Neglected Diseases Liver Fluke Induces Banchob Sripa*, Sasithorn Kaewkes, Paiboon Sithithaworn, Eimorn Mairiang, Thewarach Laha, Michael Smout, Chawalit Pairojkul, Vajaraphongsa Bhudhisawasdi, Smarn Tesana, Bandit Thinkamrop, Jeffrey M. Bethony, Alex Loukas, Paul J. Brindley*

Opisthorchiasis and Clonorchiasis: Major Regional Public Health Problems Liver fl uke infection caused by , O. felineus, and is a major public health problem in East Asia and Eastern Europe. Currently, more than 600 million people are at risk of infection with these trematodes [1]. O. viverrini is endemic in Southeast Asian countries, including Thailand, Lao People’s Democratic Republic, Vietnam, and Cambodia [2], and C. sinensis infection is common in rural areas of Korea and China. has been extensively studied in Thailand, where an estimated 6 million people are infected with the liver fl uke (calculated from overall 9.4% prevalence within the population in 2001) [3]. Infection with these food-borne parasites is prevalent in areas where uncooked cyprinoid fi sh are a staple of the diet. Due to poor sanitation practices and inadequate sewerage infrastructure, people infected with O. viverrini and C. doi:10.1371/journal.pmed.0040201.g001 sinensis pass parasite eggs in their faeces into natural water Koi-Pla reservoirs, where the parasite eggs are eaten by intermediate Figure 1. Preparation of a Meal of Using Uncooked Cyprinoid Fishes host snails, for example, aquatic snails of the genus Bithynia, O. viverrini (A) Fluke-infected fi sh are plentiful in the local rivers such as the Chi the fi rst intermediate host of . After hatching, free River in Khon Kaen province, Thailand. (B) Local people catch the fi sh swimming parasites, called cercariae, are released from the in nets and prepare the fi sh-based meals with local herbs, spices, and infected snails. Cercariae then locate their next intermediate condiments. (C) The fi nished dish of koi-pla accompanied by rice and vegetables. This dish is a dietary staple of many northeastern Thai host, cyprinoid fi shes, encyst in the fi ns, skin, and muscles of villagers and is a common source of infection with O. viverrini. the fi sh, and become metacercariae. The metacercariae are infective to humans and other fi sh-eating mammals upon Funding: We gratefully acknowledge support from the United States National ingestion of raw or undercooked fi sh in dishes such as koi-pla Institute of Allergy and Infectious Diseases, grant number 1UO1 AI065871, and the (Figure 1), and in turn the parasite’s life cycle is completed Sandler Family Supporting Foundation. Neither the National Institute of Allergy and Infectious Diseases nor the Sandler Foundation was involved in the preparation of (Figure 2). this article or in the decision to publish. Most people with opisthorchiasis or clonorchiasis have Competing Interests: PJB is the Principal Investigator of the National Institute of no symptoms. Only 5%–10% of infected people, in general Allergy and Infectious Diseases grant number 1UO1 AI065871, which deals with those with heavy fl uke infections, have non-specifi c symptoms liver fl uke–induced cholangiocarcinoma. such as right upper quadrant abdominal pain, fl atulence, Citation: Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, et al. (2007) Liver fl uke and fatigue [4,5]. Enlargement of the gall bladder can induces cholangiocarcinoma. PLoS Med 4(7): e201. doi:10.1371/journal.pmed.0040201 be detected by ultrasonography, and is reversed after Copyright: © 2007 Sripa et al. This is an open-access article distributed under the elimination of fl ukes by praziquantel [6]. Nonetheless, terms of the Creative Commons Attribution License, which permits unrestricted heavy, long-standing infection is associated with a number use, distribution, and reproduction in any medium, provided the original author and source are credited. of hepatobiliary diseases, including cholangitis, obstructive jaundice, hepatomegaly, fi brosis of the periportal system, Abbreviations: CCA, cholangiocarcinoma; ES, excretory/secretory; HCC, cholecystitis, and cholelithiasis [7–10]. Moreover, both hepatocellular ; NO, nitric oxide experimental and epidemiologic evidence strongly implicates Banchob Sripa and Chawalit Pairojkul are with the Department of Pathology; Sasithorn Kaewkes, Paiboon Sithithaworn, Thewarach Laha, and Smarn Tesana are liver fl uke infection in the aetiology of one of the liver cancer with the Department of Parasitology; Eimorn Mairiang is with the Department of subtypes—cholangiocarcinoma (CCA), or cancer of the bile Radiology; Vajaraphongsa Bhudhisawasdi is with the Department of Surgery; and ducts [2,11]. Bandit