Reduction of carcinogens in fermented fish (pla-ra and pla-som) by heating.

Background and Aim: The risk factors for cholangiocarcinoma (CCA) are opisthorchiasis and the intake of a combination of nitroso compounds through the consumption of traditionally fermented fish, which is very popular in areas where liver flukes are endemic. The incidence of CCA remains high because this cultural habit of rural people has been altered. Therefore, decreasing nitrate and nitrite concentrations in fermented fish are an alternative approach to reducing the risk of CCA. Thus, this study aimed to reduce nitrate and nitrite concentrations in fermented foods by heating and investigated its effect on CCA development in a hamster model. Materials and Methods: We used Association of Official Analytical Chemists method 973.31 to measure the nitrate and nitrite concentrations in both fermented fish (pla-ra [PR]) and pickled fish (pla-som [PS]) before and after boiling for 5 and 30 min, respectively. The same samples were fed to Opisthorchis viverrini (OV)-infected or -uninfected hamsters for 3 months. Thereafter, the hamsters' liver and blood were collected for analysis. Results: The levels of nitrates and nitrites in PS and PR significantly decreased following boiling for 5 and 30 min. The OV-PR and OV-PS groups showed dramatically increased numbers of inflammatory cells, fibrosis surrounding the bile duct, and focal fibrotic areas. However, after boiling the fermented dishes for 5 and 30 min, the extent of inflammatory cell infiltration and intensity of fibrosis in these groups were decreased. Conclusion: Our findings suggest that boiling reduces nitrate and nitrite toxicity in fermented dishes, as evidenced by reduced hepatic inflammation. However, regardless of heating, kidney tissues are adversely affected when fermented meals are consumed daily.

Prevalence and Associated Risk Factors of Intestinal Parasitic Infections: A Population-Based Study in Phra Lap Sub-District, Mueang Khon Kaen District, Khon Kaen Province, Northeastern Thailand.

Intestinal parasitic infections are still a crucial problem among communities in Northeast Thailand. Misuse of antiparasitic drugs and unhealthy food behaviors are known. This study aimed to explore the prevalence, behavioral health factors, and motivation for self-treatment of anti-parasitic drugs in this area. A community-based cross-sectional study was conducted in Phra Lap sub-district, Mu Khon Kaen district, Khon Kaen province, Northeast Thailand, in 2016. A total of 419 participants were recruited to complete a self-administered questionnaire and stool examination. Binary logistic regression was used to assess the association between the risk factor and parasitic infection. Forty-two participants (10%; 95%CI 7.5-13.3) were positive for at least one parasite species. In this community, the most detected intestinal parasite was Opisthorchis viverrini (5.3%), followed by Strongyloides stercoralis (3.1%). A total of 67.5% of the participants had the experience of anti-parasitic drug treatment within previous 1 year, and "Often eat raw food" was the most common reason for the use of anti-parasitic drugs. On multivariate analysis, parasitic infections were significantly associated with male gender (ORadj. 2.42; 95%CI 1.00-5.85), age ≥ 60 years (ORadj. 7.55; 95%CI 1.60-35.76), and often consuming raw food of at least one type (ORadj. 2.37; 95%CI 1.03-5.44). Given these findings, correction of the dietary habit of eating raw fish/meat, which is the most important measure, and limitation of the use of anthelmintic treatment for individuals with stools positive for ova as well as emphasis on sanitary toilets will be implemented for the prevention and control of parasitic infection in endemic communities.

Acetaminophen Overdose Enhances Early Cholangiocarcinoma in Opisthorchiasis Hamsters.

