Hamster Liver Research Articles

Liver fluke-induced hepatic oxysterols stimulate DNA damage and apoptosis in cultured human cholangiocytes

Oxysterols are cholesterol oxidation products that are generated by enzymatic reactions through cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols have been identified in bile in the setting of chronic inflammation, suggesting that biliary epithelial cells are chronically exposed to these compounds in certain clinical settings. We hypothesized that biliary oxysterols resulting from liver fluke infection participate in cholangiocarcinogenesis. Using gas chromatography/mass spectrometry, we identified oxysterols in livers from hamsters infected with Opisthorchis viverrini that develop cholangiocarcinoma. Five oxysterols were found: 7-keto-cholesta-3,5-diene (7KD), 3-keto-cholest-4-ene (3K4), 3-keto-cholest-7-ene (3K7), 3-keto-cholesta-4,6-diene (3KD), and cholestan-3β,5α,6β-triol (Triol). Triol and 3K4 were found at significantly higher levels in the livers of hamsters with O. viverrini-induced cholangiocarcinoma. We therefore investigated the effects of Triol and 3K4 on induction of cholangiocarcinogenesis using an in vitro human cholangiocyte culture model. Triol- and 3K4-treated cells underwent apoptosis. Western blot analysis showed significantly increased levels of Bax and decreased levels of Bcl-2 in these cells. Increased cytochrome c release from mitochondria was found following treatment with Triol and 3K4. Triol and 3K4 also induced formation of the DNA adducts 1,N6-etheno-2′-deoxyadenosine, 3,N4-etheno-2′-deoxycytidine and 8-oxo-7,8-dihydro-2′-deoxyguanosine in cholangiocytes. The data suggest that Triol and 3K4 cause DNA damage via oxidative stress. Chronic liver fluke infection increases production of the oxysterols Triol and 3K4 in the setting of chronic inflammation in the biliary system. These oxysterols induce apoptosis and DNA damage in cholangiocytes. Insufficient and impaired DNA repair of such mutated cells may enhance clonal expansion and further drive the change in cellular phenotype from normal to malignant.

Transmission of Leishmania infantum MON-98 to hamsters by the bite of Phlebotomus langeroni Nitzulescu (Diptera: Psychodidae)

The ability of Phlebotomus langeroni to successfully acquire and transmit Leishmania infantum MON-98 to hamsters was demonstrated. Sand flies and Leishmania both originated from an infantile visceral leishmaniasis focus in El Agamy Egypt. P. langeroni females were infected by feeding on lesions of needle-inoculated hamster and on infected blood suspension using a chick-skin membrane apparatus. Infection rate of sand flies fed on membrane was 88% compared to 7.8% for flies fed on leishmanial lesion. The transmission to hamster took place by the bites of infective flies taking a second blood meal, on the 8th to 10th day post-feeding. Furthermore, successful transmission was by the bites of flies that took no blood or that took full blood meal. Whereas flies that took full blood meal were not infective as indicated by dissection. In three hamsters, lesions developed after three months. Leishmania amastigotes were demonstrated from the lesion as well as from the liver and spleen of infected hamsters.

Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein

A novel 22.8 kDa of Opisthorchis viverrini ( Ov) calcium-binding EF-hand protein ( Ov CaBP) was identified and isolated from an immunoscreening of the adult stage Ov cDNA library by using a human cholangiocarcinoma (CCA) serum. This protein was related to other calcium-binding proteins and conserved among the trematodes. Ov CaBP shared 98% amino acid identity to 22.8 kDa of Clonorchis sinensis CaBP and both were classified as a new group of CaBP EF-hand protein by multiple sequence alignment and phylogenetic tree analysis. The open reading frame of Ov CaBP was 585 bp which encoded for 194 amino acids. The N-terminal part is composed of two calcium-binding EF-hand motifs whereas the C-terminal part contains a dynein light chain motif (DLC). In addition, transcription analysis by RT-PCR revealed that it was constitutively transcribed in all stages, including metacercariae, juvenile, and adult. Furthermore, recombinant Ov CaBP protein (r Ov CaBP) was expressed as a soluble protein and antibody generated against this r Ov CaBP protein was capable of detecting Ov CaBP in the Ov somatic extracts but not in Ov ES products. This anti-r Ov CaBP serum was also used to localize Ov CaBP in Ov infected hamster's liver sections which the distribution of Ov CaBP was located in gut epithelium, miracidia in eggs and slightly in parenchyma. Moreover, r Ov CaBP protein showed a calcium-binding property in non-denaturing gel mobility shift assay.