Thinkamrop is with the Faculty of Public Health at Khon Kaen University, Khon Kaen, Thailand. Banchob Sripa, Sasithorn Kaewkes, Paiboon Sithithaworn, The pathology of clonorchiasis was recently reviewed Chawalit Pairojkul, and Vajaraphongsa Bhudhisawasdi are also with the Liver in detail by Rim [12]. Unlike O. viverrini, C. sinensis is not Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon considered a Group I carcinogen (known to be carcinogenic Kaen, Thailand. Michael Smout and Alex Loukas are with the Helminth Biology Laboratory, Immunology and Infectious Diseases, Queensland Institute of in humans) [2], despite its widespread prevalence [13]. In Medical Research, Herston, Queensland, Australia. Jeffrey M. Bethony is with the Department of Microbiology, Immunology, and Tropical Medicine, George Washington University, Washington, District of Columbia, United States of America. Paul J. Brindley is with the Department of Tropical Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America. The Neglected Diseases section focuses attention either on a specifi c disease or describes a novel strategy for approaching neglected health issues in general. * To whom correspondence should be addressed. E-mail: [email protected] (BS); [email protected] (PJB)

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Figure 2. Life Cycles of O. viverrini and C. sinensis Embryonated eggs are discharged in the biliary ducts and in the stool (1). Eggs are ingested by a suitable snail intermediate host (2); there are more than 100 species of snails that can serve as intermediate hosts. Each egg releases a miracidium (2a), which goes through several developmental stages (sporocyst [2b], redia [2c], and cercaria [2d]. The cercaria is released from the snail and after a short period of free-swimming time in water, it penetrates the fl esh of a freshwater fi sh, such as Cyclocheilichthys armatus or Puntius leiacanthus, where it encysts as a metacercaria (3). Humans are infected through ingestion of undercooked, salted, pickled, or smoked freshwater fi shes (4). After ingestion, the metacercaria excysts in the duodenum (5) and ascends the biliary tract through the ampulla of Vater. Maturation to adulthood (right, O. viverrini; left, C. sinensis) takes approximately one month (6). The adult fl ukes (measuring 10–25 mm by 3–5 mm) reside in the small and medium-sized biliary ducts. In addition to humans, carnivorous animals can serve as reservoir hosts. (Adapted from http://www.dpd.cdc.gov/DPDx/HTML/Opisthorchiasis.htm.) this review we therefore focus on opisthorchiasis, where studies showed that the incidence of CCA in the fi ve the link between fl uke infection and CCA is more robust major regions of Thailand varied by at least 12-fold and than for clonorchiasis. Further, the article reviews the had a strong positive correlation with the prevalence of O. molecular pathogenesis of opisthorchiasis and associated viverrini infection, as measured by anti-O. viverrini antibody cholangiocarcinogenesis, particularly nitrative and oxidative titres in the general population (Figure 3) [20,21]. Similar DNA damage and clinical manifestations of CCA that were correlations have been observed in populations in Laos either not addressed or available in our previous review [14]. [22,23]. (HCC) shows no such relationship. Liver Fluke Infection and Cholangiocarcinoma Several similar surveys of Thai villagers showed a strong Second to tobacco use, infections are the most important association between the intensity of O. viverrini infection, preventable source of human malignancies [15–17] (Table parasite-specifi c antibody response, and abnormalities of the 1). The association between the occurrence of CCA and the biliary tract, including suspected CCA [8,24]. A linear trend presence of liver fl ukes has been known for about 50 years of the frequency of suspected CCA and O. viverrini faecal egg [18]. count was observed, where an odds ratio of 14.1 was found In Thailand, the fi rst evidence for such an association in a group with elevated egg counts [25]. Moreover, elevated came from hospital case series. CCA was observed in a anti-O. viverrini antibody titres increase the risk of appearance high percentage of liver cancers from northeast Thailand, of CCA by up to 27-fold [26]. where the prevalence of O. viverrini infection is higher than The geographical pattern of liver fl uke infection in elsewhere in this country [19]. More formal correlation Thailand is not uniform, with the greatest prevalence

PLoS Medicine | www.plosmedicine.org 1149 July 2007 | Volume 4 | Issue 7 | e201 Table 1. Malignancies Attributable to Infectious Agents Pathogen Type Pathogens Malignancies Non-Malignant Diseases Percent of 2002 Cancers

