Studies On The Cytotoxicity And Toxicological Mechanism Of Microcystin On HepG2 Cells

In the recent years, cyanobacterial blooms frequently occur worldwide in fresh waters due to the water eutrophication. Cyanobacterial blooms not only cause water pollution such as in drinking water resource but also bring about a potential health hazard to aquatic organisms and humans due to their cyanotoxins. Among these cyanotoxins, microcystins(MCs) are a family of cyclic heptapeptide hepatotoxins those have wide distribution in fresh water and high toxicity on organisms. Now there have been more than 100 MCs variants identified. MCs have a broad spectrum biological-toxicity and they are chemically stable in natural water due to their cyclic structure and novel amino acids. Therefore, their effects on human health are especially concerned. For this reason, studies on the toxicological mechanism of MCs on animal or human hepatocytes have been received more and more concerns in recent years. Although there have been many discoveries and new insights about MCs-toxicology, some issues such as molecular mechanism and pathway of MCs-hepatotoxicity are still elusive and not completely known due to the multi-pathway process of MCs toxicity. Therefore, there are still many problems to be solved, which may be beneficial for risk assessment of MCs on human health.In the present study, we choose microcystin-LR(MC-LR), which is found to be the most common and potent variant in MCs, to determine the cytotoxicity of the toxin on the human hepatocellular carcinoma(Hep G2) cells in order to elucidate the toxicity mechanism of MCs. This study aimed to further enrich and improve the toxicity mechanism theory of MCs and evaluate the possible risk of MCs on human health. Research contents and main results obtained in this study:(1) Cytotoxicity of MC-LR on Hep G2 cells The viability of Hep G2 cells was determined by MTT, the cell cycle and apoptosis were assayed by flow cytometry, and the cellular and organelles shape was observed under inverted fluorescent microscope so as to determine and evaluate the toxicity of MC-LR on Hep G2 cells. The results showed that 0.1 n M- 10 μM MC-LR failed to induce cytotoxicity in Hep G2 cells after 24 h of exposure and no difference in the cell cycle and apoptosis was found between the MC-LR-treated groups and the control group assayed by flow cytometry. In this study, the result of MTT assay also showed that 0.1- 50 n M MC-LR-exposure didn’t affect the cell viability of Hep G2 cells after 96 h of exposure. However, the results of cytotoxicity tests showed that high concentration MC-LR(20- 100 μM) had remarkable cytotoxicity on Hep G2 cells, which was found to be a time- and dose- dependent pattern. Then we choose higher concentrations of MC-LR(0.5, 5, 10, 30, and 50 μM) to exposure the Hep G2 cells for long time. The result showed that high concentration of MC-LR could significantly influence cellular shape and cause injury to Hep G2 cells observed under inverted fluorescent microscope. Meanwhile, MC-LR-exposure at higher concentration led to nuclear damage and induced apoptosis of Hep G2 cells detected by Hoechst 33342 staining. Lysosomal injury in a concentration-dependent pattern was also observed in Hep G2 cells determined by Acridine orange staining. Additionally, using Rhodamine-123 staining, we found that MC-LR-exposure at higher concentration caused obvious injury to mitochondria of Hep G2 cells, although lower concentration of MC-LR only led to slight damage to mitochondria. These results indicate that MC-LR cytotoxicity is concentration-dependent, i.e., MC-LR-exposure at low concentration has no cytotoxicity on Hep G2 cells while high concentration of MC-LR causes apoptosis of Hep G2 cells.(2) Altered cellular metabolism and the drug resistance gene expression of Hep G2 cells caused by MC-LR Firstly it is essential for the present study to determine whether MC-LR entered Hep G2 cells and whether the expressions of OATP1B1 and OATP1B3 could be detected in the cells now that MC-LR-exposure at relatively lower concentrations(0.1 n M- 10 μM) failed to alter the viability of Hep G2 cells. The results of q PCR revealed that OATP1B1 and OATP1B3 assuredly expressed in Hep G2 cells. Moreover, we found that MC-LR could enter Hep G2 cells in a concentration-dependent pattern by using western blot assay. MC-LR-exposure at lower concentrations(0.1 n M- 10 μM) affected the expressions of some key genes responsible for metabolization of toxicants in phase I or phase II system of Hep G2 cells such as CYP450 and GST, suggesting that MC-LR-exposure may disturb the metabolic destabilization of Hep G2 cells. Therefore we believe that the detoxification enzyme systems such as CYP450 and GST may paly a key role in the metabolization and detoxification of MC-LR in Hep G2 cells. For this reason, we conducted an induction experiment by using CYPs-inducers omeprazole(OME), ethanol(ETH), and