metabolite effect preferential on specific non-lethal chromosome, elimination of telomere has been reported to be a selective chromosome, it is a sensitive chromosome preferential effect on and damage in the chromosome will be detected more selectively. Researches on benzene ranged from its genotoxicity, chromosome aberration and carcinogenesis mechanism. Recent work on benzene toxicity has provided insight into its gene level from chromosome damage.The telomerase, is a ribonucleoprotein comprised by RNA and protein that functions as a reverse transcriptase, it consists of three components, telomerase RNA component (TERC), telomerase-associated protein (TELPI) and catalytic subunit that possesses reverse transcriptase activity (TERT). Telomerase uses its RNA component as a template for the addition of G-rich repeats to the 3'end of telomeric DNA, thereby compensating for the telomere loss, fusion, and preventing homologous recombination to avoid shortening associated with cell division. This escape cells from apoptosis. Telomerase activity is undetectable or express low level in most somatic cells and normal tissues, but activated and expressed high level in germ cell, embryonic cell, hemopoietic stem cell, myeloid cell and most carcinoma such as neoplasma and hematologic malignancies. Telomeres, the protective caps of eukaryotic chromosomes are the physical ends of eucaryotic chromosomes, prevent the chromosome from end-to-end fusion, recombination, and degradation, which lead to chromosome instability and cell death. The gradual loss of telomere repeats in cells appears to be a major component of cell senescence and tissue ageing, limit cellular proliferation and develop to tumor. Telomerase function as elongating telomeric RNA and bypass crisis, allowing indefinite replicating to immortal cells. Cells senescence, apoptosis and immortalization resulting in critical telomere shortening, telomerase is activated and then triggering the carcinogenesis and neoplasma, telomerase activity is strong expressed in 80~90% of human cancers, telomerase activity rise with the acuteness and aggressiveness of tumor and carcinoma. It is well accepted that the re-activation of telomerase is the common final pathway of malignant tumor occurrence and telomerase plays an important role in tumorigenesis. Telomerase is expressed to detect tumors as a specific and popular effect biomarker and early biomarker. Since regulation of telomerase activity and telomere length are critical in maintaining stability of chromosome and benzene is known for its tumorigenesis in human cells, alterations in telomerase activity and telomere may be associated with the chromosomal damage exerted by benzene.Selenium, a trace element, is nourished to cells, it functions nutrition by antioxidant effects and clearing free radical on human bodies. Low dose selenium can activate telomerase activity, enlongate telomere length and increase cells life span. It has protection role on preventing tumor initiation, development, meanwhile, it has prevention of ageing, specific cancers and antitumorigenic effects in postinitiation phases of cancer.Benzene is toxic to cells after metabolized by enzymes, therefore, we select animals to construct models to observe its toxicity and appreciate influence performance. Healthy Kunming mice were selected to perform on, they are randomly divided into several groups. We designed different benzene treated groups with different doses to exert on mice, defined different doses of benzene combined with selenium to treat on mice, meanwhile, negative control and reagent control was established. After treated with different groups, we observed the indications, obtained peripheral blood then isolated lymphocytes, and then TRAP (telomere repeat amplification protocol assay) was used to detect telomerase activities in lymphocytes. According to the telomerase activity expression in different treated groups, we observed their different influence on telomerase activity, analyzed benzene and selenium treated groups varied on influence, we found out their correlation. In the present paper, we shed light on the telomerase activity changes by benzene toxic influence on mice lymphocyte and the regulation function of selenium in vivo, and to further understand the molecular mechanism of benzene induced neoplasma and tumorigenesis, aim to find out a new biomarker of the early effect and biomarker of susceptibility for benzene exposure from telomerase system.PartⅠEffects of benzene on mice lymphocytes telomerase activity and regulations of selenium in vivoObjective: We researched on the telomerase activity changes by benzene toxic influence on mice lymphocytes and the regulation function of selenium in vivo, and to further understand the molecular mechanism of benzene induced neoplasma and tumorigenesis, aim to find out a new biomarker of the early effect and biomarker of susceptibility for benzene exposure from telomerase system. Methods: 40 male healthy Kunming mice aged from 6~8 weeks, weighted from 25-30g were randomly divided into 8 groups, with 5 animals in each group. Three benzene treated groups were designed with different doses, separately they were 100.0 mg/kg benzene, 200.0 mg/kg benzene and 400.0 mg/kg benzene; three benzene combined with selenium treated groups were established with different doses, they were 200.0 mg/kg benzene + 0.75 mg/kg selenium, 200.0 mg/kg benzene + 1.5 mg/kg selenium and 200.0 mg/kg benzene + 3.0 mg/kg selenium individually; two control groups were also set up, they were negative control and reagent control. Abdominal injection was adopted to treat mice, injection lasted for 5 days in succession, after last injection and 48h later, 1 ml heparinized PBMC was obtained from eyeball removal, lymphocytes were then isolated by kit. To quantitative detect telomerase activity, TRAP-ELISA Kit was used to assay. In the first step, telomerase adds telomeric repeats (TTAGGG) to the 3'end of the biotinlabeled synthetic P1-TS-primer. In the second step, primers P1-TS and P2 were used by PCR to amplify elongation products with telomerase 6 nucleotide increments. Then, after PCR, 5μl PCR product was denatured and hybridized to a digoxigenin-(DIG)-labeled probe, the mixture was immobilized to a