Toxic Effects Of Waterborne Cadmium Exposure On The Liver And The Lipid Metabolism Of Rare Minnows (Gobiocypris Rarus)

Cadmium(Cd) is one of the most toxic heavy metals in the world. As the progress of industry and agriculture, environmental including the aquatic system was heavily polluted by Cd. Because the un-degradation, long half life in the cells, Cd can accumulate in various tissues, and thus causes damage. In the present study, the widely used animal rare minnow(Gobiocypris rarus) was used as the experimental fish. The techniques of histology, histochemistry, TEM, physiology, molecular biology, transcriptome were used to evaluate the effects of acute and chronic waterborne Cd exposure on the morphology of liver, organelles of hepatocytes, lipid droplet deposition, physiological indexes and expression of related genes involved in the lipid metabolism, hormones and they conducted signals, and to find out the potential mechanisms. In the first part, the effects of acute(2.0, 2.5, 2.8 mg/L Cd for 0-96 h) waterborne Cd exposure on the morphology of liver, ultrastructural damage and the lipid droplet deposition in the hepatocytes were studied. In the second part, the full length cDNA of Beclin 1 gene was cloned, after the bioinformatic analysis, spatiotemporal expression pattern and the expression in various tissues after acute and chronic waterborne Cd exposure in both adults and adolescents were examined. In experiment three, the physiological and biochemical indexes including the lipids content in both serum and liver, combined with the expression of genes related to formation of lipid droplets, transportation and metabolism of lipid were investigated. In experiment four, endocrine disruption of acute and chronic waterborne Cd exposure on the H-P-I axis and its downstream signals were tested. In experiment five, differentially expressed genes were screened after the 75 μg/L Cd treated for 5w through the de-novo transcriptome analysis. Some lipid metabolism related genes were tested in other Cd treated groups. Combined with the transcriptome data, the GH family hormones and proinflammatory factors were also detected to analyze the potential mechanism of Cd caused disruption on the growth and metabolism.The main results are listed as follows:1) The mortalities of 0-100 μg/L Cd exposure for 5w were 2.56%, 0%, 27.91%, 37.5%, 57.5% and 68.18%, respectively. Fullness of fish in the 75 and 100 μg/L Cd groups were significantly(P<0.05) declined. After a 12 w exposure, fish in >25 μg/L groups showed distinct(P<0.05) decrease of body weight, net body weight and visceral mass.2) Acute waterborne Cd exposure(2.0-2.8 mg/L) caused severe damage on the liver. The hepatocytes showed ballooning degeneration, vacuolization or condensed shape. 5 μg/L Cd exposures caused no significant morphological alteration of the liver, while hepatocytes of fish in 25-100 μg/L groups were vacuolized, and the vacuole size became larger in higher Cd concentration group. Tumors were found in liver of fish treated by 50 and 75 μg/L Cd for 5w. Chronic waterborne Cd(5-100 μg/L) exposure caused lipid droplets deposition in hepatocytes, however, without dose dependent manner.3) Acute waterborne Cd exposure(2.0-2.8 mg/L) led to degradation of glycogen, organelles necrosis, increase of myeloid body, rough endoplasmic reticulum(RER) hyperplasia or decrease, mitochondrial swelling, connections opening, increase of Kupffer’s cells, cell membrane damage, infiltration of erythrocyte and so on. After the 72-96 h exposure, lipid droplets deposition in the hepatocytes increased. Nuclear lipid droplets were the first time to be observed in hepatocytes afte aucte Cd exposure. In general, different treatment duration caused some different histopathological alterations. Similar results were found in fish treated by chronic waterborne cadmium exposure, such as ballooning degeneration or condense of hepatocytes, degradation of glycogen, increase of autophagysome, RER hyperplasia in the low dose group, smooth endoplasmic reticulum(SER) hyperplasia in the high dose group, dismiss of the cell membrane and enlargment of intracelluar space. It’s the fisrt time observe RER with multiple concentric lamellar bodies and fingerprint-like lamellar bodies in heapatocytes after Cd treatment.4) The full length cDNA of Beclin1 of rare minnow was 1940 bp. It shared high similarities with that of other fish and higher vertebrates. This gene encodes a prepeptide composed of 447 amino acid residues, with BH3, CCD and ECD domains; however, the CCD domain is shorter than that of mammals. After acute waterborne Cd exposure, Beclin1 was significantly(P<0.05) upregulated in the gill and intestine, and delclined in the brain. After chronic waterborne Cd exposure, this gene was distinct(P<0.05) upregulated in the brain, while rise in the liver. The expression of Beclin1 in the larvae after waterborne Cd(5-100 μg/L) exposure was duration and dose dependent.5) After the 2.0 mg/L Cd exposure, serum HDL-C, T-CHO, TG and ATP concentrations were raised, HDL-C, T-CHO, TG, LDH and lactate in the liver were increased as well; however, the activity concentration of CS was declined. After the chronic waterborne Cd exposure, all the lipids concentration in the serum decreased or unchanged. TG in the liver was distinctly(P<0.05) declined, while T-CHO in the 100 μg/L group was increased.6) Coincidence with the deposition of lipid droplet, APOAI, which encodes a key protein of back-liver lipoprotein: HDL, PLIN2, CAV2, CPTI and CPTII, which