[Natural Water Chemistry Change in the Surface Water of Chengdu and Impact Factors].

To investigate the impact of megacities on the chemistry of surface waters, monthly sampling and monitoring were conducted in the Chengdu section of the Minjiang and Tuojiang River basin, corresponding to the upper reaches of the Yangtze River since the spring of 2019, including the influent and effluent water samples from 57 sewage treatment plants in Chengdu. All the samples were analyzed for major ions and other water chemistry parameters, and compared with the historical data of the Minjiang and Tuojiang River. The results showed that the Chengdu surface water still presented a natural chemistry with medium-low total dissolved solids(TDS), and calcium bicarbonate chemistry type, which is the natural consequence of the weathering of carbonate rocks in the basin effected by the weathering of silicates and evaporites. The natural water chemistry of the surface waters in Chengdu presented monthly variation, i.e.,the concentration of major ions and TDS was higher in the dry season compared to the wet season, reflecting the variations of point source. Spatially, the concentration of major ions and TDS downstream of the city was higher than those in the upper reaches, and the concentration in the tributary was higher than that in the mainstream, which may reflect urban influence. Further analyses, such as simulation calculations, indicated that urban activities were the major driving factor for the chemistry change in the surface waters in Chengdu, which is evidenced by the significant contribution of the sewage discharge to the elevated Cl- and Na+ and the ratio of hardness/alkalinity>1 from anthropogenic acid gas emissions. A comparison with the water chemistry of the Minjiang and Tuojiang River in the 1960s indicated that, the current Cl-/Na+ ratio has significantly increased, which has been evidenced by a salinization trend. As a megacity nearest to the source of the Yangtze River, the impact of Chengdu on the natural water chemistry of the Yangtze River system and its environmental effects deserves more attention.

[Changes in Water Chemistry and Driving Factors in the Middle and Lower Reaches of the Beijing-Hangzhou Grand Canal].

To reveal the Beijing-Hangzhou Grand Canal natural water chemistry characteristics and the influence of human activities, river samples from Xuzhou to Jiaxing were collected in 2019-2020. Simultaneously, the water chemistry data of the canal from 1959 to 1962 and 1975 to 1977 in the Suzhou, Wuxi, and Changzhou sections and the recent social and economic data of the major cities along the canal were collected and analyzed. The results showed that the type of hydrochemistry in the study area was mainly influenced by the weathering of carbonate rocks in the basin, but K++Na+ accounted for 40.39% of the cation equivalent concentration, which was higher than that in ordinary surface water, thereby indicating that the natural hydrochemistry of the canal had been significantly affected by human factors. Spatially, the major ion mass concentrations, total hardness, and total alkalinity of the Grand Canal from Xuzhou station to the downstream area tended to decrease overall, but the parameters at Wuxi and Suzhou stations increased significantly. It was found that Na+ and SO42- were increased by approximately 16 and 12 times and total dissolved solids was increased by nearly 3 times by analyzing the 60 years of water chemistry of the Suzhou, Wuxi, and Changzhou sections. The current (Ca2++Mg2+)/HCO3- ratio in the Suzhou, Wuxi, and Changzhou sections is generally greater than 1, which is significantly higher than that from 1959 to 1962, thereby reflecting the results of human activities. According to the analysis of the social and economic development of the Grand Canal, this change was the result of the accelerated weathering of carbonate rocks in the basin caused by the sulfur oxides discharged by human activities. Further statistical analysis showed that urban domestic sewage and industrial wastewater discharge were the main driving factors causing chemical salinization of natural water in the Grand Canal. This study can provide a scientific basis for coordinating urban development and protecting the water ecological environment of the Grand Canal Basin.

Regulation of hMSH2 and hMLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1.

Aberrant DNA methylation is now recognized as an important epigenetic alteration occurring early in human cancer. To directly study the role of DNA methyltransferase 1 (DNMT1) in the regulation of expression of tumor-related genes in human colon cancer cells, we stably transfected expression constructs containing sense or antisense DNMT1 into the human colon cancer cell line, SW1116. The expression level of mismatch repair genes (MMR), human mut-L homologue 1 (hMLH1) and human Mut S homologue 2 (hMSH2), was monitored by real-time RT-PCR. The methylation status of hMLH1 and hMSH2 promoters was determined by bisulfite modification and methylation-specific PCR (MSP). The protein levels of DNMT1, hMSH2 and hMLH1 were determined by Western analysis. The results show that DNMT1 protein expression was increased or decreased in transfected cell lines containing sense or antisense DNMT1 constructs, respectively. In cells expressing the sense DNMT1 construct, the expression of hMLH1 and hMSH2 was down-regulated through hypermethylation of their respective promoters. Furthermore, antisense DNMT1 expression induced promoter demethylation and up-regulated transcription of hMSH2 (P<0.05) and hMLH1 (P=0.064) in SW1116 cells.

Expression of Dnmt1, demethylase, MeCP2 and methylation of tumor-related genes in human gastric cancer.

