cyanoginosin-lr and Body-Weight

Microcystin-leucine-arginine (MC-LR) can cause male reproductive disorder. However, the underlying mechanism are not yet entirely elucidated. In this study, we aimed to investigated the effects of MC-LR on the integrity of blood-testis barrier (BTB) and the related molecular mechanisms. Both in vivo and in vitro experiments revealed that MC-LR caused disruption of BTB and gap junctions between Sertoli cells respectively, which was paralleled by the alteration of connexin43 (Cx43). Our data demonstrated that MC-LR decreased gap junction intercellular communication (GJIC) and impaired Cx43 expression by activating the phosphatidylinositol 3-kinase/Akt cascades. In addition, a possible protective effect of Icariin (ICA), a flavonoid isolated from Chinese medicinal herb, against MC-LR toxicity was investigated. The ICA prevented the degradation of GJIC and impairment of Cx43 induced by MC-LR via suppressing the Akt pathway. Together, our results confirmed that the expression of Cx43 induced by MC-LR was regulated in vivo and in vitro, which was involved in the destruction of BTB. Additionally, ICA seems to be able to mitigate the MC-LR toxic effects.

Topics: Animals; Body Weight; Cell Line; Cell Survival; Connexin 43; Drugs, Chinese Herbal; Flavonoids; Gap Junctions; Male; Marine Toxins; Mice; Microcystins; Proto-Oncogene Proteins c-akt; Sertoli Cells; Signal Transduction

To explore the effects of microcystin-LR (MC-LR), a hepatotoxin, on the incidence of liver lipid metabolism abnormality, and the potential molecular mechanisms of action, healthy male Balb/c mice were intraperitoneally injected with MC-LR at doses of 0, 5, 10, and 20 μg/kg/d for 14 days. Hepatic histopathology and serum lipid parameters of mice were determined, and the changes of mRNA and protein expression of endoplasmic reticulum (ER) stress signaling molecules related to the lipid metabolism abnormalities in the livers of mice were investigated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting, respectively. The results indicated that 5-20 μg/kg/d MC-LR altered serum lipid parameters and caused hepatic steatosis. MC-LR treatment at 10 or 20 μg/kg/d changed mRNA and protein expression of ER stress signaling molecules, including upregulation of mRNA and protein expression of activating transcription factor 6 (ATF6), pancreatic ER eukaryotic translation initiation factor 2α (eIF-2α) kinase (PERK), and eIF-2α. MC-LR exposure at 10 or 20 μg/kg/d also altered mRNA and protein expression of downstream factors and genes of ER stress signaling pathways, including the downregulation of sterol regulatory element binding protein 1c (SREBP-1c) and fatty acid synthase (FASn), and upregulation of acetyl-coenzyme A carboxylase α (ACACA) and glycogen synthase kinase 3β (Gsk-3β). Our results reveal that ER stress plays a significant role in hepatic lipid metabolism abnormalities in mice exposed to MC-LR.

Topics: Animals; Body Weight; Endoplasmic Reticulum Stress; Lipid Metabolism; Liver; Male; Marine Toxins; Mice; Mice, Inbred BALB C; Microcystins; Organ Size; Proteins; RNA, Messenger; Signal Transduction

There is growing evidence that adverse effects of microcystin-LR (MC-LR) are closely related to oxidative stress processes, free radicals and DNA damage, and involve major gene transcript changes. This study, utilizing gene expression analysis and plasma chemistries was the first to measure the effects of MC-LR in whitefish (Coregonus lavaretus L.), a feasible organism for pollution monitoring in aquatic systems. Fish were injected with different concentrations of MC-LR (0, 10 and 100 microg/kg of body weight) and then sacrificed at either 0, 8, 24, 48 or 72 h later, and their liver tissue were harvested for detailed investigation. Specifically, we were interested whether MC-LR is capable of: (i) modulating expression of two genes, tumor suppressor gene p53 and cdkn1a, p53 direct transcription target, and (ii) inducing apoptosis in whitefish liver. To study these effects, we developed a real-time qPCR assays useful for measuring both p53 and cdkn1a gene transcript levels in liver. To obtain necessary information for the study, either full-length p53 cDNA of whitefish (Wf-p53) was determined, using molecular cloning and rapid amplification of cDNA ends (RACE), or as for Wf-cdkn1a, specific primers were designed based on highly conserved regions of cdkn1a in fish. The Wf-p53 was found to share the same characteristics with a known p53 mRNA sequence of other vertebrates. Whitefish p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. The injection study showed that MC-LR at a higher dose, i.e. 100 microg/kg body weight, up-regulated expression of p53 and cdkn1a genes in whitefish liver, as reflected by the continuous increase in their mRNA levels through the whole experiment. Furthermore, DNA fragmentation was observed in liver cells of whitefish after 24h of exposure to MC-LR (100 microg/kg) that suggests the possibility of apoptosis. Finally, the study confirmed previous observations of severe injury of the liver and loss of normal organ functions as revealed by elevated levels of blood AspAT, AlaAT, and hepatosomatic index (HSI).

