Secretion of Thioredoxin Peroxidase Protein of Cat Liver Fluke Opisthorchis felineus during Modeling of Experimental Opisthorchiasis.

Mechanisms of thioredoxin peroxidase secretion by Opisthorchis felineus were studied in vivo and in vitro. Specific antibodies were obtained and used for western blotting and immunohistochemical detection in Syrian hamster model of opisthorchiasis. Secreted thioredoxin peroxidase protein was accumulated in the worm incubation medium under conditions of oxidative stress and in bile duct cells of hamsters with chronic opisthorchiasis.

Andrographolide Ameliorates Beta-Naphthoflavone-Induced CYP1A Enzyme Activity and Lipid Peroxidation in Hamsters with Acute Opisthorchiasis.

BACKGROUND: Opisthorchis viverrini (OV) infection generates oxidative stress/free radicals and is considered as a primary cause ofcholangiocarcinoma since it primarily triggers sclerosing cholangitis. OBJECTIVE: In this study, the impacts of andrographolide on acute opisthorchaisis in ß-naphthoflavone (BNF)-exposed hamsters were investigated. MATERIAL AND METHOD: Ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities and Thiobarbituric acid reaction substances (TBARS) assay of andrographolide in acute opisthorchiasis in the BNF-exposed hamsters were assessed. RESULTS: The results showed that andrographolide ameliorated the hepatic CYP1A1 and CYP1A2 activities by decreases of the specific enzymatic reactions of EROD and MROD, respectively, in the BNF-exposed hamsters. Moreover, andrographolide lowered the formation of malondialdehyde in the livers and brains of the hamsters. CONCLUSION: These observations revealed the promising chemo-protective and antioxidant activities of andrographolide via suppression of the specific EROD and MROD reactions and lipid peroxidation against acute opisthorchiasis in the BNF-exposed hamsters.

Hepatic cytochrome P450 2A6 and 2E1 status in peri-tumor tissues of patients with Opisthorchis viverrini-associated cholangiocarcinoma.

Endogenous nitrosation due to chronic inflammation is enhanced in opisthorchiasis and plays a crucial role in the development of cholangiocarcinoma (CCA). Hepatic cytochrome P450 (CYP) family enzymes, especially CYP2A6 and CYP2E1, are involved in the metabolism of procarcinogens; these two enzymes metabolize endogenous nitrosamines to carcinogenic N-dimethylnitrosamine (NDMA). CYP2A6 activity is increased in patients infected with Opisthorchis viverrini. Our aim was to determine whether the expression and function of CYP2A6 and 2E1 in the livers of patients with O. viverrini-associated cholangiocarcinoma (CCA) was altered compared to livers without CCA. Livers of CCA patients (n = 13 cases) showed increased enzyme activities, protein and mRNA levels of CYP2A6 whereas the enzyme activity and protein levels of CYP2E1 were markedly decreased (P < 0.05). CYP2E1 mRNA levels were not altered. Large numbers of inflammatory cells and increased iNOS expression was found in areas adjacent to the tumor. The data provide evidence to support the concept that enhanced CYP2A6 activity and diminished CYP2E1 activity probably involve to the progression of CCA.

The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases.

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection.

Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini.

The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63 kDa. The predicted protein has similarity to previously characterized thioredoxins with 26-51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1 at mRNA and protein levels was observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory-secretory products released by adult O. viverrini. Immunohistochemistry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not in normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host-parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation.

Oxidative and nitrative stress in Opisthorchis viverrini-infected hamsters: an indirect effect after praziquantel treatment.

Praziquantel causes adverse effects after short-term treatment. To examine the mechanism of these effects, we studied the distribution of Opisthorchis viverrini antigens and the expression of inducible nitric oxide synthase (iNOS), nuclear factor-kappaB (NF-kappaB), and antioxidant enzymes in O. viverrini-infected hamsters during short-term praziquantel treatment. Praziquantel-induced dispersion of parasite antigens produced a recruitment of inflammatory cells. NF-kappaB and iNOS mRNA expression was significantly elevated and associated with their immunoreactivity in the bile duct epithelium and inflammatory cells. Plasma nitrate, end products of nitric oxide, and malondialdehyde level increased significantly. Expression of mRNA for antioxidant enzymes (superoxide dismutases, catalase, and glutathione peroxidase) also increased significantly, which suggests host defense against oxidative stress. These results suggest that short-term praziquantel treatment induces inflammation and resulting oxidative and nitrative stress through O. viverrini antigen release. Data in this study can be used as a basis to understand potential side effects of praziquantel treatment in humans.