streptavidin-coated microplate, finally, the probe was visualized by virtue of peroxidase metabolizing TMB to form a colored reaction product. The absorbance of the samples at 450 nm was measured with a reference wavelength of 690 nm. The telomerase activity was valued by A450nm– A690nm units. Telomerase activity differences were compared between different groups, effects of benzene on telomerase and regulations of selenium were analyzed. Data was analyzed with one-way ANOVA by SPSS13.0. Results: The telomerase activity of lymphocytes from negative control group and reagent control group located in normal range which was judged as negative. It indicated that normal mononuclear cells do have demonstrated a low but marginal level telomerase activity. Among lymphocytes from mice treated with benzene at different doses, the telomerase activity was positive in all groups. The mean values were increased and higher than negative control and reagent control groups, at the dose of 100.0 mg/kg it was significantly increased compared to control groups with a P<0.01. It indicating that benzene activated lymphocytes telomerase expression. For cases of benzene combined with selenium at different concentrations, the telomerase activity was positive in all groups, the mean values were higher than negative control and reagent control groups. Compared with 200 mg/kg benzene, they were much increased, at the dose of 200 mg/kg benzene + 0.75 mg/kg selenium it was significantly enhanced with a P<0.05. Compared with negative control and reagent control groups, they were remarkably increased in all groups, at the dose of 200 mg/kg benzene + 0.75 mg/kg selenium it has a significance of P<0.01. It demonstrated that selenium activated telomerase activity. Conclusions: It is presented in our paper that, telomerase activity is increased after treated by benzene at different concentrations demonstrated that mice lymphocytes are activated and proliferated in vivo, telomerase activity might be a sensitive early biomarker of lymphocytes proliferation by benzene. Selenium can up-regulate mice lymphocytes telomerase activity. It is suggested that telomerase can be used as a biomarker of the early effect for benzene exposure to induce neoplasma and tumorigenesis. PartⅡRapid detection of Enterovirus by Real Time Quantitative RT-PCRObjective: To develop a method of rapid detection of Enterovirus by using Real Time Quantitative RT-PCR. Methods: Enterovirus gene sequences were downloaded from Genebank, using TaqMan technique, we designed a set of universal primers and TaqMan probe according to the highly reserved sequence of enterovirus gene and foreign literature. Firstly, the specificity of real time RT-PCR was detected. PolioⅠ, PolioⅡ, PolioⅢ, EV71, CA16 and CA24 standard virus were used as positive standards, Rotavirus and Norwalk virus were used as controls, a set of negative control was established to examine specificity. Then primers, probe and mixture concentrations and reactive condition were optimized. Secondly, standard curves of real time RT-PCR were established. Use EV71 as standard, we constructed plasmid standard by common primers. After quantified by detected absorbance at 260nm and 280nm, standard plasmid was serially diluted by ten-fold (108-104 copies/μl) and detected by real time RT-PCR we developed to construct quantitative standard curve. Meanwhile, a titrated viral culture from the EV71 strain was used as standard to detect sensitivity curve, according to the TCID50 the cultured virus stock was serially diluted ten-fold from 10 to 107 times in sterile water, real time RT-PCR we developed was used to construct sensitivity curve by setting a negative control. Lastly, to test the clinical specimens, we collected 2006 suspected enterovirus infection patients'feces samples in Shenzhen city, a total of 89. The specimens were tested by conventional RT-PCR using common primers, then we used real time RT-PCR to detect the samples. Compared with the two results, we testified the real time RT-PCR application in clinical diagnoses. Results: The real time RT-PCR we developed has high specificity, PolioⅠ,PolioⅡ,PolioⅢ,EV71,CA16 and CA24 positive standards had fluorescence signal increased, Rotavirus, Norwalk virus and negative control didn't observed fluorescence value, it was tested no false positive results. We successfully established quantitative standard curve by plasmid standards constructed by EV71 strain, the detection limit of the standard curve was 105 copies /μl, the slope rate of the curve was -3.474 with an intercept in Y axis was 53.67, the data showed a strong linear relationship with a R2 at 0.999, the efficiency of PCR amplification in our method was 94%. After viral titrated culture the TCID50 of EV71 strain was based on to construct sensitivity standard curve, the lowest concentration of our system was 5.0 TCID50, the slope rate of the curve was -3.230 with an intercept in Y axis was 16.36, the R2 of the standard curve was 0.991, and the amplification efficiency of our method was 104.0%. It proved that we constructed standard curves with a high correlation coefficient in each concentration. Amplification on templates was completely in PCR reactions. On detection of clinical specimens, 89 stool samples from suspected enterovirus infection patients were tested by conventional RT-PCR then by our real time RT-PCR. The result of conventional RT-PCR detected 56 positive hand-foot-and-mouth diseases (HFMD), the detection rate was 62.92%. By using real time RT-PCR it detected 72 positive, real time RT-PCR has a detection rate much higher than conventional RT-PCR, the detection rate was 70.9%. It proved the two methods have a high coincidence at 79.78%. It can be applied to routine screening work on suspected enterovirus infection patients in clinical. Conclusions: We have successfully developed a Real Time RT-PCR to rapid detect enterovirus using universal primers. It is triumphing in high sensitivity, good specificity, high efficiency, strong stability, accuracy and not labor-consuming. When screening clinical specimens it has a high detection rate and a good coincidence, proved that it can be used to rapid detect enterovirus, applied to routine screening specimens from suspected enterovirus infection in clinical.