Viruses Human papilloma virus: 13 Ano-genital, mouth, pharynx, and cervical cancer Warts, condyloma 5.2 carcinogenic strains Hepatitis B or C viruses Hepatocellular carcinoma Hepatitis, 4.9 Epstein-Barr virus Nasopharyngeal carcinoma, Burkitt, Hodgkin, and Infectious mononucleosis 1.0 immunoblastic lymphoma Human immunodefi ciency virus with Kaposi sarcoma, non-Hodgkin lymphoma, and AIDS 0.9 human herpes virus 8 other AIDS-associated cancers Human T cell lymphotrophic virus Adult T cell leukaemia 0.03 type 1 Bacteria H. pylori Gastric carcinoma, MALT lymphoma Gastritis, peptic ulcers 5.5 Helminths (blood Bladder carcinoma 0.1 fl uke) O. viverrini (liver fl uke) Cholangiocarcinoma Opisthorchiasis 0.02 Total 17.8

According to the International Agency for Research on Cancer. Adapted from [15,16]. doi:10.1371/journal.pmed.0040201.t001 occurring in the north (19.3%) and northeast (15.7%), glandular tissue, making them adenocarcinomas. Details of compared with the central (3.8%) and southern regions the gross morphology, histology, mode of spreading, and (0%) [3]. Khon Kaen province in northeast Thailand has the clinical manifestations are outlined in Figure 4 [36,37]. highest incidence of this type of cancer in the world [27,28]. [36,37] In Thailand, despite widespread implementation of chemotherapy with praziquantel in the past, the prevalence Pathogenesis of Liver Fluke-Induced CCA of O. viverrini in some endemic areas approaches 70%. Mechanical injury and fl uke metabolic products. Mechanical Moreover, in Thailand, liver cancer is the most prevalent injury from the activities of feeding and migrating fl ukes of the fatal neoplasias, and rates of CCA in regions where contributes to biliary damage in the human host. Both the parasite is endemic are unprecedented. By way of oral and ventral suckers of the fl uke hook onto the biliary comparison, CCA is responsible for about 19% of liver epithelium, resulting in tissue damage even early in cancers in the United States, compared with 71% in Khon infection [38]. As the parasite matures, the lesion becomes Kaen, Thailand, representing the highest incidence of CCA more pronounced and ulcerates. Fluke eggs become in the world in this region [28]. entrapped in the periductal tissue through the ulcer and induce granulomatous infl ammation around the eggs. The Clinical Manifestations granulomata are readily visualized in experimental hamster Most primary malignant liver cancers are of two main infections and occasionally in human cases with bile duct histologic types distinguished by their cellular origin. HCC obstruction. derives from hepatocytes, the main cells in the liver, and The liver fl uke secretes or excretes metabolic products, is the most common form of liver cancer throughout the some of which are highly immunogenic, from the tegument world. There are several important risk factors that have been and excretory openings into the bile or culture medium in demonstrated with varied mechanisms of action; 75%–80% vitro [39,40]. Apart from inducing host immune responses, of cases are attributable to hepatitis B and C infection the metabolic products themselves may be toxic to or interact [29,30]. CCA is derived from cholangiocytes, which form with the biliary epithelium [41]. Indeed, mouse fi broblasts the epithelial lining of both intrahepatic and extrahepatic co-cultured with O. viverrini adult worms (but physically bile ducts, except for those of the gallbladder. This form separated in Transwell plates) underwent more than 4-fold of liver cancer has a lower operable rate than HCC and greater proliferation than cells in media alone [42]. most cases are currently untreatable except for complete Gene microarrays were recently used to explore liver transplantation [31]. Compared to HCC, CCA is a transcriptional changes induced in these murine fi broblasts considerably less common form of liver cancer, except in that were co-cultured with O. viverrini, and the fl uke regions within East Asia where infection with O. viverrini, and molecules induced over-expression of several mRNAs to a lesser extent, C. sinensis, is widespread [32]. encoding growth-promoting proteins such as transforming CCA represents a formidable diagnostic and treatment growth factor [43]. Moreover, we observed that human CCA challenge [33]. The wide variation in CCA biology and the cell lines underwent excessive proliferation upon stimulation rarity of the condition (which represents only 3% incidence with O. viverrini soluble excretory/secretory (ES) products (B. of all gastrointestinal cancers) have signifi cantly increased