Topics: Alanine Transaminase; Amino Acid Sequence; Animals; Apoptosis; Aspartate Aminotransferases; Base Sequence; Biomarkers; Blood Glucose; Body Weight; Cloning, Molecular; Cyclin-Dependent Kinase Inhibitor p21; Databases, Genetic; DNA Fragmentation; DNA Primers; DNA, Complementary; Liver; Marine Toxins; Microcystins; Molecular Sequence Data; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salmonidae; Tumor Suppressor Protein p53

Microcystin-LR (MCLR) is the most common hepatotoxic cyanotoxin produced primarily by Microcystis aeruginosa. In this study, young F344 rats were intraperitoneally injected with a single dose (25, 50, 100, and 150 microg/kg) of MCLR to explore possible toxic effect and toxic response indicators. Acute toxic symptoms, including body weight loss and death, were monitored for 7 days. Mortality reached 100% (9/9) in rats treated with a single MCLR dose of 150 microg/kg. Histopathological examination showed spot necrosis in the liver of animals treated at low doses, while massive hemorrhage and widespread necrotic foci occurred at higher doses, indicating extensive liver damage. Protein phosphatase 2A (PP2A) expression showed a dose-dependent decrease in the liver. Immunohistochemical localization indicated that nuclear PP2A was affected first, followed by cytoplasmic PP2A. In addition, there was a significant increase in sphingolipid levels at higher doses, indicating the involvement of a ceramide-mediated apoptotic pathway. Expression of apoptosis and cell cycle regulatory proteins like Bax, Bcl2, and Bad showed a dose-dependent decrease. This study demonstrated that treatment with a single dose of MCLR caused liver damage, increased sphingolipid levels, and decreased PP2A expression, which ultimately down-regulated the expression of Bcl2 family proteins.

Topics: Animals; Apoptosis; Body Weight; Cell Cycle Proteins; Dose-Response Relationship, Drug; Hepatocytes; Male; Marine Toxins; Microcystins; Mortality; Protein Phosphatase 2; Rats; Rats, Inbred F344; Time Factors

Microcystin-LR (MCLR) is an acute hepatotoxicant and suspected carcinogen. Previous chronic studies have individually described hepatic morphologic changes, or alterations in the cytoskeleton, cell signaling or redox pathways. The objective of this study was to characterize chronic effects of MCLR in wild-type mice utilizing gene array analysis, morphology, and plasma chemistries. MCLR was given daily for up to 28 days. RNA from the 28-day study was hybridized onto mouse genechip arrays. RNA from 4 hours, 24 hours, 4 days, 1 day, and 28 days for selected genes was processed for quantitative-PCR. Increases in plasma hepatic enzyme activities and decreases in total protein, albumin and glucose concentrations were identified in MCLR-treated groups at 14 and 28 days. Histologically, marked hepatokaryomegaly was identified in the 14-day MCLR group with the addition of giant cells at 28 days. Major gene transcript changes were identified in the actin organization, cell cycle, apoptotic, cellular redox, cell signaling, albumin metabolism, and glucose homeostasis pathways, and the organic anion transport polypeptide system. Using toxicogenomics, we have identified key molecular pathways involved in chronic sublethal MCLR exposure in wild-type mice, genes participating in those critical pathways and related them to cellular and morphologic alterations seen in this and other studies.

Topics: Animals; Blood Chemical Analysis; Body Weight; Carcinogens; Chemical and Drug Induced Liver Injury; DNA, Complementary; Gene Expression; Genes, p53; Liver; Marine Toxins; Mice; Mice, Knockout; Microcystins; Oligonucleotide Array Sequence Analysis; Organ Size; Reverse Transcriptase Polymerase Chain Reaction; RNA; Toxicogenetics