rifampicin(RIF) to verify whether these enzymes are involved in the above process. The result of induction test showed that the viability of Hep G2 cells was influenced by MC-LR-exposure at 5 μM following CYPs-inducer treatment in which ETH was proved especially to be a very effective CYP2E1 inducer. Based on this result, we conducted another inhibition test to verify our assumption by using CYPs-inhibitor chlormethiazole(CMZ). The result of inhibition test showed that MC-LR-exposure exerted toxicity on Hep G2 cells, caused damage on mitochondria, and induced apoptosis of Hep G2 cells via promoting CYP2E1 expression and induced excessive ROS in Hep G2 cells. In addition, we also found that co-incubation with ROS scavenger L-Ascorbic acid and MC-LR could decreased ROS level and effectively prevented the apoptosis of Hep G2 cells.(3) Effects of long-term of MC-LR-exposure at low concentration on Hep G2 cells Recent studies have showed that long-term exposure of MC-LR at low concentration promotes cell proliferation and induce hepatitis and liver cancer in humans. In order to investigate this mechanism, we conducted a chronic toxicity test of MC-LR-exposure on Hep G2 cells at 0.1- 30 n M for 83 d under the laboratory conditons. Western blot assay revealed that MC-LR could enter Hep G2 cells even if at the concentration of 0.1 n M, but it failed to alter the vitality of Hep G2 cells determined by CCK-8 test. However, the results of DCF fluorescence assay showed that the intracellular ROS levels in the 30 n M MC-LR-treated Hep G2 cells were higher than that of the control cells, and 5 and 10 n M of MC-LR-exposure totally increased the activity of SOD in Hep G2 cells. This result indicates that exposure of MC-LR at low concentration also induced excessive ROS in Hep G2 cells and SOD might play a key role in eliminating intracellular ROS. Additionally, long-term exposure of MC-LR at low concentration also remarkably promoted the expressions of NF-κB p65, TNF-α, IL-1β, and IL-6 in the Hep G2 cells.(4) Apoptosis of Hep G2 cells caused by high concentration of MC-LR Our previous studies have indicated that the MC-LR-induced apoptosis in Hep G2 cells may be initiated by excess ROS. Then we want to determine the possible signaling pathway responsible for the apoptosis induced by MC-LR by conducting an acute toxicity test in Hep G2 cells using relatively higher concentrations of MC-LR(0.5, 5, 10, 30, and 50 μM). The results of DCF fluorescence assay showed that intracellular ROS levels in Hep G2 cells exposed to 10- 50 μM of MC-LR were significantly higher than that of the control cells. MC-LR-exposure promoted the expressions of HSP70 and HSP90 while decreased activities of SOD and CAT and GSH content in Hep G2 cells. Moreover, MDA level in the toxin-treated group was remarkably higher than that of control group, indicating that MC-LR-exposure causes lipid peroxidation in the treated cells. These results indicate that MC-LR-exposure at high concentration induces excessive ROS in Hep G2 cells that consequently leads to oxidative stress and promotes lipid peroxidation in the cells. The results of western blot and q PCR showed that MC-LR-exposure at various concentrations(0.5-50 μM) promoted the expressions of cyclooxygenase-2 activity, cytochrome c, and Smac/Diablo in Hep G2 cells, suggesting that MC-LR-induced apoptosis may be mediated by mitochondrial pathway. Moreover, we also found that p53 expression was significantly up-regulated and the phosphorylation of p53 was enhanced in the MC-LR-treated cells. Furthermore, MC-LR-exposure promoted the expressions of p21/WAF1, Fas, and Fas L. Meanwhile, Bad, cleaved-Bid, Bcl-2, and Bax protein levels were also altered. Notably, our results also showed that PUMA expression was generally promoted in the MC-LR-treated cells while survivin content maintained unchanged or decreased in the treatment groups at earlier period of experiment(3- 6 h), but increased at the later time(12- 24 h) when compared to the control group. In addition, MC-LR-exposure generally promoted the transcriptions or enzymic activities of caspase-3 and caspase-9 in Hep G2 cells in a concentration-dependent manner and caspase-8 m RNA level and enzymic activity was also promoted in the high concentration of MC-LR group, suggesting that MC-LR-induced apoptosis may be mediated caspase pathway. We also found that expressional alteration of c-myc was largely dependent on the concentration or exposure-time of MC-LR, and the protein level of c-Myc were generally increased by MC-LR exposure, especially high concentration of the toxin could promote the phosphorylation level of c-Myc(phosphor S62). Meanwhile, MC-LR-exposure also generally promoted the transcriptions or proteins of c-fos and c-jun in Hep G2 cells, suggesting that MC-LR may induce DNA damage, exert genotoxicity on Hep G2 cells, and thus promote hepatocellular carcinom.