Opisthorchiasis which exerted by infection of Opisthorchis viverrini is strongly related to the incident of cholangiocarcinoma (CCA) in many Southeast Asian countries northeastern of Thailand. The O. viverrini infection is primarily caused by raw fish consumption, and repeated exposure to liver fluke. Meanwhile, acetaminophen is usually medicated to relieve pain in particularly people in northeast Thailand. OBJECTIVE: This study therefore aimed at investigating effects of acetaminophen on pathogenesis in hamsters for opisthorchiasis. METHODS: There were 4 groups of hamsters: i) uninfected hamster (N); ii) sole acetaminophen administration (N-Ac); iii) sole O. viverrini infection (OV); and iv) combination of O. viverrini infection and acetaminophen (OV-Ac) on pathology of hamsters for 1 month post infection. For analysis of histopathological changes through hematoxylin and eosin, Sirius red and immunohistostaining for Cytokeratin 19 (CK-19), Proliferating cell nuclear antigen (PCNA) and CA 19-9, serum's hamsters were used detected for liver function tests and tumor-related genes expression. RESULTS: After 1 month under these treatments, the OV-Ac showed significantly higher CCA risk, including inflammatory cells were aggregations around bile duct, new bile duct and fibrosis in subcapsular hepatic tissues, than other treatments. These pathological parameters were positively correlated with immunohistochemical staining derived from CK-19, PCNA and CA 19-9. In addition, OV-Ac had significantly higher liver function tests (ALT). CONCLUSION: Combined intake of liver fluke-contaminated raw fishes and acetaminophen rendered more severity of CCA than sole consumption of the contaminated raw fishes.

Efficacy of Dipterocarpus alatus oil combination with Rhinacanthus nasutus leaf and Garcinia mangostana pericarps against canine demodicosis.

BACKGROUND AND AIM: Canine demodicosis is a skin disease that is a major global health problem in dogs. Ivermectin is a drug of choice for treatment, but it may cause toxicity in dogs carrying multidrug resistance mutation-1 gene mutations. Hence, alternative herbal medicines are used instead of the drug, such as Dipterocarpus alatus oil (YN oil), Rhinacanthus nasutus leaf (WC), and Garcinia mangostana pericarps (MG) extracts. This study aimed to determine the efficacy of D. alatus oil, R. nasutus leaf, and G. mangostana pericarp extracts on canine demodicosis in vivo. MATERIALS AND METHODS: Twenty-five mixed-breed dogs with localized demodicosis were examined. Dogs were diagnosed with demodicosis through deep skin scraping and screened with the inclusion criteria. Five dogs of each group were treated in five treatment groups (ivermectin, YN oil, YN oil+WC, YN oil+MG, and YN oil+WC+MG) for 1 month. The individual dogs were clinically evaluated, and the dermatological lesions were monitored daily for 60 days. RESULTS: Dermatological lesion improvement was predominantly observed in the group of dogs treated with YN oil+WC. This was evidenced by the disappearance of the hyperpigmentation and lichenification on day 28 post-treatment and alopecia on day 56 post-treatment. Moreover, no allergic or clinical signs were observed during treatment. CONCLUSION: YN oil+WC is an alternative herbal medicine that could be used for the treatment of localized canine demodicosis.

Effect of Temperature on the Killing of Opisthorchis viverrini Eggs In Vitro.

Contaminated liver fluke egg in the environment has led to the high prevalence of human opisthorchiasis associated with cholangiocarcinoma in Southeast Asia. To find the effective lessening methods of Opisthorchis viverrini eggs in the contaminated environment, we investigated the temperature conditions for killing of these trematode eggs in vitro. Numerous O. viverrini eggs were obtained in the proximal part of uteri of adult worms from experimental hamsters. Mature eggs with miracidium were allocated by experimental groups (2 control: positive and negative and 4 treatment: 50, 60, 70, and 80°C) with 0.85% saline, and treated by the experimental plan. Eggs in each experimental groups were observed under the confocal microscope after stain with Propidium Iodide (PI) to evaluate the effect of temperatures. Eggs in 70 and 80°C groups were all killed after over 10 min heated. Majority of eggs in 60°C (10, 15, and 30 min heated), 70 and 80°C (5 min heated) groups were inactivated. However in 50°C group, below half of eggs were to be killed in all time lapse (10, 15 and 30 min). In order to prevent O. viverrini infection and cholangiocarcinoma, direct treatment of sewage by heating at 70 or 80°C at least 10 min is essential. Therefore, treatment of O. viverrini eggs at a high temperature is a potential method for controlling egg contamination in sewage.