Entamoeba histolyticaelectrondense granules secretion in vitro and in vivo: Ultrastructural study

Electron dense granules (EDGs) were identified by transmission electron microscopy in Entamoeba histolytica trophozoites recovered from hamster liver lesions. Abundant granules were present in trophozoites recovered after 15 min of liver inoculation. Variation in the size and morphology of these EDGs was also observed. Numerous granules were present in the plasma membrane when these parasites were incubated for 5 min with MDCK monolayers. Release of these EDGs was suggested by the presence of granules in contact with the surface of the target cell plasma membrane. Parasite phagocytic invaginations were observed after 10 min of parasite-monolayer interaction. In these structures, scarce granules were seen. Granules secretion was corroborated by obtaining of a pellet of these small structures from the incubation of trophozoites with collagen supernatant. Collagenase and gellatinase activity of this pellet was identified in SDS-PAGE gels. EDGs were also present in amebic hamster liver lesions. Our observations corroborate that these granules are secreted and suggest that may participate in the cytopathic effect of E. histolytica both in vitro and in vivo.

Chemopreventive Potential of 18β-glycyrrhetinic Acid: An Active Constituent of Liquorice, in 7,12-dimethylbenz(a)anthracene Induced Hamster Buccal Pouch Carcinogenesis

Chemoprevention, a useful and attractive approach in experimental oncology, helps to investigate the cancer preventive potential of natural products and synthetic entities. Present study evaluated the chemopreventive potential of glycyrrhetinic acid in 7,12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Oral tumor was developed in the buccal pouch of golden Syrian hamsters by painting with DMBA three times a week for 14 weeks. The tumor incidence and the status of phase I and phase II detoxification enzymes were assessed in hamsters treated with DMBA alone and DMBA+glycyrrhetinic acid treated hamsters. One hundred percent tumor formations, which were histopathologically confirmed as well-differentiated squamous cell carcinoma, were observed in hamsters treated with DMBA alone. Also, the status of detoxification enzymes were markedly altered in the liver and buccal mucosa of hamsters treated with DMBA alone. Oral administration ofglycyrrhetinic acid at a dose of 45 mg kg(-1) body weight to hamsters treated with DMBA completely prevented the tumor formation as well as restored the status of detoxification enzymes. Present study thus demonstrated the chemopreventive potential of glycyrrhetinic acid in DMBA induced oral carcinogenesis.

Pravastatin activates PPARα/PPARγ expression in the liver and gallbladder epithelium of hamsters

Our earlier study with cultured gallbladder epithelial cells demonstrated that statins (HMG-CoA reductase inhibitors) activate the expression of PPARalpha and PPARgamma, consequently blocking the production of pro-inflmmatory cytokines. The present study used hamsters to investigate the effects of pavastatin on PPARalpha/PPARgamma expression in the liver and gallbladder epithelium, and to determine whether pravastatin suppresses cholesterol crystal formation in the gallbladder. A total of 40 Golden Syrian male hamsters (4 weeks old) were randomly assigned to four groups (basal diet control; basal diet+pavastatin; high cholesterol diet; high cholesterol diet+pravastatin). All hamsters were 11 weeks old at the end of the experiment. The liver, gallbladder and bile were harvested. Immunohistochemical staining and Western blotting for PPARalpha and PPARgamma were performed in the liver and gallbladder. A drop of fresh bile was examined for cholesterol crystals under a microscope. In the gallbladder and liver of the hamsters, pravastatin activated the PPARalpha and PPARgamma expression of gallbladder epithelial cells and hepatocytes, and particularly the response of PPARgamma was much stronger than that of PPARalpha. Pravastatin suppressed the formation of cholesterol gallstones or crystals in the gallbladder. Pravastatin is an effective medication to activate PPARs (especially PPARgamma) in the liver and the gallbladder epithelium of hamsters, and contributes to the prevention of gallstone formation.

