Studies On The Hepatotoxicity And Molecular Mechanism In Mouse Caused By Microcystin-LR

The occurrences of cyanobacterial blooms in aquatic environments are increasing with progressive eutrophication of water bodies which is of increasing concern as many cyanobacteria are known to produce a diverse range of toxins(cyanotoxins). Toxic cyanobacterial blooms can be found in eutrophic to hypereutrophic lakes, ponds, and rivers throughout the world and are responsible for illness and death of wild and domestic animals.Globally, the most frequently occurring and widespread cyanotoxins in freshwater blooms are the cyclic heptapeptides – microcystins(MCs). MCs consist of over 90 congeners and the most common distribution and abundant content congeners in natural aquatic body are MC-LR, MC-RR and MC-YR. The present study aimed to investigate the effects of MC-LR on histopathology, gene and protein expressions, and biochemical enzyme activities in the liver of mouse. HE staining method was employed to observe the change of tissue structure in mouse liver, Real-time quantitative PCR(Q-PCR), western blot, and spectrophotometer were used to detect their expressions, levels and enzyme activities, the RNA-Seq approach was used to determine the gene expression profile,,and the ELISA method was used to investigate inflammatory response associated proteins. The results obtained in this study were as following.1. Subchronic MC-LR exposure induced apoptosis in mouse liverThe result of acute toxicity test showed that intraperitoneal LD50 of MC-LR in mouse was 70 μg/kg(56.74-75.6 μg/kg). Then, according to the LD50, we used the relatively lower doses of MC-LR(3.75, 7.5, and 15 μg/kg) to investigate the effects of MC-LR on the liver of mouse exposed to MC-LR for 28 days. It was found that 7.5 and 15 μg/kg of MC-LR significantly increased the serum ALT and AST activities after 21 and 28 days of exposure,and serum AKP activity was also increased in all treatment groups after 28 days of exposure,indicating that MC-LR caused liver injury in mouse. Moreover, MC-LR also induced the pathological change in the liver of mice after 28 days of MC-LR-exposure, caused the inflammatory cell infiltration in the liver of mouse in 7.5 and 15 μg/kg MC-LR groups, andinduced apoptosis in mouse liver in 15 μg/kg of MC-LR group. In order to further investigate the mechanism of MC-LR-induced apoptosis, we detected the changes of Bcl-2, Bax, cyt c,caspase 3, and caspase 9 proteins in mouse liver after 28 days of MC-LR-exposure. The results showed that Bcl-2 protein level decreased while Bax, cyt c, caspase 3, and caspase 9protein levels increased in mouse liver, indicating that MC-LR-induced apoptosis in mouse liver was mediated by the pathway Bax–cyt c–caspase 9–caspase 3.2. Oxidative damage induced by MC-LR and the related pathway Nrf2 in mouse liverThe present study also investigated the oxidative damage induced by MC-LR and the related pathway Nrf2 in mouse liver after 28 days of MC-LR-exposure. The results showed that Nrf2 protein expression increased in mouse liver after exposure to MC-LR from 14 days to 28 days, indicating that MC-LR promoted the expression of Nrf2. Exposure to MC-LR significantly increased the activities of superoxide dismutase(SOD), catalase(CAT), and glutathione peroxidase(GPX), and quinone oxidoreductase 1(NQO1), cysteine ligase catalytic subunit(GCLC), and heme oxygenase 1(HO-1) protein contents at 7 days compared to the control group, suggesting the increased capacity to scavenge reactive oxygen species(ROS) was induced in mouse liver. With the extension of exposure time, the glutathione S-transferase(GST) enzyme activity, GCLC and HO-1 protein contents were still higher than that of the control group, but the activities of SOD, CAT and GPx, NQO1 protein and T-AOC were significantly decreased, indicating that the antioxidant ability decreased in MC-LR-treated mouse liver. ROS was significantly increased in 7.5 and 15 μg/kg treatment groups for 21 days and all treatment groups for 28 days when compared to the control group.Moreover, MC-LR also increased the MDA content in mouse liver at 28 days, indicating that MC-LR has induced oxidative stress and lipid peroxidation in the liver of mouse. This result suggests that the antioxidative enzymes and proteins regulated by Nrf2 may play an important role in protecting the cells from the toxicity of MC-LR.3. Effects of MC-LR on cytochrome P450 metabolic enzymes in mouse liverCytochrome P450(CYP) is an important member of phase I metabolizing enzymes.Among the