bioRxiv preprint doi: https://doi.org/10.1101/606772; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 PLoS NTD 2 3 Helminth infection-induced carcinogenesis: spectrometric insights from the 4 liver flukes, Opisthorchis and Fasciola 5 6 Maria João Gouveia1,2,3, Maria Y. Pakharukova4,5, Banchob Sripa6, Gabriel Rinaldi7,♯, Paul J. 7 Brindley7, Viatcheslav A. Mordvinov4, Fátima Gärtner2,3,8, José M. C. da Costa1,9, Nuno 8 Vale2,3,8,10* 9 10 1 Center for the Study of Animal Science, CECA-ICETA, University of Porto, Praça Gomes Teixeira, 11 Apartado 55142, 4051-401 Porto, Portugal 12 2 i3S, Instituto de Investigação e Inovação em Saúde, University of Porto, Rua Alfredo Allen, 208, 4200- 13 135 Porto, Portugal 14 3 Department of Molecular Pathology and Immunology, Institute of Biomedical Sciences Abel Salazar 15 (ICBAS), University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal 16 4 Laboratory of Molecular Mechanisms of Pathological Processes, Institute of Cytology and Genetics, 17 Siberian Branch of the Russian Academy of Science, 10 Lavrentiev Avenue, 630090 Novosibirsk, Russia 18 5 Department of Natural Sciences, Novosibirsk State University, 2 Pirogov Street, 630090 Novosibirsk, 19 Russia 20 6 Department of Pathology, and Tropical Diseases Research Laboratory, Faculty of Medicine, Khon Kaen 21 University, Khon Kaen, 40002, Thailand 22 7 Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected 23 Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, 24 D.C., 20037, USA 1 bioRxiv preprint doi: https://doi.org/10.1101/606772; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 25 8 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Júlio 26 Amaral de Carvalho 45, 4200-135 Porto, Portugal 27 9 National Health Institute, R&D Unit, Dr. Ricardo Jorge (INSA), Rua Alexandre Herculano, 321, 4000- 28 055 Porto, Portugal 29 10 Laboratory of Pharmacology, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, 30 Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal 31 32 # Current address: Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 33 1SA, UK 34 35 * Corresponding author at: Nuno vale, Laboratory of Pharmacology, Department of Drug Sciences, Faculty 36 of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Tel.: 37 +351220428606; Fax: +351226093390. 38 E-mail address: [email protected] (N. Vale) 39 40 41 42 43 44 45 46 47 48 2 bioRxiv preprint doi: https://doi.org/10.1101/606772; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 49 Abstract 50 Chronic infections with the flatworm parasites Opisthorchis viverrini, Clonorchis sinensis and 51 Schistosoma haematobium are classified as group 1 biological carcinogens, i.e. definitive causes 52 of cancer. In addition, we reported findings that support the inclusion of Opisthorchis felineus in 53 this list of biological carcinogens. By contrast, infections with close phylogenetic relatives 54 including Fasciola hepatica have not been associated with carcinogenesis. Earlier reports revealed 55 of oxysterol metabolites of Opisthorchis liver fluke origin conjugated with DNA bases, suggesting 56 that the generation of these DNA-adducts may underlie the mutagenicity and carcinogenicity of 57 the infection with these food-borne pathogens. Here we employed liquid chromatography-mass 58 spectrometry (LC-MS/MS) to investigate, compare and contrast spectrograms of soluble extracts 59 from F. hepatica adult worms from bile ducts of cattle with those from O. viverrini and O. felineus 60 from experimentally-infected hamsters. F. hepatica displayed a complex spectrophotometric 61 profile. F. hepatica and Opisthorchis spp. shared several common compounds including oxysterol- 62 like metabolites, bile acids and DNA-adducts, but the spectrometric profiles of these Opisthorchis 63 species included far fewer compounds than F. hepatica. These findings support the postulate that 64 oxysterol-like metabolites of parasite origin can initiate carcinogenesis and they point to a 65 molecular basis for the inconsistencies among major groups of liver flukes concerning infection- 66 induced malignancy. 