In vitro and experimental therapeutic studies of the calcium channel blocker bepridil: Detection of viable Leishmania (L.) chagasi by real-time PCR

The need for novel and efficacious drugs against neglected parasitic diseases, such as Leishmaniasis and American Trypanosomiasis, is certainly apparent. In this work, we evaluated the in vitro potential of the calcium channel blocker bepridil against Leishmania spp. and Trypanosoma cruzi parasites and exploited an experimental assay using a hamster model with Leishmania (L.) chagasi, with a real-time PCR method for therapeutic evaluation. Bepridil was in vitro effective against promastigotes and intracellular amastigotes of L. (L.) chagasi, with 50% inhibitory concentration (IC50) values of 3.81 and 21.55μM, respectively. Leishmania (L.) amazonensis, L. (L.) major and L. (V.) braziliensis promastigotes and T. cruzi trypomastigotes were also susceptible to bepridil, with in vitro selectivity toward parasites and IC50 values in the range of 3 to 7μM. The mammalian cytotoxicity using LLC-MK2 cells resulted in an IC50 value of 62.67μM. However, bepridil showed lack of activity at 12mg/kg in the experimental hamster model infected with L. (L.) chagasi parasites. However, the real-time PCR was a promising tool for the accurate and fast quantification of RNA of living parasites in the liver and spleen of infected hamsters after treatment, eliminating time-consuming light microscopy evaluations.

Reduction of serum free fatty acids and triglycerides by liver-targeted expression of long chain acyl-CoA synthetase 3

ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that consists of 5 isozymes responsible for cellular fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies have demonstrated that expression of ACSL3 mRNA and protein in liver was specifically increased after feeding hamsters with a fat- and cholesterol-enriched diet, providing the first in vivo evidence for the regulated expression of ACSL3 in liver tissue. The aim of the current study was to further investigate the role of ACSL3 in regulating hepatic lipid metabolism in vitro and in vivo. We utilized an adenoviral-mediated gene delivery approach to exogenously express hamster ACSL3 in hamster liver as well as in HepG2 cells. Transduction of HepG2 cells with Ad-hamACSL3 adenovirus elevated total cellular ACSL enzyme activity, which was accompanied by a significant reduction of cellular contents of triglycerides and total phospholipids. Immunostaining and confocal microscopy studies revealed that ACSL3 was localized to endoplasmic reticulum and mitochondria. In vivo, infection of hamsters with Ad-hamACSL3 led to sustained expression of ACSL3 mRNA and protein in liver two weeks after infection. Importantly, compared with Ad-GFP control virus infected hamsters, we observed significantly lower free fatty acids and triglycerides plus modest reduction of phospholipids in the serum of Ad-hamACSL3 infected animals. Furthermore, triglyceride levels were significantly reduced in Ad-hamACSL3 infected hamster liver. Altogether, these results provide important and physiologically relevant evidence that strengthens the link between ACSL3 expression and hepatic reduction of triglycerides and fatty acids.

Antidyslipidemic Activity of Hot‐water Extracts from Leaves of Cinnamomum osmophloeum Kaneh

The antidyslipidemic activity of hot-water extracts of Cinnamomum osmophloeum leaves (COE) were evaluated on hamsters fed a high-fat diet. Oral administration of COE to hyperlipidemic hamsters reduced the total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL-C) levels. Compared with hyperlipidemic hamsters, the plasma TC and TG levels of hamsters fed with COE at a dosage of 100 mg/kg body weight for 5 and 10 weeks were significantly reduced to 12.63% and 34.25%, and 33.88% and 36.88%, respectively. Plasma LDL-C was also reduced to 27.77% after 10 weeks feeding with the same regimen. Standard diagnostic tests indicated that the extracts did not cause damage to hamster liver or kidneys. Based on these results, it is concluded that COE possesses antidyslipidemic activity. The composition of COE was characterized. Two main compounds, kaempferol 3-O-β-D-apiofuranosyl-(1 → 2)-α-L-arabinofuranosyl-7-O-α-L-rhamnopyranoside (1) and kaempferitrin (2) were identified in the hot-water extracts. Their contents were 7.56% and 9.95%, respectively.

Coumarin protects 7,12-dimethylbenz(a)anthracene-induced genotoxicity in the bone marrow cells of golden Syrian hamsters