AIM: To explore the effect of DNA methyltransferase, demethylase and methyl-CpG binding protein MeCP2 on the expressions and methylation of hMSH2 and proto-oncogene in human gastric cancer. METHODS: Paired samples of primary gastric cancer and corresponding para-cancerous, non-cancerous gastric mucosae were obtained from surgically resected specimens of 28 patients. Transcription levels of Dnmt1, mbd2, MeCP2, p16(INK4A), hMSH2 and c-myc were detected by using real-time PCR or RT-PCR. Promoter methylation of p16(INK4A), c-myc and hMSH2 genes was assayed by methylation-specific PCR (MSP) and sequencing (mapping). Their relationships were analyzed by Fisher's exact test using the software SPSS. RESULTS: The average mRNA level of Dnmt1 gene from cancerous tissue was higher and that of mbd2 gene from cancerous tissue was lower than that from non-cancerous tissue, respectively. mbd2 was lower in cancerous tissue than in non-cancerous tissue in 14 (50.0%) of patients but higher in 3 cases (10.7%) of non-cancerous gastric tissue (P<0.001). c-myc expression was up-regulated in cancer tissues (P<0.05). The up-regulation of mbd2 was found in all patients with hypomethylated c-myc. The transcriptional levels of p16(INK4A) and MeCP2 genes did not display any difference between gastric cancerous and matched non-cancerous tissues. There were down-regulation and hypermethylation of hMSH2 in cancer tissues, and the hypermethylation of hMSH2 coexisted with down-regulated transcription. However, the transcription level of the above genes was not associated with biological behaviours of gastric cancers. CONCLUSION: The up-regulation of proto-oncogene may be the consequence of epigenetic control of gene expression by demethylase, and mbd2 is involved in the regulation of hMSH2 expression in human gastric cancer.

Histone acetylation regulates p21WAF1 expression in human colon cancer cell lines.

AIM: To investigate the effect of histone acetylation on regulation of p21WAF1 gene expression in human colon cancer cell lines. METHODS: Two cell lines, Colo-320 and SW1116 were treated with either trichostatin or sodium butyrate. Expressions of p21WAF1 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Acetylation of two regions of p21WAF1 gene-associated histones and total cellular histones were examined by chromatin immunoprecipitation assay and Western blotting. RESULTS: Trichostatin or sodium butyrate re-activated p21WAF1 transcription resulted in up-regulated p21WAF1 protein level in colon cancer cell lines. Those effects were accompanied by an accumulation of acetylated histones in total cellular chromatin and p21WAF1 gene-associated region of chromatin. CONCLUSION: Histone acetylation regulates p21WAF1 expression in human colon cancer cell lines, Colo-320 and SW1116.

[Effect of eukaryotic expression plasmid DNA methyltransferase gene on methylation status and transcription level of DNA mismatch repair genes in human colon cancer cell line].

OBJECTIVE: To analyze the effect of eukaryotic plasmids containing sense or antisense DNA methyltransferase (Dnmt1) genes on the methylation status and transcription level of DNA mismatch repair (MMR) genes and microsatellite instability (MSI) in human colon cancer cell line. METHODS: Human colorectal cells of the SW1116 line were cultured. Recombinant plasmids containing sense Dnmt1 (HMT) or antisense Dnmt1 (THM) gene, pCMV-HMT and pCMV-THM, were constructed. Then pCMV-HMT, pCMV-THM, and pcMV blank plasmid were transfected into SW1116 cells respectively by using lipofectAMINE. The expression of Dnmt1 protein was examined by Western blotting. The transcription levels of hMLH1 and hMSH2 genes were detected by using real-time (RT-PCR). The status of methylation in promoters of hMLH1 and hMSH2 genes were examined with methylation specific PCR (MSP). The MSI of DNA in SW1116 cells was evaluated by silver-stained polyacrylamide gel electrophoresis. RESULTS: Both the expressions of the hMLH1 and hMSH2 gene mRNAs were remarkably decreased in the SW1116-HMT cells in comparison with those in the untransfected cells. The expression of hMSH2 gene mRNA in the SW1116-THM cells was remarkably increased in comparison with that in the untransfected cells. No significant difference in the expressions of the hMLH1 and hMSH2 gene mRNAs was found between the SW1116 cells transfected with blank pCMV and the untransfected SW1116 cells. MSP showed that the methylation level in the regions of hMLH1 and hMSH2 promoters was remarkably increased in the SW1116 cells transfected with sense Dnmtl plasmid. However, in the SW1116 cells the hMSH2 promoter region was changed from partially-methylated into de-methylated, and the hMLH1 promoter region remained non-methylated. MST test showed that extra bands indicating MSI were seen only in the D2S123 groups. CONCLUSION: Dnmt1 regulates the expression and methylation status of MMR genes and affects MSI in human colon cancer cell line SW1116.

5-Aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16INK4A gene in human gastric cancer cell lines.

BACKGROUND: To investigate the effects of DNA methylation on the expression of tumor-associated genes and the cell cycle in human gastric cancer cells. METHODS: Two gastric cancer cell lines (MKN-45 and HGC-27) were treated with DNA methyltransferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC). The expressions of p16INK4A, p21WAF1, p53, p73, c-Ha-ras and c-myc genes mRNA were detected by using reverse transcription PCR (RT-PCR). DNA methylation status of p16INK4A gene promoter was assayed by bisulfite modification and sequencing. The cell cycle was analyzed by using flow cytometry (FCM). RESULTS: 5-aza-dC induced the demethylation of p16INK4A gene promoter. The expression of p16INK4A mRNA was obviously up-regulated by treatment with 10 micro mol/L (MKN-45 cells) or 5 micro mol/L (HGC-27 cells) of 5-aza-dC for 24 hours. However, 5-aza-dC treatment failed to regulate the expressions of p21WAF1, p53, p73, c-Ha-ras and c-myc genes in MKN-45 and HGC-27 cells. Furthermore, 5-aza-dC induced the cell cycle arrest in G1 phase in HGC-27 cell, but not in MKN-45 cell. CONCLUSIONS: DNA methylation regulates the transcription of p16INK4A but not p21WAF1 and proto-oncogenes in human gastric cancer cell lines MKN-45 and HGC-27.