Sripa et al., unpublished data). These studies clearly indicate the diffi culty of any studies involving this cancer, because of that metabolic products of Opisthorchis contain mitogen-like broad variations in presentation and low study participant activity and can induce cell proliferation. This is in agreement numbers [34]. The disease’s rarity, combined with its lack of with earlier fi ndings of hyperplasia of biliary epithelial cells in early symptoms and a range of possible alternative diagnoses, opisthorchiasis [38,44]. contributes to the challenge of identifying and investigating It is intriguing that O. viverrini would secrete a protein that this deadly cancer [35]. Practically all cases are derived from promotes cell proliferation and eventually cancer, since the

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Figure 3. Incidence of CCA and O. viverrini in Thailand from 1990–2001 Increasing intensity of red represents increasing prevalence of O. viverrini, while increasing number of dots represents increasing cancer rates. In general, higher O. viverrini prevalence correlates with a higher CCA burden, although sporadic anthelmintic therapy has infl uenced this relationship. It should be noted that even one spot represents signifi cant cancer rates anywhere else in the world. *Truncated age-standardised incidence from 35–64 years. **Age-standardised incidence of CCA throughout registered regions. Adapted from [20]. benefi t to the parasite is unclear. One possibility might be damage to the infected biliary epithelium. Moreover, excess that over-production of biliary cells may provide an abundant NO and other reactive oxygen intermediates produced food source for the fl ukes, which graze on epithelial cells by infl ammatory cells during infection might exert direct and/or their secreted mucins, though it has been proposed cytotoxic and mutagenic effects and cause increased cell that C. sinensis ingests host blood through the damaged proliferation [49–51]. Increased formation of 8-nitroguanine mucosa [12]. (8-NO2-G) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8- Immunopathology. It has long been suspected that oxodG) is associated with various pathological conditions host immune responses and immunopathologic processes [52,53], and 8-oxodG is considered to be mutagenic. mediate hepatobiliary damage in opisthorchiasis [38,45,46]. Recently, we reported diffuse nitrative and oxidative DNA Sripa and Kaewkes [40] showed that infl ammation around damage (8-nitroguanine and 8-oxodG) in biliary epithelium infected hamster bile ducts was a consequence of the of hamsters infected with O. viverrini [48], and these DNA host’s cellular response to Opisthorchis antigens. Marked lesions still remained in the epithelium at least 180 days infi ltration of infl ammatory cells at the periportal areas of post-infection. Moreover, repeated infections with liver infected hamster liver was associated with the presence of fl uke result in enhanced biliary DNA damage [54]. This may parasite antigens in the bile duct epithelium as detected by be explained by the fact that repeated infection increased immunohistochemistry. Small bile ducts, the fi rst-order ducts inducible nitric oxide synthase expression in the bile duct (where fl ukes do not reside because the diameter of the epithelium. The DNA damage in infected biliary cells is ducts is too small), were also positive for O. viverrini antigens proven to be a result of the infl ammatory response caused by and were markedly infl amed. Opisthorchis antigens were also O. viverrini because 8-nitroguanine and 8-oxodG disappeared observed in macrophages, epithelioid cells, and giant cells of after praziquantel treatment [55]. Usually genotoxic events the granuloma [40]. with DNA damage lead to either DNA-mismatch repair Biliary cell damage induced by O. viverrini likely also stems mechanisms or, if the damage is beyond repair, to cell death from the actions of oxygen radicals such as nitric oxide through apoptosis. NO not only induces DNA damage (NO) released from effector cells activated by infl ammatory but has been reported to mediate DNA repair inhibition cytokines [47,48]. These radicals can induce oxidative DNA [56,57]. Moreover, NO has also been demonstrated to inhibit