(5) Transcriptome analysis of MC-LR-cytototoxicity on Hep G2 cells In order to further investigate the cytotoxicity mechanism of MC-LR, the pattern of transcriptomic profiles of Hep G2 cells were determined after 24 h of MC-LR-exposure. The results showed that the total numbers of genes with significant changes in expression from MC-LR-treated cells were detected relative to that of the control, for example, 532 significant genes in 0.5 μM of MC-LR group, 32 in 5 μM group, 30 in 10 μM, and 984 in 50 μM of MC-LR group. GO analysis reveals that genes responding to exposure at low concentration of MC-LR(0.5 μM) were related to cell migration, cell motility, localization of cell, cellular component movement, regulation of phosphorylation, and regulation of phosphate metabolic process. However, in the higher concentration group(50 μM MC-LR), the differentially expressed genes were significantly enriched 94 classes of biological processes in Hep G2 cells, for example, regulation of cellular metabolic, regulation of cellular biosynthetic process, regulation of gene expression, regulation of phosphorylation, regulation of kinase activity, immune system process, regulation of MAPKKK cascade, cell proliferation, regulation of cell differentiation, cell migration, cell motility, cell death and apoptosis. Moreover, KEGG pathway analysis showed that MC-LR-exposure caused signaling pathway alteration, such as inositol phosphate metabolism, phosphatidylinositol signaling system, NF-κB signaling pathway, p53 signaling pathway, and MAPK signaling pathway.(6) Effects of MC-LR on the mi RNA expression profiles in Hep G2 cells In the recent years, there have been accumulating reports regarding the possible regulatory faction of micro RNAs in the cytotoxicity of MC-LR. Therefore, we also conducted the high-throughput sequencing of mi RNA expression profiles in Hep G2 cells after 24 h of MC-LR-exposure to affirm whether micro RNA is involved in the cytotoxicity of MC-LR. The results showed that totally 21 mi RNAs were found to be significantly altered(5 up-regulated but 16 down-regulated) in 10 μM of MC-LR treated cells when compared to the control cells. However, in 50 μM of MC-LR group, totally 37 mi RNAs displayed altered expressions(20 upregulated and 17 downregulated) compared with the control group. In these two groups, there were 37 566 and 39 174 target genes were predicted, respectively. We also found that MC-LR-exposure promoted the expressions of has-mi R-149-3p, has-mi R-449c-5p, and has-mi R-454-3p while suppressed the expressions of has-mi R-4286, has-mi R-500a-3p, has-mi R-500a-5p, and has-mi R-500b-5p in both 10 μM and 50 μM groups when compared to the control group. Moreover, the result of q PCR confirmed the above result, suggesting that these mi RNAs may be involved in MC-LR-hepatotoxicity and may play an important role in the hepatitis and liver cancer caused by MC-LR. The target genes for differentially expressed mi RNAs in MC-LR treatment groups were significantly enriched to totally 23 classes of GO in which 3 were significantly enriched in both 10 and 50 μM of MC-LR groups. Moreover, the results of KEGG pathway analysis showed that MC-LR-exposure-involved altered signaling pathway such as MAPK signaling pathway, biosynthesis of secondary metabolites,pyrimidine metabolism, and purine metabolism were possibly negatively regulated by mi RNAs and might play an important role in the toxicity process of MC-LR in Hep G2 cells. Additionally, the analysis result of mi RNA expression profile predicted and screened 110 new candidates of mi RNAs which will be useful for further enriching the resource of human mi RNA database, and may provide the basis for the future functional study.Conclusions:(1) The cytotoxicity of MC-LR on Hep G2 cells depends on the exposure concentration and action time of MC-LR. Higher concentration of MC-LR induces apoptosis of Hep G2 cells in which mitochondria and lysosome might be involved.(2) MC-LR-exposure exerted toxicity on Hep G2 cells and induced apoptosis of Hep G2 cells via promoting CYP2E1 expression and induced excessive ROS in Hep G2 cells.(3) Long-term of MC-LR-exposure at low concentration induced inflammatory reaction of Hep G2 cells.(4) High concentration of MC-LR caused the apoptosis of Hep G2 cells initiated by excessive ROS and mediated via mitochondrial pathway. PUMA and survivin may play an important role in this process.(5) High concentration of MC-LR also can activate Fas/Fas L that mediates the apoptosis of Hep G2 cells.(6) Multiple genes and proteins were involved in MC-LR cytotoxicity and multiple signaling pathways work together or antagonism in it.(7) mi RNA might play an important negatively regulated role in the cytotoxicity of MC-LR and the hepatitis or liver cancer induced by MC-LR.Our results uncover the possible reasons for the human hepatitis and liver cancer caused by MC-LR at toxicological and molecular levels, which may be valuable for risk assessment of MC-LR and human health protection.