various CYP isoenzymes, CYP1, CYP2, and CYP3 are three major families of theCYP superfamily that are involved in metabolism and biotransformation a wide variety of xenobiotic chemicals. The present study investigated the response of phase I metabolism enzymes, such as CYP1A1, CYP2E1, and CYP3A11, in mouse liver after exposure to 2, 4,and 8 μg/kg MC-LR for 7 days. The results showed that MC-LR significantly inhibited the ethoxyresorufin-O-deethylase(EROD) activity in mouse liver. However, the expression of CYP1A1 mRNA was only suppressed after 1 and 3 days of exposure to 2 μg/kg MC-LR.Moreover, the change of CYP1A1 protein level was inconsistent with EROD level. These results suggest that the inhibition of EROD activity by MC-LR may be regulated at post transcriptional and post translational level. MC-LR increased the CYP3A11 mRNA level and decreased its protein content in the liver of mouse. Furthermore, MC-LR significantly inhibited the erythromycin N-demethylase(ERND) activity in mouse liver in the whole exposure time and exposure groups. MC-LR significantly increased CYP2E1 mRNA and protein level and aniline hydroxylase(ANH) activity in mouse liver in whole experimental groups, suggesting that CYP2E1 may be involved in the metabolism of MC-LR. In addition,MC-LR significantly increased serum ALT and AST activities and liver ROS level in CYP2E1- overexpressed mice induced by acetone, which indicates that CYP2E1 may be able to produce excessive ROS in the metabolism of MC-LR.4. Digital gene expression profiling analysis of gene expression in the liver of mouse exposed to MC-LRTo explore the molecular mechanism of MC-LR-hepatotoxicity, we analyzed the digital gene expression profiling in mouse liver exposed to 10 and 40 μg/kg MC-LR for 24 h based on RNA sequencing technology. Gene Ontology(GO) and Kyoto Encyclopedia of genes and Gnomes(KEGG) were used to analyze the differential expression genes(DEGs). The results showed that 402 significantly up-regulated genes and 38 down-regulated genes were found in mouse liver exposed to 10 μg/kg of MC-LR and 72 significantly up-regulated genes and 76down-regulated genes in 40 μg/kg of MC-LR group. GO enrichment analysis of biological process identified 10 significantly enriched GO terms in 10 μg/kg of MC-LR treatment group.Among them, innate immune response(GO:0045087), defense response(GO:0006952) and response to bacterium(GO:0009617) were associated with immune related GO terms. Nosignificant enrichment GO term was found in 40 μg/kg of MC-LR group. KEGG analysis showed that 47 significantly enriched pathways were involved in biological pathways in 10μg/kg of MC-LR group, in which there were 17 pathways associated with immune and inflammatory responses. KEGG analysis also revealed five enrichment pathways in 40 μg/kg of MC-LR group. The analysis of digital gene expression profiling suggests that the inflammatory response may play an important role in the hepatotoxicity of MC-LR on mouse.5. Inflammatory response in mouse liver induced by MC-LRThe present study also investigated the inflammatory response induced by MC-LR after exposure to 5, 10, 20, and 40 μg/kg of MC-LR for 7 days. The results showed that the expressions of IL-6, IL-18 and MIF were induced in mouse liver after MC-LR exposure.MC-LR inhibited the expression of anti-inflammatory cytokines IL-2 and increased the expressions of IL-4 and IL-10, indicating that MC-LR affected the pro-inflammatory/anti-inflammatory balance of mouse liver and caused damage to mouse liver. It was found that MC-LR promoted the protein expressions of the toll like receptor 6(TLR6) and TLR9 in all treatment groups, suggesting that TLR6 and TLR9 may play an important role in MC-LR-induced inflammatory response in mouse liver. Moreover, high concentration of MC-LR(40 μg/kg) significantly increased the expression of TLR1, TLR2,TLR11 and TLR13, suggesting that MC-LR at higher dose may induce/activate TLRs and mediate inflammatory reaction.Conclusion:1. Subchronic exposure of MC-LR caused liver damage and induced apoptosis in mouse liver via the mitochondrial pathway.2. Nrf2 pathway may play an important role in protecting mouse liver from MC-LR toxicity.3. CYP2E1 may participate in the metabolism of MC-LR and excessive ROS may be produced in this process, which may result in oxidative damage to mouse liver.4. MC-LR may affect the balance of pro-inflammatory/anti-inflammatory balance in mouse liver, which can cause the inflammation response. Moreover, the inflammation response induced by MC-LR may be mediated by TLRs.