A Large Scale Study of the Epidemiology and Risk Factors for the Carcinogenic Liver Fluke Opisthorchis viverrini in Udon Thani Province,Thailand

Opisthorchis viverrini infection and cholangiocarcinoma are serious problems in South East Asia. This study aimed to find the prevalence of opisthorchiasis in various hosts in Udon Thani Province. Total fecal samples were collected from 14,766 participants. The epidemiological data collected and analysed included prevalence and intensity of infection. Odds ratios (OR) were calculated to determine the associations between cross sectional data and to predict possible risk factors. The prevalence of O. viverrini infection in Udon Thani Province averaged 15.3% (eggs per gram (epg.) = 48.9 and range; 12-1,320), with differences between villages (range; 3.8%-79.8%). An age-dependence for infection was observed to increase from ages 25 to 50 years and then decrease for older participants. A univariate analysis identified risk parameters including age (p = 0.040; OR = 3.9 (95% CI = 1.2-7.5)), education (p<0.0001; OR = 7.3 (95% CI = 1.8-21.6)) and eating habits (p = 0.032; OR = 1.6 (95% CI = 0.5-3.7)). Interestingly, most participants were not aware of treatments such as praziquantel (p< 0.0001; OR = 3.5 (95% CI = 1.4-11.6)), had no history of parasitic treatment (p = 0.486; OR = 1.5 (95% CI = 0.5-3.5)) and had eaten raw fish (p = 0.04; OR = 7.4 (95% CI = 1.5-18.6)). Liver fluke infection in dogs (18.1%, epg. = 44.7, range; 32-96) was significantly higher than in cats (11.0%, epg. = 117.8, range; 44-372) (p<0.05). A positive association between O. viverrini infection in dogs and their owners was found. In addition, cyprinid fish dominantly infected by metacercaria including Henicorhynchus siamensis (27.7%), Cyclocheilichthys repasson (21.9%), Hampala dispar (14.1%), and Barbonymus gonionotus (6.9%). This study provides basic information required for the development of future effective and sustainable strategies to reduces infection rates, mainly by providing health education and encouraging behavioural changes.     

Involvement of MMP‐9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1‐dependent DNA damage in a hamster model

Peribiliary fibrosis caused by chronic infection with Opisthorchis viverrini (OV) is a risk factor of cholangiocarcinoma (CCA) in northeastern Thailand. Matrix metalloproteinases (MMPs) are enzymes capable of degrading and remodeling the extracellular matrix in the process of fibrosis and carcinogenesis. We examined MMPs expression and their role in fibrogenesis and cholangiocarcinogenesis in hamsters treated with OV and N‐nitrosodimethylamine (NDMA). We assessed the time profiles of MMPs, inducible nitric oxide synthase (iNOS), Rac1, α‐smooth muscle actin (α‐SMA) and DNA lesions (8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine, 8‐oxodG) in relation to fibrosis and CCA development. Histopathology revealed OV and NDMA synergistically induced peribiliary fibrosis time‐dependently, and CCA occurred at 3 months, whereas OV or NDMA alone induced less fibrosis. Hydroxyproline levels in the liver and plasma were positively associated with the expression of collagen I and α‐SMA. MMP‐9 expression was significantly increased and correlated with the accumulation of myofibroblast, fibrosis levels and cholangiocarcinogenesis. MMP‐9 activity was correlated with iNOS, and immunocolocalization was observed in inflammed tissues, early and invasive CCA. OV and NDMA synergistically induced MMP‐9 expression in association to Rac1. In addition, Rac1 was colocalized with iNOS, and 8‐nitroguanine, in inflammed tissues and CCA. Formation of 8‐nitroguanine and 8‐oxodG increased with tumor progression. The results suggest that MMP‐9 expression is associated with the accumulation of peribiliary fibrosis in conjunction to the induction of iNOS and Rac1 that may potentiate DNA damage and cholangiocarcinogenesis.     

Protective effect of melatonin against Opisthorchis viverrini‐induced oxidative and nitrosative DNA damage and liver injury in hamsters

Abstract: The liver fluke, Opisthorchis viverrini, is the risk factor of cholangiocarcinoma, which is a major health problem in northeastern Thailand. Production of reactive oxygen and nitrogen species during the host’s response leads to oxidative and nitrosative stress contributing to carcinogenesis. We investigated the protective effect of melatonin against O. viverrini‐induced oxidative and nitrosative stress and liver injury. Hamsters were infected with O. viverrini followed by oral administration of various doses of melatonin (5, 10, and 20 mg/kg body weight) for 30 days. Uninfected hamsters served as controls. Compared to the levels in O. viverrini‐infected hamsters without melatonin treatment, the indoleamine decreased the formation of oxidative and nitrosative DNA lesions, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 8‐nitroguanine, in the nucleus of bile duct epithelium and inflammatory cells, in parallel with a reduction in 3‐nitrotyrosine. Melatonin also reduced the expression of heme oxygenase‐1 and cytokeratin 19, nitrate/nitrite levels, and bile duct proliferation in the liver. Alanine transaminase activity and the levels of 8‐isoprostane and vitamin E were also dose dependently decreased in the plasma of melatonin‐treated hamsters. Melatonin reduced the mRNA expression of oxidant‐generating genes [inducible nitric oxide synthase, nuclear factor‐kappa B (NF‐κB), and cyclooxygenase‐2] and proinflammatory cytokines (TNF‐α and IL‐1β), accompanied by an increase in the expression of antioxidant genes [nuclear erythroid 2‐related factor 2 (Nrf2) and manganese superoxide dismutase]. Thus, melatonin may be an effective chemopreventive agent against O. viverrini‐induced cholangiocarcinoma by reducing oxidative and nitrosative DNA damage via induction of Nrf2 and inhibition of NF‐κB‐mediated pathways.     

Plasma hydroxyproline, MMP-7 and collagen I as novel predictive risk markers of hepatobiliary disease-associated cholangiocarcinoma

Chronic opisthorchiasis caused by Opisthorchis viverrini infection is characterized by advanced periductal fibrosis leading to hepatobiliary diseases (HBD), including cholangiocarcinoma (CCA). We aimed to determine fibrotic markers to differentiate HBD status including opisthorchiasis, benign biliary disease (BBD) and CCA. Candidate fibrotic markers in plasma of healthy individuals (n = 14) and patients with opisthorchiasis (n = 32, pre- and post-treatment with praziquantel), BBD (n = 31), CCA (n = 37) and other types of tumors (n = 14) were measured by ELISA and zymography. Plasma levels of hydroxyproline (HYP), collagen I, MMP-7 and TIMP2 in opisthorchiasis patients were significantly higher than those in healthy individuals, and MMP9/TIMP2 balance may be associated with tissue resorption after praziquantel treatment. HYP and TIMP-2 levels were significantly correlated with periductal fibrosis status evaluated by ultrasonography. Plasma HYP level of CCA patients was the highest among HBD patients (p < 0.05). ROC curves revealed HYP, MMP-7 and collagen I levels significantly distinguished opisthorchiasis, BBD and CCA (p < 0.001). Odd ratio (OR) analysis demonstrated these markers in opisthorchiasis were predictable for BBD risk (p < 0.05; OR = 28.50, 10.12 and 4.63 for collagen I, MMP-7 and HYP, respectively), and the risk was reduced by praziquantel treatment. Interestingly, only plasma HYP level in BBD was predictable for CCA risk (OR = 3.69; p = 0.020). In conclusion, plasma HYP, collagen I and MMP-7 may be useful as novel predictive markers of opisthorchiasis-related BBD, and HYP may be used as a diagnostic marker for CCA.     

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.