Study On The Toxic Effects Of MC-RR And Cyanobacterial Bloom Extract On Mouse Liver By Oral Administration At Biochemical Level

The frequent occurrences of the toxic cyanobacterial (specifically Microcystisaeruginosa) bloom are becoming a global environmental issue. Microcystin produced bycyanobacteria in diverse water systems is a potent specific hepatotoxin and has beendocumented to induce hepatotoxicity and tumor promotion. There are more than eightyreported microcystins and microcystin-RR (MC-RR) is one of the most widelydistributed variants among the MC family. One of the mechanisms of toxicity of thesetoxins is their inhibition on protein phosphatases 1 and 2A, leading to increased proteinphosphorylation, which is directly related to their cytotoxicity and tumor-promotingactivity. Acute microcystin poisoning in mammals is characterized by disruption ofhepatic architecture due to phosphorylation of cytoskeletal proteins leading to massiveintrahepatic haemorrhage and death in few hours and chronic uptake of microcystinsresults in generalized hepatocyte degeneration with necrosis, progressive fibrosis andmononuclear leukocyte infiltration. At present, the exact mechanisms by which MCinduce hepatotoxicity have not been fully elucidated. Several pieces of evidencesstrongly suggest that apoptosis induced by MC may play a significant role in thepathogenesis of MCs-induced toxicity in mammals, but its detailed mechanism is notclarified so far. It has been documented that oxidative stress and mitochondrial injure are the main elements involved in MC-triggered apoptosis, which means mitochondrialpathway may play a key role in the apoptosis induced by MCs. Nowadays anotherapoptotic pathway, which is iniated by the extress ER stress exceeding the homeostasisof ER, has been proposed by some researchers, however there is little knowledge aboutthe role of ER pathway in the MC- induced apoptosis.Among the study on MC variants, the toxic effects of MC-RR have not beenstudied extensively, especially in mammals and via oral exposure. Though the toxicityof MC-RR is less than MC-LR, but its toxic effect should be paid attention to sinceaccumulated evidences have shown that MC-RR is the dominant toxin produced bycyanobacterial bloom in some water bodies, especially in China.Until now, there are varieties of researches related to the toxicity of purifiedmicrocystins, but little is reported on the toxic effects of cyanobacteria crude extract,whose components are most similar to the natural environment water bodies (wherecyanobacteria bloom occurred), but all the organisms are living in a complexcircumstance where multiple effect factors exist. To study the complicated toxic effectof cyanobacteria is very important for us to evaluate the risk of human and animalsexposed to these toxins.In this work, an in vivo approach was applied to administrate MC-RR andcyanobacterial bloom extract orally to mice and multiple indicators were measured. Theaim of this work is to investigate the toxic effect of MC-RR and cyanobacteria bloomextract on mice liver. In current work, the two kinds of preparation of microcystinhave been administered orally to ICR mice for 7 days with different dosages. In MC-RRtreated mice, apoptotie cell death in liver was detected by TUNEL assay, and theexpression levels of Bcl-2, Bax and p53, GRP 78 and CHOP which have been reportedto be related to apoptosis and ER stress were determined via western-blot. The activityof PP2A was measured using the serine-threonine phosphatase assay system, and PP2AA subunit and C subunit expression is measured by western blot, the oxidative stress isdetermined by ROS and MDA assay, and the calpain activity is investigated todetermine its role in MC-RR induced apoptosis. In cyanobacteria bloom extract-treatedmice, the expression levels of Bcl-2, Bax, GRP 78 and CHOP, HSP 70, PP2A-A subunit were determined via western-blot ; the oxidative stress is determined by ROS and MDAassay.The results were showed as below:1. MC-RR exposure has induced significant apoptosis in mice liver.2. MC-RR exposure has no significant effect on ROS lever in mice liver, except for93μg/kg treated group. There is no distinct change on MDA content in anyMC-RR treated group.3. MC-RR exposure has no distinct effect on PP2A activity or its A/C subunitexpression level in mice livers.4. MC-RR exposure significantly increased the expression level of p53 and Bax anddecreased the level of Bcl-2 in mice livers. The ratio of Bax/Bcl-2 was increaseddistinctly.5. MC-RR exposure significantly increased the expression level of liSP70 and GRP78,and the level of CHOP had not changed obviously..6. MC-RR exposure has no distinct effect on calpain activity in mice livers.7. Cyanobacteria bloom extract exposure significantly increased the expression levelof Bax and PP2A A subunit in a dose-dependent manner, but there was no distincteffect on Bcl-2 level in mice liver. However, the ratio of Bax/Bcl-2 was increasedobviously.8. Cyanobacteria bloom extract exposure has no obviously effect on oxidative stresssystem in mice livers, based on ROS level and MDA content in mice liver.9. Cyanobacteria bloom extract exposure obviously increased the expression level ofHSP70 and GRP78, but there was no distinct effect on CHOP expression level inmice livers. Conclusions:There is no significant change in PP2A activity or subunit expression level after themice were orally exposed to MC-RR for 7 days. In this study, the inhibition of PP2Aand the induced oxidative stress is not a major mechanism of the MC-RR toxic effect.The apoptosis in MC-RR treated mouse livers is proportional to MC-RR dosage, andaccompanied with increased expression level of p53 and Bax and decreased level ofBcl-2. This suggests that the apoptosis is closely related to the mitochondrial pathway.MC-RR exposure can induced moderate ER stress and the ER apoptotic pathway hasnot initiated.Cyanobacteria bloom extract can induce moderate ER stress and increase theexpression level of Bax, and HSP70. The obvious change of PP2A A subunit indicatedthat the toxic effects exerted by cyanobacteria bloom may be related with its interferingon signal pathways regulated by PP2A.This study concludes that both pure MC and cyanobacteria bloom have negativerole on mice, even at a low dosage. The toxicity of oral exposure to MC can not benegligible. The toxic effect of cyanobacteria bloom is not exactly the same with puretoxin. A comprehensive assessment of its toxicity is necessary and will be our futurework.