Aim : Aim of the present study was to evaluate the antigenotoxic effect of coumarin by measuring the frequencies of micronuclei and the degree of DNA damage in the bone marrow cells of hamster treated with 7,12-dimethylbenz(a)anthracene (DMBA). Materials and Methods: Genotoxicity was induced in experimental hamsters by single intraperitoneal injection of DMBA (30 mg/kg b.w.). The frequency of micronucleated polychromatic erythrocytes (MnPCEs) and DNA damage were assessed in the bone marrow cells of experimental hamsters. The status of lipid peroxidation, antioxidants and phase I and II detoxification agents were utilized as biochemical end points to assess the dose-dependent antigenotoxic potential of coumarin in DMBA-induced genotoxicity. Results: Increase in micronuclei frequency was accompanied by increase in tail length, percent of tail DNA, tail movement and olive tail movement in the bone marrow cells of hamsters treated with DMBA alone. A significant increase in the levels of thiobarbituric acid reactive substances, antioxidants and phase I and II detoxification agents were also noticed in hamsters treated with DMBA alone. Oral pretreatment of coumarin at a dose of 100 mg/kg b.w to hamsters treated with DMBA significantly decreased the frequency of MnPCEs and DNA damage in the bone marrow cells. Also, coumarin restored the status of biochemical variables in the plasma and liver of hamsters treated with DMBA. Conclusions: Present study demonstrated the antigenotoxic effect of coumarin in DMBA-induced genotoxicity. The antigenotoxic potential of coumarin is probably due to its antioxidant potential and modulating effect on detoxification cascade during DMBA-induced genotoxicity.

Effect of phosphatidylcholine–cholesterol liposomes on Entamoeba histolytica virulence

Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.

Effect of cigarette smoke on mutagenic activation of environmental carcinogens by cytochrome P450 2A8 and inactivation by glucuronidation in hamster liver

To elucidate the mechanism underlying suppression of N-nitrosobis(2-oxopropyl)amine (BOP)-induced hamster pancreatic carcinogenesis by cigarette smoke (CS), hepatic levels of microsomal cytochrome P450 (CYP) enzymes, mutagenic activation of environmental carcinogens and three types of uridine diphosphate-glucuronyltransferase (UDPGT) and sulphotransferase (ST) activities were assayed in male Syrian golden hamsters and F344 rats exposed to CS. Immunoblot analyses of microsomal CYP proteins revealed induction of constitutive CYP1A2 (2.6-fold increase) and 2A8 (4.0-fold increase) and induction of CYP1A1 and constitutive CYP1A2 (3.9-fold increase) in rats following exposure to CS for 4 weeks using a Hamburg type II smoking machine. CS exposure enhanced mutagenicities of four heterocyclic amines in the presence of liver S9 in both species, whereas the mutagenicities of aflatoxin B(1) (AFB(1)), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were significantly increased by CS in hamsters but not in rats. However, no CS-induced alterations in the mutagenic activities of other carcinogens, including BOP and other pancreatic carcinogens, were observed in either species. Application of several CYP inhibitors revealed that the mutagenic activities of MeAαC, AFB(1) and NNK in the hamster liver S9 were partly associated with CYP2A8, whereas those of the three pancreatic carcinogens were selectively associated with CYP2B. CS enhanced UDPGT activities towards 4-nitrophenol (4-NP) (1.9- to 2.0-fold) but did not affect those of bilirubin, testosterone UDPGTs and three STs in both species. Together with the previous findings that BOP does not induce tumourigenesis in rats and that the glucuronidation of β-oxypropylnitrosamines is higher in rats than in hamsters, suppression of BOP-induced pancreatic carcinogenesis by CS might be attributed to increased detoxification by 4-NP UDPGT and not decreased CYP2B activation. This is the first demonstration of the induction of CYP2A protein by CS; CYP2A protein polymorphisms have been associated with oral and pulmonary carcinogenesis in smokers.

Immunogenicity and Protective Efficacy in Mice and Hamsters of a β-Propiolactone Inactivated Whole Virus SARS-CoV Vaccine

The immunogenicity and efficacy of β-propiolactone (BPL) inactivated whole virion SARS-CoV (WI-SARS) vaccine was evaluated in BALB/c mice and golden Syrian hamsters. The vaccine preparation was tested with or without adjuvants. Adjuvant Systems AS01(B) and AS03(A) were selected and tested for their capacity to elicit high humoral and cellular immune responses to WI-SARS vaccine. We evaluated the effect of vaccine dose and each adjuvant on immunogenicity and efficacy in mice, and the effect of vaccine dose with or without the AS01(B) adjuvant on the immunogenicity and efficacy in hamsters. Efficacy was evaluated by challenge with wild-type virus at early and late time points (4 and 18 wk post-vaccination). A single dose of vaccine with or without adjuvant was poorly immunogenic in mice; a second dose resulted in a significant boost in antibody levels, even in the absence of adjuvant. The use of adjuvants resulted in higher antibody titers, with the AS01(B)-adjuvanted vaccine being slightly more immunogenic than the AS03(A)-adjuvanted vaccine. Two doses of WI-SARS with and without Adjuvant Systems were highly efficacious in mice. In hamsters, two doses of WI-SARS with and without AS01(B) were immunogenic, and two doses of 2 μg of WI-SARS with and without the adjuvant provided complete protection from early challenge. Although antibody titers had declined in all groups of vaccinated hamsters 18 wk after the second dose, the vaccinated hamsters were still partially protected from wild-type virus challenge. Vaccine with adjuvant provided better protection than non-adjuvanted WI-SARS vaccine at this later time point. Enhanced disease was not observed in the lungs or liver of hamsters following SARS-CoV challenge, regardless of the level of serum neutralizing antibodies.

