Research Marked Liver Tumorigenesis by Helicobacter hepaticus Requires Perinatal Exposure Bhalchandra A. Diwan,1 Marek Sipowicz,2 Daniel Logsdon,3 Peter Gorelick,3 Miriam R. Anver,4 Kazimierz S. Kasprzak,2 and Lucy M. Anderson 2 1Basic Research Program, SAIC-Frederick, Inc., Frederick, Maryland, USA; 2Laboratory of Comparative Carcinogenesis, National Cancer Institute–Frederick, National Institutes of Health, Department of Health and Human Services, Frederick, Maryland, USA; 3Laboratory Animal Sciences Program, and 4Pathology Histotechnology Laboratory, SAIC-Frederick, Inc., Frederick, Maryland, USA BACKGROUND: Although severe hepatitis and liver tumors occur in a high percentage of A/J male mice naturally infected with Helicobacter hepaticus, these effects have not been observed after injec- tion of adult mice with the bacteria. OBJECTIVES: We tested the hypothesis that perinatal exposure to the bacteria is required for liver tumorigenesis. METHODS: A/J female mice were infected by intragastric (ig) or intraperitoneal (ip) treatment with 1.5 × 108 H. hepaticus before pregnancy. We examined offspring at progressive time intervals, including some kept until natural death in old age. A/J, BALB/c, and C57BL/6 weanling male mice were similarly treated ig with the bacteria and observed for up to 2 years. RESULTS: After ip bacterial infection of A/J females, 41% of their male offspring developed hepatitis and 33% had hepatocellular tumors, including 18% with hepatocellular carcinoma. Treatment by the ig route resulted in a similar incidence of hepatitis in offspring (35%) but fewer total liver tumors (8%) and carcinomas (4%). By contrast, ig instillation of H. hepaticus in weanling A/J, C57BL/6, or BALB/c mice resulted in low incidence of hepatitis (0–20%) and few liver tumors, despite presence of bacteria conﬁrmed in feces. CONCLUSIONS: Results indicate that a high incidence of liver tumors in mice infected with H. hepaticus requires perinatal exposure. Contributing perinatal factors could include known high sensitivity of neonatal liver to tumor initiation, and/or modulation of immune response to the bac- terium or its toxins. Mechanisms of human perinatal sensitivity to such phenomena can be studied with this model. KEY WORDS: adult exposure, Helicobacter hepaticus, hepatocellular tumors, infection and cancer, perinatal exposure. Environ Health Perspect 116:1352–1356 (2008). doi:10.1289/ehp.11493 avail- able via http://dx.doi.org/ [Online 13 June 2008] Helicobacter hepaticus is a murine micro- aerophilic bacterium ﬁrst noted in association with unexplained cases of hepatitis and liver tumors in our animal colony (Ward et al. 1994b) and subsequently isolated and charac- terized as a new species (Fox et al. 1994). Koch’s postulates were fulﬁlled with regard to causation of persistent chronic hepatitis (Fox et al. 1996b). H. hepaticus selectively colonizes the hepatic bile canaliculi of mice, causing a morphologically distinctive pattern of chronic active progressive hepatitis, and also persis- tently infects the large bowel. H. hepaticus infection is readily established in mice by intra- gastric (ig) or intraperitoneal (ip) treatment with bacterial suspensions, and this method is commonly used to study the disease. In our initial study of A/J male mice natu- rally infected with H. hepaticus, we observed hepatitis of increasing severity with age, with most mice exhibiting hepatitis by 1 year (Rice 1995; Ward et al. 1994a). Liver tumors occurred in 6% of the mice at 1 year of age, in 50% at 15 months, and in 92% at 18 months. This high incidence of liver tumorigenesis was striking, in view of human gastric cancer and lymphoma associated with infection by Helicobacter pylori. A later study with another sample of the naturally infected A/J males gave 1352 similar quantitative results, with liver tumors in 40% of the males at 12–15 months of age (Diwan et al. 1997). Similarly, another labora- tory reported liver tumors in 75% of naturally infected A/J males at 15–18 months of age (Fox et al. 1996a). Thus, the model seemed promising as an experimental system for study of bacterial carcinogenesis. However, studies with experimental infec- tion by injection of cultured bacteria have yielded lower liver tumor incidences. In an investigation involving ig treatment of mice with H. hepaticus at 10 weeks of age, and including both sexes and a selection of recom- binant inbred strains, liver preneoplasia/neopla- sia incidence at 14 months of age was, at most, 20% (Ihrig et al. 1999). Avenaud et al. (2003) exposed A/J mice at 3–4 weeks of age to a rela- tively low titer of H. hepaticus (107) ig and examined small groups of mice at ages up to 17 months. Although all mice tested positive for the bacterium, hepatitis was relatively mild, and no liver tumors were observed. Possible insight into the reason for the low yield of liver tumors in these studies was offered by Rogers et al. (2004), who exposed A/J mice to H. hepaticus at various perinatal stages and at 12 weeks of age and evaluated the livers at time points up to 12 months of age. Although the limited numbers of mice in each group con- strained statistical certainty, the results sug- gested that exposure before 12 weeks of age was required for development of hepatitis and liver dysplasia: none of the four male mice treated at 12 weeks had these lesions, whereas such changes occurred in 30–70% of those exposed by treatment of the dams at conception or on fetal day 10, or of offspring at 3 weeks of age. We therefore tested whether progressive hepatitis and a high incidence of liver tumors with aging requires perinatal exposure, as would occur in naturally infected mouse colonies. Our ﬁnding that such is, in fact, the case may have bearing on the potential impor- tance of human perinatal exposure to infec- tious agents on later health consequences. A preliminary report of this work has appeared in abstract form (Diwan et al. 2001). Materials and Methods H. hepaticus culturing, treatment, and detec- tion. We used H. hepaticus, Frederick strain 1A (passage 7), which was isolated contemporane- ously from the same colony of mice as those providing the tissues used for cloning and genetic analysis of the bacterium and deposi- tion in the American Type Culture Collection (Fox et al. 1994, 1996b). We grew the bacteria under microaerophilic conditions at 37°C on plates of Brucella agar with horse blood and trimethoprim, vancomycin, and polymyxin B (TVP) (Remel Co., Lenexa, KS) for 3 days. This bacterial strain has subsequently been shown to be detected by H. hepaticus–speciﬁc polymerase chain reaction (PCR) primers and to be negative for all PCR-based tests for all Address correspondence to L.M. Anderson, NCI at Frederick, Building 538, Room 205B, Ft. Detrick, Frederick, MD 21702 USA. Telephone: (301) 846- 5600. Fax: (301) 846-5946. E-mail: andersol@mail. ncifcrf.gov We thank J. Ward for pathology consultations. This work was supported in part by the Intramural Research Program of the National Cancer Institute (NCI), National Institutes of Health, and with federal funds from the NCI under contract NO1-CO-12400. The content of this publication does not necessar- ily reﬂect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement of the U.S. government. The authors declare they have no competing ﬁnancial interests. Received 19 March 2008; accepted 12 June 2008. VOLUME 116 | NUMBER 10 | October 2008 • Environmental Health Perspectives