| INTRODUCTION: Conventional surface water treatment processes are not sufficient to totally remove cyanobacterial toxins, and chronic exposure to cyanobacterial toxins via drinking water may have tumour-promotion effect. Certain disinfections by-products (DBPs) in drinking water produced during the process of water chlorination are found to be mutagenic. Coexistence of DBPs and cyanobacterial toxins in drinking water may lead to potential synergistic effects during carcinogenesis.OBJECTIVE: To investigate the joint actions of cyanobacterial toxins Microcystins (MCs) and organic extractions of water (OEWs) during carcinogenesis, and explore its potential mechanism.METHODS: Cyanobacterial toxins Microcystins (MCs) were isolated and purified from cyanobacterial samples collected during outbreaks of cyanobacterial water bloom. Isomers of MC-LR and MC-YR were quantitatively analyzed with HPLC (High Performance Liquid Chromatography). The effects of MCs on metabolic cooperation in V79 (HPRT+/HPRT-) and on cell cycle and related gene expressions in SHE cells (Syrian Hamster Embryo cell) were investigated in vitro. Organic extractions of water (OEWs) were extracted from tap water sample, and then mutagenicity screening tests including Ames test, micronuclei test (MN), unscheduled DNA synthesis induction (UDS) and HPRT mutation assay were used to detect the mutagenicities of OEWs. SHE cells transformation system in vitro was also used to evaluate the potential carcinogenecities of MCs and OEWs. At the same time, a two-stage hepatocarcinogenesis rat model (Solt-Farber Model) was established using OEWs and MCs applied as initiator and promoter, respectively. The histopathological changes in liver were observed, and expressions of GSTPi and other genes involved in MAPKs (mitogens activated protein kinase) signals pathway such as TCF/ELK-1, c-fos and c-jun during hepatocarcinogenesis were detected by means of immunohistochemical staining, Western-blot, RT-PCR (reverse-transcription PCR) and ISH (in situ Hybridization). DNA binding activity of AP-1 and GEPI binding activity of nuclei proteins were analyzed by EMSA (electrophoretic mobility shift assay) as well. The relationships among expressions of TCF/ELK-1, c-fos, c-jun, and GSTPi were analyzed.RESULTS: The results showed that MCs possessed strong acute toxicity with liver as the target organ, the LD50 was estimated to be 106.7mg/kg (89.1~127.6 mg/kg, mass of dry algal cells / b.w). The inhibited metabolic cooperation in vitro was seen in V79 (HPRT+/HPRT-) treated with MCs. Up-regulated expressions of c-fos and c-jun as well as promoted cell proliferation were also observed in SHE cell co-incubated with MCs in vitro. It was also indicated that OEWs were mutagenic and could induce SHE cell transformation in vitro. MCs alone could not induce SHE cell transformation, but could promote the cell transformation induced by OEWs.The two-stage hepatocarcinogenesis bioassay results showed that OEWs and MCs could induce hyperplastic and precancerous foci as well as nodule in livers of rats. MCs alone could up-regulate the expressions of pELK-1, c-jun, and c-fos, and enhance DNA binding activity of AP-1 and GEPI binding activity of nuclei protein in hepatocytes of rats. Further promoted up-regulation of related gene expressions were also seen in this study when rats were initiated with OEWs before the administration of MCs (p<0.05). It was also indicated that OEWs alone could induce expressions of GSTPi mRNA and protein in livers. While compared to OEWs, MCs alone could not induce the expression of GSTPi, but could promote the expressions of GSTPi mRNA and protein induced by OEWs (p<0.05). CONCLUSIONS: 1. MCs could inhibit cell GJIC (gap junctional intercellular communication) and promote cell proliferation through up-regulation of MAPKs signals pathway.2. MCs possessed tumor-promoting activity during experimental hepatocarcinogenesis in rats.3. OEWs were mutagenic and possessed initiation activity during hepatocarcinogenesis in rats. 4.
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