Simplified Techniques for Killing the Carcinogenic, Opisthorchis Viverrini Metacercariae in Cyprinid Fish

Consumption of fluke-free fish is the most important factor in controlling Opisthorchis viverrini (OV) infection in endemic areas such as northeast Thailand and thereby reducing the risk of cholangiocarcinoma. Cooking fish is the best way to avoid infection; however, the cultural practice of eating raw or fermented fish is difficult to change. We investigated the food preparation process, using freezing, heating and fermentation to kill OV metacercariae in fish. Uncooked cyprinid fish infected with OV were divided into three groups: refrigerated at 4 oC for 24, 48 or 72 h (control group); frozen at -20 oC for 24, 48 or 72 h; or heated by microwaving (at 400 or 800 W) or boiling at 90 oC for 1, 5 or 10 min. Moreover, pickled (fermented) fish were divided into two groups: refrigerated at 4 oC (control) or frozen at -20 oC for 24 or 48 h. The infectivity of recovered metacercariae was confirmed by infecting hamsters with OV and then evaluating the recovery of adult worms after 1 month. We found that a heating process, by boiling or microwaving at 400 or 800 W for at least 5 min, could kill OV metacercariae, and freezing pickled fish at -20 oC for 48 h could kill OV metacercariae in all sizes of fish. The present study found that heating and freezing processes, as well as the fermentation process under optimal conditions, could kill OV metacercariae in a timely manner. This knowledge is valuable for implementation in endemic areas to control OV infection and cholangiocarcinoma.

Application of Pineapple Juice in the Fish Digestion Process for Carcinogenic Liver Fluke Metacercaria Collection

Pepsin is common digestive enzyme used for fish digestion in the laboratory to collect trematode metacercariae. In a field study, to survey the infected fish is needed a huge yield of pepsin and it is very expensive. Therefore, our purpose of this study was to investigate the candidate enzyme from pineapple juice which has a digestive enzyme called bromelain, a mixture of proteolytic enzymes, to digest fish in order to harvest metacercariae. Fish were divided into 2 groups: one group in which metacercariae were harvested using acid pepsin as a control and other groups in which the fish was digested using fresh pineapple juices. The results showed that pineapple juice is able to digest fish similarly to pepsin. The Pattavia pineapple juice had the highest number of metacercariae similar to the control. For Trat Si Thong pineapple juice,we found the number of metacercariae was less than control. This result suggests that the Pattavia pineapple juice was optimal juice for fish digestion to metacercaria collection and can be used instread of pepsin acid.

In Vitro and In Vivo Inhibitory Effects of α-Mangostin on Cholangiocarcinoma Cells and Allografts

We investigated the anti-cholangiocarcinoma effect of α-mangostin from Garcinia mangostana pericarp extract (GM) in a human cholangiocarcinoma (CCA) cell line and a hamster CCA allograft model. In vitro, human CCA cells were treated with GM at various concentrations and for different time periods; then cell-cycle arrest and apoptosis were evaluated using flow cytometry, and metastatic potential with wound healing assays. In vivo, hamster allografts were treated with GM, gemcitabine (positive control) and a placebo (negative control) for 1 month; tumor weight and volume were then determined. Histopathological features and immunostaining (CK19 and PCNA) characteristics were examined by microscopy. The present study found that α-mangostin could: inhibit CCA cell proliferation by inducing apoptosis through the mitochondrial pathway; induce G1 cell-cycle arrest; and inhibit metastasis. Moreover, α-mangostin could inhibit CCA growth, i.e. reduce tumor mass (weight and size) and alter CCA pathology, as evidenced by reduced positive staining for CK19 and PCNA. The present study thus suggested that α-mangostin is a promising anti-CCA compound whose ready availability in tropical countries might indicate use for prevention and treatment of CCA.

Carcinogenic Liver Fluke and Others Contaminated in Pickled Fish of Northeastern Thailand

Twenty provinces in northeastern Thailand were investigated for fluke metacercariae contamination in pickled fish, or pla-som, during January ­June 2016. A total of 129 pickled fish shops were randomly chosen. Samples were digested with acid-pepsin and those found to be infected with metacercariae were fed to hamsters to test for metacercariae infectivity. The results demonstrated that only 20.2% of the pla-som samples were infected with fluke metacercariae (mc), at various levels (1 to 268 mc/kg). All recovered fluke metacercariae were inactive, degenerated and could not develop to adults in the animal model. In conclusion, the fluke mc infection status in pla-som was correlated with the prevalence of fluke infection in this region known for high O.viverrini and cholangiocarcinoma development. Clearly, systematic control of the fluke life cycle is needed. Whether pickling is an effective preventive measure needs further assessment.