Opisthorchis felineus infection provokes time-dependent accumulation of oxidative hepatobiliary lesions in the injured hamster liver.

Opisthorchiasis caused by food-borne trematode Opisthorchis felineus is a substantial public health problem, with 17 million persons infected worldwide. This chronic disease is associated with hepatobiliary inflammation, cholangiocyte dysplasia, cholangiofibrosis, intraepithelial neoplasia, and even cholangiocarcinoma among chronically infected individuals. To provide first insights into the mechanism by which O. felineus infection causes precancerous liver lesions, we investigated the level of oxidative stress (lipid peroxidation byproducts and 8-hydroxy-2'-deoxyguanosine) as well as the time course profiles of chronic inflammation and fibrogenesis markers in the dynamics of opisthorchiasis from 1 month to 1.5 years postinfection in an experimental model based on golden hamsters Mesocricetus auratus. For the first time, we showed that O. felineus infection provokes time-dependent accumulation of oxidative hepatobiliary lesions in the injured liver of hamsters. In particular, over the course of infection, lipid peroxidation byproducts 4-hydroxynonenal and malondialdehyde were upregulated; these changes in general correlate with the dynamics of hepatic histopathological changes. We detected macrophages with various immunophenotypes and elevated levels of CD68, COX2, and CD163 in the O. felineus-infected animals. Meanwhile, there was direct time-dependent elevation of TNF-α (R = 0.79; p < 0.001) and CD163 protein levels (R = 0.58; p = 0.022). We also provide quantitative data about epithelial hyperplasia marker CK7 and a marker of myofibroblast activation (α smooth muscle actin). Our present data provide first insights into the histopathological mechanism by which O. felineus infection causes liver injuries. These findings support the inclusion of O. felineus in Group 1 of biological carcinogens

Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke <i>Opisthorchis felineus</i>

<div><p>The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (<i>Opisthorchis viverrini</i>, <i>Clonorchis sinensis</i> and <i>Schistosoma haematobium</i>) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke <i>Opisthorchis felineus</i>, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in <i>O</i>. <i>felineus</i> tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in <i>O</i>. <i>felineus</i>. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.</p></div

Transcriptional responses for mRNA encoding CYP following <i>in vitro</i> exposure of <i>Opisthorchis felineus</i> to xenobiotics.

<p>Analysis by real-time PCR with normalization based on the expression stability value (M-value); normalization was undertaken using <i>Ub</i> and <i>MrpL16</i> genes as endogenous internal controls (M < 1.2). Triplicate real-time PCRs were run for each sample. <b>A</b>. An adult worm; <b>B</b>. Newly excysted metacercariae (NEM). <b>C.</b> CYP mRNA level after <i>in vitro</i> treatment of <i>O</i>. <i>felineus</i> for 20 h with hemoglobin (DDPCR). CYP gene expression levels were quantified and values were simultaneously normalized to reference gene <i>MrpL16</i> expression using QuantaLife (Bio-Rad, USA). The data are shown as the normalized ratio of CYP to <i>MrpL16</i> ± S.D. Each run was made in duplex. Results of three independent experiments are presented.</p

Suppression of expression of CYP in adult <i>Opisthorchis felineus</i> by RNA interference (RNAi).

<p><b>A.</b> Transcript levels were determined using EVA-green real-time RT-PCR. The <i>MrpL16</i> gene was used for normalization. The control group (no dsRNA treatment) was compared to the negative control group transformed with <i>LUC</i> dsRNA. Three biological samples with technical duplicates were used for analysis. The data are presented as means ± SD. <b>B.</b> Primer positions for analysis of CYP gene expression. <b>C.</b> Control adult <i>O</i>. <i>felineus</i>, eight days after electroporation. EC: excretory channel, EB: excretory bladder. <b>D.</b> Worms eight days after of the knockdown of CYP mRNA. <b>E.</b> Percentage of worms with phenotypic changes in excretory system after three days of RNA interference (data of three independent experiments). Wild type–untreated worms; mock–worms subjected to electroporation without dsRNA; LUC–worms that received <i>LUC</i> dsRNA (non specific control); CYP–worms that received CYP dsRNA; keto—worms were treated with ketoconazole for three days. <b>F, G.</b> Worms five days after the knockdown of CYP (<b>F</b>) and LUC (<b>G</b>) mRNA were treated for 20 h with pentoxyresorufin, and images acquired using multiple fluorescence filters using optical sectioning by means of the AxioImager fluorescence microscope (Zeiss). The resorufin (<b>R</b>) forming in the excretory bladder is indicated with an arrow. Representative images are shown. <b>H.</b> Adult <i>O</i>. <i>felineus</i> after three days of treatment with 40 μM ketoconazole.</p

<i>In situ</i> activity of CYP.

<p>Fluorescence micrographs of <i>Opisthrochis felineus</i> after exposure <i>in vitro</i> for 20 h to several substances. <b>A.</b> A control fluke (multiple fluorescence filters: FITC, rhodamine, DAPI). <b>B.</b> Adult worms were treated with methoxyresorufin. <b>C.</b> Adult worms were treated with benzoxyresorufin. <b>D.</b> Adult worms were treated with pentoxyresorufin (PR). The resorufin (<b>R</b>) that was formed in fluke tissues is indicated with an arrow (FITC, rhodamine fluorescence filters). <b>E.</b> Adult worms were treated with penzoxyresorufin (rhodamine). <b>F.</b> Excretory granules of the resorufin formed in <i>O</i>. <i>felineus</i> treated with pentoxyresorufin. Monochrome images were acquired using a rhodamine filter (5F is a part of image 5E). <b>G.</b> A worm was treated with PR and ketoconazole (rhodamine filter).</p

Kaplan-Meier survival curves and pentoxyresorufin metabolizing activity in worm tissues after RNA interference.

<p><b>A.</b> A set of Kaplan-Meier survival curves (out of three independent experiments) is shown. Statistical difference in survival log-rank (Mantel-Haenszel) test between each pair of samples was calculated. The survival curves show significance when either the wild type (p<0.0001) or LUC (p<0.001) or mock control (p = 0.002) is compared to CYP group and no difference is observed when LUC group and mock control are considered (p = 0.7)('survival'(v.2.38) R package). <b>B.</b> Analysis of pentoxyresorufin (PR) metabolizing activity. Worms five days after the knockdown were treated for 20 h with pentoxyresorufin, and images were acquired using multiple fluorescence filters using optical sectioning by means of the AxioImager fluorescence microscope (Zeiss). The total size of the resorufin particles in each worm was measured. Data were expressed as a fold difference compared to the wild-type (not exposed to dsRNA) control. Data are presented as means ± S.D. Results are averaged from three independent experiments. Significance at <i>p</i> < 0.005, ***; <i>p</i> < 0.01, ** (<i>F</i>-test).</p

Consortium high level timelines/activities.

<p>1. Siberian State Medical University, Tomsk, Russian Federation, 2. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands, 3. Department of Parasitology and Leiden Parasite Immunology Group, Leiden University Medical Center, Leiden, the Netherlands, 4. George Washington University Medical Center, United States, 5. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russian Federation, 6. Institute of Tropical Medicine, University of Tübingen, Germany, 7. Khon Kaen University, Khon Kaen, Thailand, 8. Pfizer LLC, Moscow, Russian Federation, 9. ReMedys Foundation, Geneva, Switzerland, 10. Royal Brompton Hospital, United Kingdom; Research Institute for Medical Genetics, Tomsk, Russian Federation, 11. Swiss Tropical and Public Health Institute, Basel, Switzerland.</p