Microcystin-LR (MCLR) is a potent hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. The histology of acute lethal toxicity has been well characterized, but histology is limited regarding sublethal exposure. Balb/C mice were given a single sublethal dose of MCLR (45 microg/kg) and euthanized at 2, 4, 12, and 24 hours after exposure. Centrilobular to midzonal hepatocellular hypertrophy with loss of cytosolic vacuolation consistent with glycogen depletion occurred at 2 hours. At 4 hours, central lobular hepatocytes exhibited eccentric areas of eosinophilic cytoplasmic condensation that were partially aggregated around the outer nuclear membrane. The areas were weakly positive for cytokeratin and somewhat resembled the Mallory bodies of alcoholic human hepatitis. Small numbers of apoptotic hepatocytes were seen at 24 hours. The toxin was detectable by immunohistochemistry (IHC) as early as 2 hours and was colocalized with the areas of hepatocellular hypertrophy. Intense nuclear staining occurred at 4 hours; this was no longer evident after 12 hours. Strong staining of apoptotic bodies occurred at 24 hours. Mice that received two daily doses had a marked increase in apoptotic hepatocytes in the centrilobular areas. Lesions at four and seven doses consisted of marked hepatocytomegaly and karyomegaly with parenchymal disarray and cytosolic vacuolation. IHC revealed diffuse staining throughout the liver parenchyma consistent with toxin accumulation. An anti-MCLR monoclonal antibody detected bands at the 40-kDa mark in nuclear extracts that were identified as protein phosphatases 1 and 2A by western blotting, consistent with a covalent interaction between MCLR and nuclear protein phosphatases.

Topics: Animals; Bacterial Toxins; Body Weight; Cyanobacteria; Dose-Response Relationship, Drug; Liver; Male; Marine Toxins; Mice; Mice, Inbred BALB C; Microcystins; Organ Size; Peptides, Cyclic; Phosphoprotein Phosphatases

The purpose of this study was to relate dose-dependent hepatotoxicity stemming from prolonged exposure to sublethal concentrations of the cyclic heptapeptide microcystin-LR (Mcyst) to hepatic Mcyst concentrations and protein phosphatase activity. Mcyst is a potent inhibitor of protein phosphatase types 1 and 2A (PP1 and PP2A). Twenty male Sprague-Dawley rats were infused continuously with 0, 3, 6, or 9 micrograms Mcyst/day for 28 days using intraperitoneal mini-osmotic pumps containing highly purified toxin or saline. At the end of 28 days, dose-dependent increases in several serum biochemical tests including sorbitol dehydrogenase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, and bile acids had occurred. Serum albumin decreased in a dose-dependent fashion. Liver activity of both PP1 and PP2A decreased in a dose-dependent manner, but with a relatively greater effect on PP2A than PP1. Liver cytosol Mcyst concentrations, measured by direct competitive ELISA, also increased in a dose-dependent manner, although at a higher rate than would be predicted from the incremental increase in dose given. This disproportional increase is suggestive of the bioaccumulation of Mcyst with increasing dose. Histopathological abnormalities included hepatocellular apoptosis and cytosolic vacuolation of principally zone 3 hepatocytes. Immunohistochemical stains revealed Mcyst predominantly within pericanalicular regions of zone 3 hepatocytes. It was concluded that prolonged exposure to sublethal concentrations of Mcyst results in multiple dose-dependent hepatotoxic effects that correspond to decreased hepatic serine/threonine protein phosphatase activity and increasing cytosolic Mcyst concentrations. The disproportional increase of hepatic Mcyst concentrations observed may suggest the bioaccumulation of toxin and an increasing relative risk of hepatotoxicity with increasing dose.

Topics: Animals; Blood Chemical Analysis; Body Weight; Chemical and Drug Induced Liver Injury; Cytosol; Enzyme Inhibitors; Immunohistochemistry; Injections, Intravenous; Liver; Male; Marine Toxins; Microcystins; Organ Size; Peptides, Cyclic; Phosphoprotein Phosphatases; Rats; Rats, Sprague-Dawley

In 26 hr laboratory trials a dose of 1000 micrograms/kg microcystin-LR (MC-LR) caused 100% mortality in rainbow trout, while no mortality was observed at doses of 400 micrograms/kg or less. The liver to body mass ratio increased in fish exposed to the toxin which was likely due to water retention in the liver. In contrast to mammalian studies, hemorrhage of the liver was rare in fish. Exposure to MC-LR caused widespread hepatocellular swelling and lysis of hepatocyte plasma membranes, resulting in liquifactive necrosis (organelles floating in a milieux of cellular debris). Kidney lesions in the fish consisted of coagulative tubular necrosis with a dilation of Bowman's space. Lesions observed in the liver and kidney of fish exposed to MC-LR were considerably different than those previously reported for mammals.

Topics: Animals; Bacterial Toxins; Body Weight; Dose-Response Relationship, Drug; Hemorrhage; Injections, Intraperitoneal; Kidney Tubules; Liver; Marine Toxins; Microcystins; Microscopy, Electron; Oncorhynchus mykiss; Organ Size; Peptides, Cyclic; Poisoning