Proteomic Analysis of Hepatic Tissue of <i>Cyprinus carpio</i> L. Exposed to Cyanobacterial Blooms in Lake Taihu, China

<div><p>With the rapid development of industry and agriculture and associated pollution, the cyanobacterial blooms in Lake Taihu have become a major threat to aquatic wildlife and human health. In this study, the ecotoxicological effects of cyanobacterial blooms on cage-cultured carp (<i>Cyprinus carpio</i> L.) in Meiliang Bay of Lake Taihu were investigated. Microcystins (MCs), major cyanobacterial toxins, have been detected in carp cultured at different experimental sites of Meiliang Bay. We observed that the accumulation of MCs in carp was closely associated with several environmental factors, including temperature, pH value, and density of cyanobacterial blooms. The proteomic profile of carp liver exposed to cyanobacterial blooms was analyzed using two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The toxic effects of cyanobacterial blooms on carp liver were similar to changes caused by MCs. MCs were transported into liver cells and induced the excessive production of reactive oxygen species (ROS). MCs and ROS inhibited protein phosphatase and aldehyde dehydrogenase (ALDH), directly or indirectly resulting in oxidative stress and disruption of the cytoskeleton. These effects further interfered with metabolic pathways in the liver through the regulation of series of related proteins. The results of this study indicated that cyanobacterial blooms pose a major threat to aquatic wildlife in Meiliang Bay in Lake Taihu. These results provided evidence of the molecular mechanisms underlying liver damage in carp exposed to cyanobacterial blooms.</p></div

EPR signal intensity of PBN-•OH in the liver of <i>C. carpio</i> from lab cultures and <i>in-situ</i> experiment sites.

<p>The data are shown as the means ± SD (n = 4). The same letter indicates no significant difference between the groups. Different letters indicate significant differences between the groups with <i>p</i><0.05.</p

Expression level and classification of differentially expressed proteins in the liver of <i>C. carpio</i> collected from different <i>in situ</i> experiment sites.

<p>T1: Xukou Bay; T2: Meiliang Bay.</p

MC content (ng g⁻¹ DW) in the organs/tissues (muscle, gill, intestine, liver) of fish.

<p>The MC content was determined using ELISA (n = 4). Lab-CC represents carp (<i>Cyprinus carpio</i>) cultured in laboratory for the control group. S1-CC, S3-CC, S4-CC, and S5-CC represent the experimental groups for carp cultured in net cages located at experiment sites S1, S3, S4 and S5, respectively. Eastern-CA represents carp (<i>Carassius auratus</i>) captured in the eastern part of Lake Taihu (weight of 49.16±10.89 g; length of 15.69±1.28 cm), Eastern-PF represents <i>Pelteobagrus fulvidraco</i> captured in the eastern part of Lake Taihu (weight of 24.44±5.90 g; length of 13.80±1.15 cm), ML Bay-CA represents carp captured in Meiliang Bay (weight of 33.4±4.24 g; length of 12.75±0.35 cm), ML Bay-PF represents <i>Pelteobagrus fulvidraco</i> captured in Meiliang Bay (weight of 69.50±40.10 g; length of 18.9±3.54 cm), and ML Bay-EI represents <i>Erythroculter ilishaeformis</i> captured in Meiliang Bay (weight of 19.95±2.90 g; length of 15.50±0.71 cm).</p

The physic-chemical parameters of the water from different experiment sites.

a<p>n.a. = not analyzed.</p>b<p>n.d. = un-detectable.</p><p>The water was sampled every three days from July 11–24, 2009. All data are presented as the means of five water samples.</p

Representative graphs and bar charts of hepatic mRNA and protein expressions of iNOS, eNOS, and Collagen І in each group of rats.

<p>The hepatic protein expression of iNOS and eNOS were detected in the control group of rats, with little collagen I expression (A). With the consumption of high-fat diet, the iNOS and collagen I expressions were gradually increased (B, C, F, G), while eNOS expression was gradually decreased (D, E). Compared with rats in the 24^th week group, simvastatin treatment could up-regulate eNOS expression (A, D, E) and down-regulate iNOS expression (B, C), followed by improved hepatic fibrosis, which represented by decreased collagen I expression (F, G). (^#<i>P</i><0.05, compared with the control group; * <i>P</i><0.05, compared with 24^th week group.).</p

Masson staining of hepatic tissues in each group of rats.

<p>Slightly sinusoidal fibrosis appeared only in part of model rats until 16^th week (B). At the end of 24^th week, all model rats showed hepatic fibrosis in sinusoids, partly in portal area (C). These changes were improved in the liver of simvastatin-treated rats (D). A: Control ×200; B: 16^th week ×200; C: 24^th week ×200; D: Simvastatin-treated group ×200.</p

Representative graphs and bar charts of the mRNA and protein expressions of iNOS, eNOS, α-SMA, and Collagen І in LX-2 cells cultured with L-NAME and simvastatin.

<p>L-NAME served as a NOS inhibitor, it inhibited both iNOS and eNOS expression (A~E) and induced more α-SMA, and Collagen І expressions (F~I). Simvastatin had antagonistic effect on the activation of LX-2 cells induced by L-NAME via increasing the eNOS expression (D, E). (^#<i>P</i><0.05, compared with the control group; * <i>P</i><0.05, compared with LX-2 cells treated with L-NAME; ^ <i>P</i><0.05, compared with LX-2 cells treated with simvastatin.).</p

Functional classification of up-regulated (A)/down-regulated (B) proteins from the liver of <i>C. carpio</i> in the T2 group.

<p>Functional classification of up-regulated (A)/down-regulated (B) proteins from the liver of <i>C. carpio</i> in the T2 group.</p

Classification of the differentially expressed proteins identified in the liver of field treatment fish compared with the fish in laboratory.

a<p>Unique spot number of the position where the spot is displayed in the master gel.</p>b<p>Accession number according to the NCBI rat database.</p>c<p>Number matched peptides.</p>d<p>Protein scores greater than 64 were successfully identified.</p>e<p>Fold-change between the T2, T1 and C groups.</p><p>*<i>p</i><0.05 was considered statistically significant and</p><p>**<i>p</i><0.01 was extremely significant.</p><p>A positive value signifies up-regulation and a negative value signifies down-regulation. C: laboratory (Control), T1: Xukou Bay, T2: Meiliang Bay.</p>f<p>The identified proteins were grouped according to their functions based on the Gene Ontology and KEGG databases.</p