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Figure 4. Characteristics of CCA Morphologically this cancer falls into three subtypes: mass forming, periductal infi ltrating, and intraductal. This classifi cation is independent of the location of the affected bile duct, which may be intrahepatic (within the liver) or extrahepatic (outside the liver, excluding the gallbladder). The line between these subtypes tends to blur as the malignancy matures. Tumour dimensions, mode of spreading, and clinical manifestations are noted. Adapted from [36,37] by Giovanni Maki. apoptosis downstream of cytochrome c [58]. All of these patients from sites endemic for O. viverrini in Thailand versus manifestations facilitate carcinogenesis. non-endemic areas in Japan [63]. Of particular interest was Characterisation of the host immune responses—including the selective up-regulation in Thai patients with CCA of genes the cytokine phenotypes—to O. viverrini and their roles in involved in xenobiotic metabolism, implying that these genes immune/infl ammatory cell-induced tissue damage, as well may be involved in the detoxifi cation of possible carcinogens as their relationship with host susceptibility, will likely yield such as nitrosamines [63]. CCA can be reliably induced a more complete understanding of the immunopathologic in hamsters experimentally infected with O. viverrini and process involved in liver fl uke–induced CCA. exposed to sub-carcinogenic doses of nitrosamine, but not Multi-factorial aetiology of CCA. Liver fl uke–induced CCA in liver fl uke–infected hamsters not exposed to nitrosamine is therefore generally accepted to be the result of chronic [11]. infl ammation [53,58,59]. Several mechanisms by which O. Apart from exogenous carcinogens, however, endogenous viverrini infection may enhance cholangiocarcinogenesis nitrosation caused by liver fl uke infection has also been have been proposed above, and are summarised in Figure investigated in animals and humans. Experimental 5. Indeed, it is almost certain that a combination of those Opisthorchis infection in hamsters can induce NO synthase pathologies described above (mechanical damage, parasite expression by immune effector cells in infl amed areas secretions, and immunopathology) culminate in CCA after surrounding the bile ducts and increased endogenous chronic infection with O. viverrini. The primary pathologic nitrosation of thiazolidine-4-carboxylic acid (thioproline) change, i.e., epithelial desquamation, may be due to [64]. Several human studies suggest that infected people have mechanical irritation caused by the liver fl uke and/or its a higher endogenous nitrosation potential than uninfected metabolic products. However, immunopathologic processes people [65,66]. Restricted diet studies clearly demonstrated may contribute to the long-standing hepatobiliary damage. an increase in endogenous generation of NO and N-nitroso During liver fl uke infection, infl ammation, periductal fi brosis, compounds as indicated by increased levels of plasma, and proliferative responses, including epithelial hyperplasia, urinary nitrate and nitrite, and by nitrosation of proline and goblet cell metaplasia, and adenomatous hyperplasia, may thioproline among infected men compared to members of represent predisposing lesions that enhance susceptibility of an uninfected control group [25]. Moreover, the difference DNA to carcinogens [11,60]. Several N-nitroso compounds was specifi cally abolished by praziquantel treatment and by and their precursors occur at low levels in fermented food co-administration of ascorbic acid with proline. Enhanced such as preserved mud fi sh paste, pla-ra, a condiment that immune responses to O. viverrini are also associated with an is a ubiquitous component of the cuisine of northeastern increase of endogenous nitrosation in infected individuals Thailand and Laos. [67]. Both exogenous and in situ nitrosamine formation may Indeed, it has been hypothesised that N-nitroso compounds lead to DNA alkylation and deamination in predisposed and (e.g., nitrosamine) are a primary carcinogen leading to infl amed tissues [49–51]. CCA in humans in this region [61,62]. We recently reported Apoptosis is rarely observed in either O. viverrini–infected dissimilar gene expression profi les in CCA tissues from hamster liver or in biliary cells that are co-cultured with

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Figure 5. Proposed Mechanisms of Opisthorchis-Derived CCA Initiation The three proposed pathways linking the parasite to CCA initiation are mechanical damage (in yellow), molecular products (in blue), and immunopathology (in green). Combined, these mechanisms result in several common elements (purple) that all lead to DNA damage. The inhibition of a normal DNA damage response is the fi nal oncogenic factor proposed to dramatically increase the likelihood of a malignant transformation. This invariably leads to progression of CCA. (Adapted from [10].) The photographs underneath the schematic show two livers with advanced CCA.