Strong induction of PCSK9 gene expression through HNF1α and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters

We investigated the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the resistance of dyslipidemic hamsters to statin-induced LDL-cholesterol (LDL-C) reduction and the molecular mechanism by which statins modulated PCSK9 gene expression in vivo. We utilized the fructose diet-induced dyslipidemic hamsters as an in vivo model and rosuvastatin to examine its effects on liver PCSK9 and LDL receptor (LDLR) expression and serum lipid levels. We showed that rosuvastatin induced PCSK9 mRNA to a greater extent than LDLR mRNA in the hamster liver. The net result was that hepatic LDLR protein level was reduced. This correlated closely with an increase in serum LDL-C with statin treatment. More importantly, we demonstrated that in addition to an increase in sterol response element binding protein 2 (SREBP2) expression, rosuvastatin treatment increased the liver expression of hepatocyte nuclear factor 1 alpha (HNF1alpha), the newly identified key transactivator for PCSK9 gene expression. Our study suggests that the inducing effect of rosuvastatin on HNF1alpha is likely a underlying mechanism accounting for the higher induction of PCSK9 than LDLR because of the utilization of two transactivators (HNF1alpha and SREBP2) in PCSK9 transcription versus one (SREBP2) in LDLR transcription. Thus, the net balance is in favor of PCSK9-induced degradation of LDLR in the hamster liver, abrogating the effect of rosuvastatin on LDL-C lowering.

Effect of high-fat diets supplemented with okara soybean by-product on lipid profiles of plasma, liver and faeces in Syrian hamsters

The main components of okara, a by-product from soybean, are dietary fibre and protein. In this study we fed okara based diets with different amounts of fibre, protein and isoflavones to determine their effect on the lipid profile in the plasma, liver and faeces of an animal model. Male golden Syrian hamsters were fed high-fat diets supplemented with okara for 3 weeks. The supplemented diets contained 13% or 20% of okara fibre (OK-13 and OK-20), low-protein okara with 13% of fibre (OK1-13), and isoflavone-free okara with 13% of fibre (OK2-13). Okara supplemented diets did not produce significant differences in the feed intake or body weight gain ( p > 0.05). The plasma levels of triglycerides, VLDL- plus LDL cholesterol and total cholesterol in hamsters fed OK-20 decreased significantly ( p < 0.05) with respect to the 20% control group (C-20). However, no significant differences ( p > 0.05) were found in LDL- and HDL-cholesterol plasma levels in all experiments. Total lipids, triglycerides, total and esterified cholesterol concentrations in liver were reduced by OK-20 diet. Regarding the hamsters fed OK-13 diets, the mean values of the total lipid, triglyceride and cholesterol in the plasma and liver decreased as compared to the control (C-13), but the differences were not statistically significant. All of the okara diets assayed increased the faecal excretion of total lipids, triglycerides, free cholesterol and total nitrogen ( p < 0.05) compared to their respective controls. Our results suggest that the main components of okara, dietary fibre and protein, could be related with the total lipids and cholesterol decrease in the plasma and liver, as well as with the faecal output increase in high-fat fed hamsters. Okara might play an interesting role in the prevention of hyperlipidemia and could be used as a natural ingredient or supplement for functional food preparation.

Long Chain Acyl-CoA Synthetase-3 Is a Molecular Target for Peroxisome Proliferator-activated Receptor δ in HepG2 Hepatoma Cells

ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

Granulomatous-like immune reaction and hepatic fibrosis induced by Schistosoma haematobium immature worms

Golden hamsters were inoculated with Schistosoma haematobium cercariae to examine histological lesions at different time points over an 18 month period of infection. Hamsters were sacrificed 26 weeks and 82 weeks after inoculation. The parasite was found in the blood and in the liver of infected animals as was expected, but we found exclusively male worms, no female worms nor eggs. Interestingly we observed unexpected hepatic lesions induced by S. haematobium adult male worms alone in the golden hamster, characteristic of schistosome eggs. Samples from liver, kidneys, lungs, bladder and gastrointestinal tract were collected during necropsy to evaluate injuries induced by S. haematobium. Notably we observed hepatitis in the liver of infected hamsters, no lesions were found in other organs. We also found liver fibrosis in infected hamsters. This study provides further experimental evidence for the role that schistosome worms, and their derived antigens, may play in the pathology of the infection and modulation of liver chronic inflammation in the murine model of schistosomiasis.

Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess

Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.

Entamoeba histolytica and E. dispar trophozoites in the liver of hamsters: in vivo binding of antibodies and complement

BackgroundHuman amoebiasis is caused by the parasitic protozoan Entamoeba histolytica that lives in the large intestine of hosts, where can produce asymptomatic colonization until severe invasive infections with blood diarrhea and spreading to other organs. The amoebic abscesses in liver are the most frequent form of amoebiasis outside intestine and still there are doubts about the pathogenic mechanisms involved in their formation. In this study we evaluated the in situ binding of antibodies, C3 and C9 complement components on trophozoites, in livers of hamsters infected with E. histolytica or E. dispar. These parameters were correlated with the extension of the hepatic lesions observed in these animals and with trophozoites survivor.MethodsHamsters were inoculated intra-hepatically with 100,000 trophozoites of E. histolytica or E. dispar strain and necropsied 12, 24, 48, 72, 144 and 192 h after inoculation. Antibodies, C3 and C9 binding to trophozoites were detected by immunohistochemistry. The estimation of the necrosis area and the number of labeled trophozoites was performed using digital morphometry analysis.ResultsIn the liver sections of animals inoculated with the amoebas, the binding of antibodies to E. histolytica trophozoites was significantly lower than to E. dispar trophozoites. Trophozoites of E. dispar were also more frequently vacuolated and high labeled cellular debris observed in the lesions. Positive diffuse reaction to C3 complement component was more intense in livers of animals inoculated with E. histolytica after 24 and 72 h of infection. C3(+) and C9(+) trophozoites were detected in the vascular lumen, granulomas and inside and in the border of necrotic areas of both infected group animals. C3(+) and C9(+) trophozoite debris immunostaining was higher in livers of E. dispar than in livers of E. histolytica. A positive correlation between necrotic areas and number of C9(+) trophozoites was observed in animals inoculated with E. dispar.ConclusionMorphological and immunohistochemical results suggest that antibodies and complement are able to bind and destroy some trophozoites in the liver of experimentally infected hamsters, perhaps selecting the more resistant parasites which are responsible by progression of amoebic abscesses. The findings indicate that E. histolytica possesses an enhanced ability in vivo to evade the immune responses compared to E. dispar, although it also causes experimental hepatic lesions.

Distribution and metabolism of four different dimethylated arsenicals in hamsters

Arsenic toxicity and distribution are highly dependent on animal species and its chemical species. Recently, thioarsenical has been recognized in highly toxic arsenic metabolites, which was commonly found in human and animal urine. In the present study, we revealed the mechanism underlying the distribution and metabolism of non-thiolated and thiolated dimethylarsenic compounds such as dimethylarsinic acid (DMA V), dimethylarsinous acid (DMA III), dimethylmonothioarsinic acid (DMMTA V), and dimethyldithioarsinic acid (DMDTA V) after the administration of them into femoral vein of hamsters. DMA V and DMDTA V distributed in organs and body fluids were in their unmodified form, while DMA III and DMMTA V were bound to proteins and transformed to DMA V in organs. On the other hand, DMA V and DMDTA V were mostly excreted into urine as their intact form 1 h after post-injection, and more than 70% of the doses were recovered in urine as their intact form. By contrast, less than 8–14% of doses were recovered in urine as DMA V, while more than 60% of doses were distributed in muscles and target organs (liver, kidney, and lung) of hamsters after the injection of DMMTA V and DMA III. However, in red blood cells (RBCs), only a small amount of the arsenicals was distributed (less than 4% of the doses) after the injection of DMA III and DMMTA V, suggesting that the DMA III and DMMTA V were hardly accumulated in hamster RBCs. Based on these observations, we suggest that although DMMTA V and DMDTA V are thioarsenicals, DMMTA V is taken up efficiently by organs, in a manner different from that of DMDTA V. In addition, the distribution and metabolism of DMMTA V are like in manner similar to DMA III in hamsters, while DMDTA V is in a manner similar to DMA V.