Infection with Opisthorchis felineus induces intraepithelial neoplasia of the biliary tract in a rodent model

Abstract

The liver fluke Opisthorchis felineus is a member of the triad of epidemiologically relevant species of the trematode family Opisthorchiidae, and the causative agent of opisthorchiasis felinea over an extensive range that spans regions of Eurasia. The International Agency for Research on Cancer classifies the infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis as group 1 agents and a major risk factor for cholangiocarcinoma. However, the carcinogenic potential of the infection with O. felineus is less clear. Here, we present findings that support the inclusion of O. felineus in the Group 1 list of biological carcinogens. Two discrete lines of evidence support the notion that infection with this liver fluke is carcinogenic. First, novel oxysterol-like metabolites detected by liquid chromatography-mass spectroscopy in the egg and adult developmental stages of O. felineus, and in bile, sera, and urine of liver fluke-infected hamsters exhibited marked similarity to oxysterol-like molecules known from O. viverrini. Numerous oxysterols and related DNA-adducts detected in the liver fluke eggs and in bile from infected hamsters suggested that infection-associated oxysterols induced chromosomal lesions in host cells. Second, histological analysis of liver sections from hamsters infected with O. felineus confirmed portal area enlargement, inflammation with severe periductal fibrosis and changes in the epithelium of the biliary tract characterized as biliary intraepithelial neoplasia, BilIN. The consonance of these biochemical and histopathological changes revealed that O. felineus infection in this rodent model induced precancerous lesions conducive to malignancy.

Figure 1.

Summary of findings from LC-MS/MS analysis of Opisthorchis felineus worms, eggs and biological fluids from hamsters infected with this liver fluke. The m/z species (mass to charge ratios) detected in adult liver flukes worms and the eggs of O. felineus are presented. M/z species common to bile and O. felineus are highlighted in green, and those common to sera and O. felineus in red. M/z species common to both O. felineus and hamster urine were not detected. Exclusive metabolites (m/z) from each one of the biofluids analyzed seemed to be associated with liver fluke infection, they may be directly related to compounds present in O. felineus that undergo metabolization after release from the parasite. Control samples were free of all m/z represented here.

Figure 2.

Biliary histological features observed in the liver biopsies from control, non-infected hamsters. (A) Normal portal unit with bile duct, hepatic arteriole, portal venule, and a clearly defined limiting plate (magnification ×200). The smaller or interlobular bile ducts are lined by cuboidal or low columnar epithelium. No evidence of inflammation (H&E staining); (A1) defines magnified area (magnification ×400) of normal portal unit. Biliary histological features observed in the liver biopsies of hamsters infected with Opisthorchis felineus. (B, B′ and C) Biliary obstruction caused by the O. felineus worm with portal area enlargement (H&E staining) (magnification ×100). (B1) Dashed line defines magnified area (magnification ×100). (A′ ) dashed lines define magnified areas as (B′1) (magnification ×400) and (C) demonstrated the biliary obstruction caused by O. felineus with portal area enlargement. Bile ducts were lined by enlarged nuclei, with pseudo-stratification, hyperchromatism and some loss of polarity, nuclear crowding, mitotic figures (C1, C′1—arrows) and low-to moderate-grade of dysplasia. Evidence of flat or micropapillary dysplastic epithelium in the bile duct; these lesions are referred as biliary intraepithelial neoplasia (BilIN). (C) Epithelium lining a large intrahepatic bile duct displays flat hyperplasia with dysplastic changes (BilIN1/2). (C1) (magnification ×400), Note increased cellularity, modestly increased pseudo-stratification, and nuclear irregularities including variation in size and polarity, cytologic atypia including presence of nucleoli and loss of polarity (BilIN2).

Figure 3.

(A) Normal portal tract free of fibrosis (stained with Masson’s trichrome) (magnification ×40). (A1 and A2) Masson’s trichrome stained tissues showing massive peri-portal fibrosis (region stained blue, magnification ×100). Fibrosis is not only the result of necrosis, collapse, and scar formation but also results from derangements in the synthesis and degradation of matrix by injured mesenchymal cells that synthesize the various components of the matrix.

Figure 4.

(A) Epithelioid granuloma with multinucleated giant cells, lymphocytes, and eosinophils in portal area, occasionally surroundings eggs. Biliary duct obstruction caused the presence of an adult O. felineus liver fluke (magnification ×40). (A1) Dashed line defines magnified area. Evidence of egg granulomata identified by white arrows surrounding by inflammatory cells (magnification ×100). (B) Granulomas with multinucleated giant cell (magnification ×100). (B1) Mononuclear and eosinophilic cell infiltration in portal regions and multinucleated giant cells surroundings eggs (arrow) (magnification ×400).

Figure 5.

Immunohistochemistry to reveal expression of Ki-67 and p53 in liver from hamsters infected with the liver fluke Opisthorchis felineus (A) (magnification ×100), Epithelium of bile ducts with high proliferative index (Ki-67) translating intraepithelial neoplasia (A1, magnification ×400x). (B and B1, magnification ×100), expression of p53 was not observed in non-neoplastic epithelium of the bile ducts, as well as in BilIN-1 and BilIN-2 (#). Eggs of O. felineus (arrow).