fl ukes in vitro (B. Sripa et al., unpublished data). This may In this regard, liver fl uke–associated CCA is preventable by be explained by the mechanisms described above and/or changes in eating habits. Unfortunately, age-old culinary the effect of bile acids [58]. Elevation of bile acids has also preferences for uncooked dishes such as koi-pla (Figure 1) do been reported in opisthorchiasis [68]. Bile acids are potent not readily allow for this possibility. Moreover, fi sh farming tumour promoters and have also been shown to block of grass carp and other susceptible species in ponds that are protein degradation of myeloid cell leukemia protein 1 routinely contaminated by untreated sewage has resulted in (MCL-1), a potent anti-apoptotic protein of the BCl-2 family, the establishment of infection in fi sh populations at large, via activation of EGFR signalling [69]. Anti-apoptosis has which, along with the involvement of animal reservoir hosts, been described in infection-associated cancers such as those makes control of liver fl uke infection even more challenging caused by Helicobacter pylori [70,71]. In the case of O. viverrini [13,72]. Nonetheless, given this extraordinary linkage infection, DNA damage is caused in biliary epithelial cells between a metazoan parasite and a tumour, characterisation while apoptotic mechanisms are dysregulated, resulting of the nature and action of carcinogens of O. viverrini or C. in genetic alterations which may become fi xed, leading to sinensis may provide fundamental insights into carcinogenesis malignant transformation. at large. In the short term, enhanced knowledge of pathogenesis, Implications for Improving Public Health particularly infl ammation and its associated DNA damage— From a public health perspective, thorough cooking of the both nitrative and oxidative—may assist in disease control, fi sh hosts effi ciently blocks infection with these parasites. provided the at-risk populations can be educated of the

PLoS Medicine | www.plosmedicine.org 1153 July 2007 | Volume 4 | Issue 7 | e201 elevated risk of CCA from repeated infections. Also, dietary 21. Srivatanakul P, Ohshima H, Khlat M, Parkin M, Sukarayodhin S, et al. (1991) Endogenous nitrosamines and liver fl uke as risk factors for changes that include natural products rich in anti-oxidants cholangiocarcinoma in Thailand. IARC Sci Publ: 88–95. may provide enhanced protection against CCA. In the longer 22. Chai JY, Park JH, Han ET, Guk SM, Shin EH, et al. (2005) Mixed infections term, it should be feasible to develop specifi c and sensitive with Opisthorchis viverrini and intestinal fl ukes in residents of Vientiane Municipality and Saravane Province in Laos. J Helminthol 79: 283–289. techniques for detection of oxidative damage markers in 23. Rim HJ, Chai JY, Min DY, Cho SY, Eom KS, et al. (2003) Prevalence serum, urine, or faeces for early cancer screening of the at- of intestinal parasite infections on a national scale among primary risk populations. We and others are now actively engaged in schoolchildren in Laos. Parasitol Res 91: 267–272. 24. Elkins DB, Haswell-Elkins MR, Mairiang E, Mairiang P, Sithithaworn P, this endeavour. et al. (1990) A high frequency of hepatobiliary disease and suspected cholangiocarcinoma associated with heavy Opisthorchis viverrini infection in Supporting Information a small community in north-east Thailand. Trans R Soc Trop Med Hyg 84: 715–719. Alternate Language Summary S1. A summary of the article translated 25. Haswell-Elkins MR, Satarug S, Tsuda M, Mairiang E, Esumi H, et al. (1994) into Thai by BS Liver fl uke infection and cholangiocarcinoma: Model of endogenous nitric Found at doi:10.1371/journal.pmed.0040201.sd001 (29 KB DOC). oxide and extragastric nitrosation in human carcinogenesis. Mutat Res 305: 241–252. 26. 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