Hepatobiliary changes, antibody response, and alteration of liver enzymes in hamsters re-infected with Opisthorchis viverrini.

We investigated pathological changes, antibody response, and liver enzymes in hamsters re-infected with Opisthorchis viverrini. Group 1 received a single dose of 50 metacercariae; Groups 2 and 3 were first dosed with of 30 metacercariae and re-infected with 20 more once or twice at three month intervals. Inflammation and liver cell necrosis were observed on 3D (day 3) for Group 3 and 7D for Group 2 in comparison with 21D for Group 1. Pathological changes included peri-ductal fibrosis, bile duct dilation, and small bile duct formation. Increased O. viverrini-specific IgG levels ranked in the order Group 3>Group 2>Group 1. Liver enzyme activity was related to inflammatory cell infiltration. Re-infection induced faster inflammation and more severe pathological changes in association with parasite-specific antibody during chronic inflammation. This study emphasizes that there is an important relationship between the gradual decreases of inflammation with a concomitant increase in fibrosis after re-infection.

Liver enzyme activity and histological changes in the liver of silver foxes (Vulpes vulpes fulva) experimentally infected with opisthorchiid liver flukes. A contribution to the pathogenesis of opisthorchiidosis.

Blood samples from silver foxes experimentally infected with Opisthorchis felineus and Metorchis bilis, respectively, were examined for the activity of liver enzymes. The average activities of aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), alkaline phosphatase and alanine aminotransferase in uninfected control animals were 20, 1.8, 57 and 44 units/l, respectively. The liver enzymes in infected foxes reacted differently, depending on dose, species of flukes and individual peculiarities. The highest individual deviation of infected from control animals was registered in the case of GLDH, reaching increases of up to 200-fold. In contrast, AST showed the lowest deviation from control values (less than 10-fold). By the end of the study period, enzyme activities had declined. The prepatent periods for M. bilis and O. felineus in foxes were 2 weeks and 4 weeks, respectively. High egg per gram values were established at the beginning of the patent period. At necropsy, chronic inflammatory reactions were found in the bile ducts and in the wall of the gall bladder. The number of flukes at the end of the study was low.

Induction of cytochrome P450 2A6 expression in humans by the carcinogenic parasite infection, opisthorchiasis viverrini.

The purpose of this study was to examine in vivo the activity of cytochrome P450 (CYP) 2A6, an enzyme capable of activating carcinogens, including N-nitrosodimethylamine, in humans with the carcinogenic liver fluke infection, opisthorchiasis viverrini, before and after treatment with the antiparasitic agent, praziquantel. Coumarin hydroxylase activity of CYP 2A6 was assessed by administering a probe drug, coumarin, and measuring its metabolite, 7-hydroxycoumarin, in urines collected between 0-2 h and 2-4 h of 106 people with varying intensities of Opisthorchis viverrini infection. Five individuals who did not excrete any detectable 7-hydroxy coumarin (and have a genetic defect probably leading to an absence of catalytic activity of the CYP 2A6 protein) were excluded from analysis. Infected people excreted an average of 22.7 mumol of 7-hydroxycoumarin in the first 2 h after taking the drug, whereas the mean of the uninfected group was 19.4 mumol; this difference did not reach statistical significance (P = 0.10). However, a highly significant increase in CYP 2A6-related activity was observed in infected individuals who also had radiological evidence of biliary fibrosis (28.1 mumol) compared to those without (19.4 mumol; P = 0.01). Reassessments of coumarin hydroxylase activity of CYP 2A6 made 2 months after praziquantel treatment showed highly significant reductions in the amount of 7-hydroxycoumarin excreted among the infected groups but no difference in the uninfected group. These results suggest that expression of CYP 2A6 is induced among chronically infected people who also have fibrosis of the intrahepatic bile duct. As already demonstrated in an animal model and now observed in humans for the first time, this increase in CYP 2A6-related enzyme activity may represent an important mechanistic link between inflammatory products of chronic liver fluke infection (e.g., DNA alkylation damage from endogenously formed N-nitrosamines) and the high risk of cholangiocarcinoma faced by infected individuals.