related to lipid droplet formation, and the lipid metabolism gene PPARr were significantly(P<0.05) upregulated at the liver after the fish treated by 2.0 mg/L exposure for 96 h. However, the gene APOB-100 which is key member of LDL that is responsible for the delivery of cholesterol to non-hepatic cells, was significantly(P<0.05) decreased. After a 5w chronic exposure, lipid contains in the serum were declined, however, T-CHO in the heaptopancreas of 100 μg/L group was raised. Only CPTII showed significantly(P<0.05) rise in fish of 100 μg /L group.7) After the acute waterborne Cd exposure, serum MSH and Cortisol rose. StAR, CYP11A1 and CYP11B1, which are key genes in the Cortisol synthesis pathway, were significantly(P<0.05) upregulated. GR, receptor of Cortisol, was decreased in the gill, intestine and muscle, while a GR regulator FKBP5 rose in the gill and intestine. Neither serum CRH, ACTH, nor the expression of CRH, POMC, CRHR and MC2 R in the brain or kidney was significantly influenced by the acute waterborne Cd exposure. 50 μg/L and higher concentration of Cd treated for 5w led to rise of serum CRH, ACTH, MSH and Cortisol(P<0.05). In the 100 μg/L group, CRH, MC2 R, StAR, CYP11A1 and CYP11B1 were distinctly upregulated in the brain or kidney of adult female rare minnows. No alteration of GR was observed, however, FKBP5 was significantly in the gill and intestine(P<0.05).8) The top 60 highly expressed genes in liver of rare minnow were involved in the following seven categories: lipid transporter and fatty acid binding, digestive enzymes and related proteins, development of gonad and fertilization, iron or calcium transporter, respiratory chain blood factors, stress and innate immune response, protein synthesis and degradation. After the 75 μg/L Cd exposure for 5w, 288 differentially expressed genes were screened, in which 192 were downregulated. Some of these genes were verified by RT-qPCR.9) Of the differentially expressed genes, 51, 33, 18 and 11 genes were related to ion binding, lipid absorption and metabolism, oxidation stress and growth, respectively. 27 genes were related to Zn, 8 were related to both Fe and Ca, respectively. Of which, the expressions of SCD, PEPC and I G F 1 t h a t a r e r e l a t e d t o t h e g r o w t h a n d m e t a b o l i s m w e r e s i g n i f i c a n t l y a l t e r e d.10) Acute and chronic waterborne Cd exposure caused GH family hormones aberrant both in mRNA and protein level, as well as the serum proinflammator. The screened genes VtgAO1 and SCD were significantly(P<0.05) downregulated in fish of all groups after chronic and acute Cd treatment.The main conclusions:1) The growth arrest and decrease of fullness of rare minnows suggested the energy storage were reduced, and organic metabolism was aberrant after the chronic waterborne Cd exposure.2) The biotoxicity of Cd on rare minnows are so complicated that it’s a dose and duration specific manner. Both acute and chronic waterborne Cd exposures caused severe damage of liver, and programmed cell death such as apoptosis and autophagy were involved in the damage. Hypofunction of hepatocytes was related to the aberration of glucose, lipid metabolism, and inflammation. Chronic Cd exposure is a powerful promoter for cell proliferation.3) Beclin1 is a highly conserved gene. It might be an important regulator in the fetal, post fetal development of rare minnows, and it might be related to the Cd induced autophagy formation. What’s more, Beclin1 could be used as a biomarker when used rare minnow larvae for the waterborne Cd risk assessment.4) After the acute waterborne exposure, the aberrant deposition of lipid droplet in the hepatocytes was related to the disruption of lipid transportation; moreover, the formation of LD was related to the PLIN2, CAV2, PPARr and CPTII conducted lipid metabolism pathways. After the chronic waterborne Cd exposure, lipid degradation of all the organs might be a factor for the decrease of fullness and visceral mass. However, the rose of T-CHO in the liver of 100 μg/L group suggested the disruption of T-CHO might be involved in the lipid deposition and aberrant metabolism.5) Acute waterborne cadmium exposure mainly affect on the synthesis and secretion of Cortisol and the downstream GR expression. And we hypothesized that, the Cd induced production of Cortisol was not mediated by CRH-ACTH signal, perhaps via MSH or some unknown hormones. The FKBP5 mediated downregulation of GR and the upregulation of 11β-HSD2 were two negative feedback of Cortisol signal, which may result the inhibition of the Cortisol toxicity. Chronic waterborne Cd exposures affect hormones secretion in H-P-I axis and their synthesis were also disrupted by 100 μg/L Cd exposure. The Cd caused production of Cortisol was mediated by the ACTH-MC2 R signal, without alteration of GR; however, the upregulation of FKBP5 might decrease the GR activity to inhibit the Cortisol signals.6) The transcriptome data revealed that the function of liver in the digestion, immune response, reproduction and protein synthesis and degradation. After the waterborne Cd exposure, the ion homeostasis, redox system and lipid absorption, digestion and metabolism were disrupted. Cd induced growth arrest might be related to the inhibition of GH-IGF signals. What’s more, S C D- P E P C m i g h t b e i n v o l v e d i n t h e C d c a u s e d l i p i d m e t a b o l i s m d i s r u p t i o n.7) VtgAO1 and SCD can be used as effective biomarkers for risk assessment of the waterborne cadmium exposure.