Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay

The potential harm of organic pollutants in drinking water to human health is widely focused on in the wodd; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference （P 〈 0.01） was observed when compared with the solvent control, The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100x; the degree of DNA damage treated by Hushu waterworks （at town level） was the most serious, the arbitrary units （AU） was 141.62±6.96, however, that of Shangyuanmen waterworks （at city level） was only 109.64±2.97. The analysis also revealed that the genotoxicity of town＇s water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

Objective To study the gene expression of metallothionein 1 （MT-1） isoforms in human peripheral blood lymphocytes （HPBLs）. Methods The expression of mRNA representing the seven active MT-I genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-IX, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT- 1 H, IF, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference （P〈0.05）. in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, IF, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1 G, MT-1 H, MT-1 F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

Human papilloma virus 16 E6 oncoprotein associated with p53 inactivation in colorectal cancer

AIM:To investigate the association between human papilloma virus(HPV) infection and colorectal cancer.METHODS:Sixty-nine patients with pathologically confirmed primary colorectal cancer including 6 stage Ⅰ,24 stage Ⅱ,21 stage Ⅲ,and 18 stage Ⅳ patients were enrolled in this study to investigate whether HPV 16 could be involved in colorectal tumorigenesis.Nested-polymerase chain reaction(nested-PCR) was used to detect HPV16 DNA in colorectal tumor tissues and further confirmed by in situ hybridization(ISH).In addition,immunohistochemistry analysis was performed to examine the E6 oncoprotein in colorectal tumors.To verify whether E6 could inactivate the p53 transcriptional function,the levels of p21 and Mdm2 mRNA expression were evaluated by real-time reverse transcription(RT)-PCR.RESULTS:Of the 69 colorectal tumors,HPV16 DNA was detected in 11(16%) by nested-PCR,and HPV16 DNA was present in 8 of the 11(73%) tumors which was confirmed by ISH.The presence of HPV16 DNA in colorectal tumors was not associated with patients' clinical parameters including age,gender,smoking status,tumor site;however,HPV16 infection was more common in stage Ⅰ patients than in late-stages patients(Ⅱ,Ⅲ and Ⅳ).We next asked whether HPV16 infection could be linked with colorectal cancer development.Immunohistochemical data indicated that 8 of the 11 HPV16 DNA-positive tumors had E6 oncoprotein expression.Moreover,we also observed that the adjacent normal tissues including endothelial cells,lymphocytes,fibroblasts,and gland cells in E6-positive tumors had E6 oncoprotein expression.In addition,3 of the 4(75%) E6-positive tumors carrying p53 wild-type had negative immunostaining,but one tumor had less p53 immunostaining.We further examined whether E6-positive and/or p53 mutated tumors reduce p53 transcriptional activity.Real-time RT-PCR analysis indicated that p21 and mdm2 mRNA expression levels in E6/p53-wildtype tumors were significantly lower than in their adjacent normal tissues;as expected,E6-positive/p53-mutated tumors had lower p21 and mdm2 mRNA expression levels compared with their adjacent normal tissues.These results clearly indicate that the E6 oncoprotein expressed in p53 wildtype tumors may reduce p21 and mdm2 expression via p53 inactivation.CONCLUSION:These results suggest that HPV16 infection may be involved in a subset of colorectal cancer,and we suggest that the transmission of HPV to the colon and rectum might occur through peripheral blood lymphocytes.

Investigation of the cytotoxicity,antioxidative and immune-modulatory effects of Ligusticum porteri(Osha) root extract on human peripheral blood lymphocytes

OBJECTIVE： Ligusticum ported is a traditional Native American herb. The roots of L. ported are traditionally used in the treatment of many diseases, however, its cytotoxicity, antioxidative and immune- modulatory effects need to be investigated. In this study, we evaluated the effects of the root extract at different doses on human peripheral blood lymphocytes （PBLs）. METHODS： The lymphocytes were incubated with different concentrations of the root extracts （0, 50, 100, 200, and 400 μg/mL） and harvested every 6 h for 2 d （P〈0.05）. The protective effect of the herb against oxidative damage was determined by inducing oxidative stress with the administration of 50 μmol/L of hydrogen peroxide （H202）. RESULTS： Treatments with L. ported at 200 and 400 pg/mL increased the viability of PBLs. The deleterious effect of H2O2 was ameliorated by 400μg/mL L. ported treatment. Addition of 400 μg/mL L. ported reduced lipid peroxidation in stressed PBLs by 94% （P〈0.05）. Treatment with 400 μg/mL of L. ported resulted in a 26.4% increase of reduced glutathione levels. Activities of superoxide dismutase and catalase increased by 17.5% and 55.2% respectively, when stressed PBLs were treated with 400 μg/mL L. ported for 2 d （P〈0.05）. Treatment with 400 μg/mL L. ported increased interferon-γand interleukin-2 expressions in H2O2-challenged PBLs （P〈0.05）, however, the root extract did not cause a significant difference in interleukin-10 levels compared to the control （P〉0.05）. CONCLUSION： The findings suggest that L involving protective effects against oxidative ported might be a potential immune-modulating agent damage.