67 (202 words) 68 69 70 71 72 73 3 bioRxiv preprint doi: https://doi.org/10.1101/606772; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 74 Author Summary 75 Several species of trematodes are parasites of the human hepatobiliary tract. Infection with 76 two of these flukes, Clonorchis sinsensis and Opisthorchis viverrini, fresh water fish-borne 77 parasites that occur in East Asia is classified as group 1 carcinogens by the International Agency 78 for Research on Cancer (IARC), i.e. definitive causes of cancer in humans. By contrast, infection 79 with a different liver fluke, Fasciola hepatica, does not lead to malignant transformation of the 80 biliary tract. Given the close phylogeny of all three parasites, this difference in carcinogenicity is 81 intriguing and, if explained, likely of value in novel therapeutic approaches. The importance of the 82 current findings is informative because they present a mass spectrometric analysis and catalog of 83 the similarities and differences between fluke of the genus Opisthorchis and F. hepatica, 84 potentially identifying carcinogenic metabolites of liver fluke origin. These metabolites can be 85 expected to provide deeper understanding of helminth infection induced malignancy. 86 (149 words) 87 88 89 90 91 92 Keywords 93 Fasciola hepatica, Opisthorchis viverrini; Opisthorchis felineus; oxysterols; DNA adducts 94 95 4 bioRxiv preprint doi: https://doi.org/10.1101/606772; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 96 Introduction 97 More than 20% of cancer in the developing world are caused by infections [1]. The World 98 Health Organization’s International Agency for Research on Cancer (IARC) recognizes the 99 infection with about 12 pathogens as group 1 biological carcinogens, i.e., definitive causes of 100 cancer. These group 1 agents include three helminth parasites, specifically the fish-borne 101 trematodes (FZT) Opisthorchis viverrini and Clonorchis sinensis and the blood fluke, Schistosoma 102 haematobium [2]. In addition, we reported findings from hamster infection that support the 103 inclusion of Opisthorchis felineus, also an FZT, to this list of biological carcinogens and definitive 104 cause of cholangiocarcinoma [3]. We hypothesised that these helminths produce and release 105 derivatives of oestrogens and oxysterols that promote oxidation of host DNA, inducing lesions, 106 adducts and mutations [1,3-6]. The findings supported the postulate that these infection-associated 107 cancers originate from a biological and/ or chemical insult followed by chronic inflammation, 108 fibrosis, and a change in the tissue microenvironment that leads to a pre-cancerous niche [7,8]. 109 Paradoxically, infections with other close phylogenetic relatives of these carcinogenic helminths, 110 also food borne trematodes of the Phylum Platyhelminthes (Table 1), have not been categorized as 111 group 1 biological carcinogens [9-15]. 112 113 114 115 116 117 5 bioRxiv preprint doi: https://doi.org/10.1101/606772; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 118 Table 1. Comparison of morphology, life cycle and pathogenesis between Fasciola hepatica and Opisthorchis 119 species. Fasciola spp. Opisthorchis spp. MORPHOLOGY · Flatworms; leaf-like; 20-30 mm · 5-10 mm (ADULT WORM) LIFE CYCLE · Eggs embryonate in water releasing miracidia, which · Eggs are ingested by freshwater snails release invade a suitable snail intermediate host. miracidia, which undergo several developmental · Cercaria released from the snail encysts as stages in the snail. metacercaria on aquatic plants. · Cercaria released form the snail encysts as · After ingestion, the metacercaria excysts in the metacercaria in fish. duodenum and migrates through the intestinal wall, · After ingestion, the metacercaria excysts in the the peritoneal cavity, and the liver parenchyma into duodenum and ascends though the ampulla de Vater biliary ducts, where development to the into