Diwan et al. transplacental. In the present study, establish- ment of the infection by exposure to feces from infected males, although resulting in hepatitis in some mice, did not lead to tumors, but maternal feces might be more effective. Passage of bacteria in the milk is theoretically possible. The mechanism of hepatic tumorigenesis after perinatal exposure may well involve immune tolerance. In genetically engineered mice heterozygous for the Apc gene (ApcMin/+) and deﬁcient in lymphocytes because of lack of Rag2, H. hepaticus infection increased both colon and mammary tumor incidence (Erdman et al. 2003; Rao et al. 2006a, 2006b). This tumorigenic effect was suppressed by CD4+CD45RBloCD25+ regulatory T cells, especially if these were obtained from H. hepaticus–infected mice. Such regulatory T cells have a role in establishing tolerance to self-antigens, which evolves during the neona- tal period (e.g., Arnold et al. 2005). One hypothesis is that H. hepaticus or a bacterial product present during the neonatal period becomes identified as “self” and so escapes effective immune surveillance. An interesting feature in the perinatal models we used in our experiments was that hepatitis occurred to sim- ilar degrees with all four modes of infection, but hepatic tumor outcomes were quite differ- ent. Thus, tumorigenesis involves something more than hepatitis. H. hepaticus expresses a cytolethal distending toxin (CDT) (Chien et al. 2000), which is essential for colonization of the intestines of normal mice (Ge et al. 2005), as well as for severe typhlocolitis in immunodeficient mice (Young et al. 2004). CDT suppresses host immune response to the bacteria (Pratt et al. 2006); both T cells and antigen-presenting cells may be affected. Of particular interest in the present context, when Ge et al. (2007) exposed A/J mice to a high titer of bacteria, both wild-type and CDT mutant H. hepaticus resulted in comparable degrees of hepatitis at 4 and 10 months of age, but only mice infected with the wild-type bac- teria showed hepatic preneoplastic foci. A number of proinﬂammatory mediators, includ- ing tumor necrosis factor-α, interleukin-6, and transforming growth factor-α, were reduced in the absence of CDT. These results confirm that hepatitis and tumorigenesis are separable, and link CDT speciﬁcally to the tumor development process through its effects on immune system balance. It may be that immune response to CDT speciﬁcally is suppressed during the acquisition of immune tolerance in the neonatal period, resulting in higher levels of CDT production, a qualitatively and/or quantitatively enhanced proinflammatory, protumorigenic environ- ment, and ultimately liver tumor development. Chronic inﬂammatory events have long been 1356 associated with the promotion phase of liver tumorigenesis in rodents. Promotion of liver tumors by H. hepaticus infection has been demonstrated (Diwan et al. 1997). Furthermore, the CDT also causes DNA damage by its DNase activity (Avenaud et al. 2004) and thus could contribute to tumor ini- tiation. It is possible that this property of CDT explains the special sensitivity of perinates, because liver tumors are initiated by geno- toxicants with higher efficiency in neonatal than in adult rodents (reviewed by Anderson et al. 2000). In this scenario, chronic hepatitis from H. hepaticus might lead to liver tumors effectively only when these have been initiated perinatally. These hypotheses may be readily tested with the model that we have estab- lished. 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In vitro and in vivo characterization of Helicobacter hepaticus cytolethal distending toxin mutants. Infect Immun 72:2521–2527. VOLUME 116 | NUMBER 10 | October 2008 • Environmental Health Perspectives