[The myeloperoxidase of the polymorphonuclear leukocytes as a marker of the stage of Opisthorchis invasion and as an indicator of the efficacy of chemotherapy]. / Mieloperoksidaza polimorfno-iadernykh leikotsitov kak marker stadii opistorkhoznoi invazii i indikator éffektivnosti khimioterapii.

A total of 152 patients with Opisthorchis infection at the acute and chronic stage of the diseases, as well as 1-3 days and 1-2 months after therapy with biltricide in a dose of 60 mg/kg body weight. Intracellular myeloperoxidase was spectrophotometrically determined. Its activity was demonstrated to be significantly varying at the different opisthorchiasis stage. At the acute stage there was a 6-fold increase in the activity of the enzyme as compared with that at the chronic stage. The therapy resulted in the drop of myeloperoxidase levels, at the same time the authors interpret the heterodirectional changes in the activity of myeloperoxidase in the early post-therapeutic period as a fact of individual anthelmintic intolerance the normalization of the parameter studied 1 month after the therapy only in 42.1% of the cases as a fact residual Opisthorchis antigen persistence.

[Granulomatous inflammation of the liver in opisthorchiasis]. / Granulematoznoe vospalenie pecheni pri opistorkhoze.

Granulomatous liver inflammation was studied in Syrian hamsters in opisthorchiasis alone and in combination with different factors, such as carcinogens, anthelmintics, immunosuppressants and pregnancy. Parasitic ova and metabolites, as well as hepatic cell necrosis are shown to be the initiating factors of granulomatous inflammation. Granulomas originate in ductal walls, hepatic stroma and parenchyma. Four granuloma types are distinguished, i.e. macrophagal, giant-cell, necrotizing and cicatricial one. The biggest granuloma area was observed in superinvasive form of helminthiasis, when infection was combined with a carcinogen (DMNA), and in pregnant female hamsters treated with droncite. It is suggested that opisthorchiasis is a granulomatous disease.

Serum glutamyl transferase and other liver function tests in Opisthorchis viverrini infection.

Serum glutamyl transferase (gamma-GT), serum total protein, albumin, aspartate aminotransferase (GOT), alanine aminotransferase (GPT), alkaline phosphatase and bilirubin were measured in 55 males and 45 females suffering from O. viverrini infection and in apparently healthy non-infected individuals. A decrease in total protein, albumin and bilirubin, as well as an increase in GOT, GPT and gamma-GT was observed in males with O. viverrini infection, whereas alkaline phosphatase remained unaffected. In female patients with O. viverrini, serum total protein and albumin also decreased, GOT and GPT increased, whereas gamma-GT remained unchanged. The difference in gamma-GT alteration between females and males is discussed with regard to the possible significance of alcohol consumption and in relation to the parasitic infection and its possible implications for malignancy, associated with liver fluke infection.

Liver procollagen prolyl hydroxylase in Opisthorchis viverrini infected hamsters after praziquantel administration.

Infection of hamsters by the human liver fluke Opisthorchis viverrini elevated liver procollagen prolyl hydroxylase activity, reflecting increased collagen biosynthesis. The increase was proportional to the intensity of infection. However, the infected liver procollagen prolyl hydroxylase activity decreased after administration of praziquantel 300 mg kg-1 body weight, and approached normal levels two weeks after treatment. In the infected hamsters, praziquantel, at a curative dose, caused a transient increase in serum aminotransferase levels and a small but persistent rise in serum alkaline phosphatase. The drug, however, did not cause changes in these enzyme activities in the uninfected hamsters.

Increased liver prolyl hydroxylase activity in hamsters infected with the human liver fluke Opisthorchis viverrini.

A parallel increase in liver collagen content and prolyl hydroxylase activity was observed in hamsters infected with the human liver fluke Opisthorchis viverrini. They were elevated at 2 weeks after infection, gradually increased to approximately 2-fold at 7-11 weeks of infection, and then declined as with duration of infection time increasing from 11 to 22 weeks.