To test the efﬁcacy of bacterial transmission through feces, we exposed pregnant mice and their offspring up to the time of weaning to feces from Helicobacter-positive male mice. Eight litters yielded a total of 32 male offspring. Incidence of males with Helicobacter hepatitis (19%) was less than that in offspring of moth- ers exposed ip (41%), but this difference was only of borderline signiﬁcance (p = 0.086), and average severity grade was similar (Table 2). We observed only one liver tumor, a hepatocellular carcinoma in a male of advanced age without hepatitis. Feces from four of the nine cages housing these mice were positive for H. hepati- cus at approximately monthly samplings between 1 month and 1 year of age. We further tested fecal transmission by mating Helicobacter-positive male offspring from the above study with uninfected females. We obtained a total of 20 male off- spring from nine litters. Although these F2 mice were terminated at earlier ages than mice in the other studies, the incidence and average grade of Helicobacter hepatitis were very simi- lar to those for the F1 males exposed directly to contaminated feces during gestation and infancy (Table 2). We found no liver tumors in the F2 males. We noted typhlitis and hyperplasia of gut- associated lymphoid tissue (GALT) in 4–19% of the male offspring in the various perinatal exposure groups, with no significant differ- ences among them. Lack of tumorigenic effectiveness of ig treatment of A/J weanling mice with H. hepaticus. We tested H. hepaticus of the same strain used for the perinatal studies above in weanling A/J mice. Chronic active hepatitis associated with experimental ig H. hepaticus infection was ﬁrst apparent after 12 weeks after exposure and occurred with low frequency at later time points, but never exceeding 20% (Table 1). We noted mild-to-moderate histopathologic changes (scores of 2–3) in 6 of 15 (40%) of the livers of experimentally infected mice at 12 weeks after exposure but Mothers and newborns F2 offspring of exposed through feces feces-exposed males 32 647 ± 22 6 (19) 2.5 ± 0.2 675 ± 62 1 (3)# 0† 1 (3) 3 (9) 6 (19) 20 458 ± 16 5 (25) 2.6 ± 0.6 441 ± 40 0 0 0 1 (5) 3 (15) Diwan et al. found to have positive fecal cultures were mated. Four females produced a total of 27 male offspring, which were housed in groups of two to four. All cages gave positive results for H. hepaticus in feces, and the num- ber of mice per cage had no obvious relation- ship to outcomes (data not shown). At an average age of about 22 months, 11 (41%) of these male offspring had Helicobacter hepatitis. Of these, 9 (33%) had at least one liver tumor, 7 (26%) had multiple tumors, and 5 (18%) had hepatocellular carcinoma (Table 2). Hepatocholangiolar tumors, an adenoma and a carcinoma, occurred in 2 mice. We found tumors in offspring of each of the mothers. All of the hepatocellular tumors occurred in livers that also had Helicobacter hepatitis, which was moderate to severe in all cases except for one, which was categorized as mild. Three livers that did not have a tumor had minimal or mild chronic active hepatitis. The four mothers were euthanized when moribund at 49–87 weeks of age; of the four livers, three had chronic inflammation of moderate intensity, but no hepatitis characteristic of Helicobacter infection. Female mice treated ig with the same H. hepaticus preparations as the ip-treated females and found to have positive fecal cul- tures were bred. Five mothers produced a total of 26 male offspring. Fecal cultures from both the mothers and the male offspring were posi- tive for Helicobacter. Among these male off- spring, 35% had Helicobacter hepatitis, an incidence similar to that in male offspring of the ip-treated females (Table 2). However, despite greater average age of these mice, the average severity of the hepatitis was less (close to significance). Only two mice (8%) had hepatocellular tumors, and no mouse had mul- tiple liver tumors. These values are signiﬁcantly different from those in the male offspring of ip-treated females. The ﬁve ig-treated mothers were euthanized at 67–105 weeks of age. We did not observe chronic inﬂammation of the liver (p = 0.048 compared with chronic inﬂam- mation in three of four ip-treated mothers). none in the controls (p = 0.0068). Subacute inﬂammation was the most common hepatic lesion (5 of 15) in these mice. The incidence of this change decreased at 24 weeks (1 of 10) and 36 weeks (1 of 15) after exposure. We observed mild-to-moderate peribiliary leuko- cyte infiltration in a small percentage of the exposed mice at ≥ 12 weeks after exposure, but in none of the controls through 54 weeks (4 of 45 vs. 0 of 47, p = 0.053). Similarly, mild-to-moderate necrosis occurred only in the exposed mice (9 of 81 vs. 0 of 82, p = 0.0015). Thus, we observed apparent hepatic effects of the H. hepaticus infection in some of the mice, but these were not striking, and typical Helicobacter-associated hepatitis was rare. Correspondingly, only two mice, one exposed and one control, presented with liver tumors at advanced ages. The control tumor, at 751 days of age, was a hepatocellular adenoma. An exposed mouse at 790 days of age presented four hepatocellular adenomas, one hepato- cholangiolar adenoma, and a hemangio- sarcoma within the liver; however, hepatitis typical of H. hepaticus was not present. We measured levels of 8-oxo-dG indica- tive of oxidative DNA damage in two to ﬁve randomly selected mice for each treatment cat- egory and time point up to 1 year; in most cases, samples from the median and left liver lobes were analyzed for each mouse. As shown in Table 1, a significant increase in hepatic 8-oxo-dG had already occurred within 2 weeks of infection. We obtained very similar results at 4 weeks. We noted a further increase at 12 weeks after exposure, where the elevated level of 8-oxo-dG was highly signiﬁcant com- pared with controls at this age and compared with exposed mice at 4 weeks. Thereafter, the level of 8-oxo-dG in the livers of the infected mice did not change significantly, whereas that in the controls increased steadily, as is expected with aging. The difference relative to the controls was still significant at 24 weeks after exposure, but not at 36 or 54 weeks. 8-oxo-dG levels were not clearly correlated with histopathologic findings: At 24 weeks postexposure, the average number of 8-oxo-dG residues in hepatic DNA from the mouse with clear Helicobacter hepatitis was 2.27/105 dG residues, compared with 2.28/105 dG in one without liver lesions. At 54 weeks after exposure, the liver with hepati- tis had 1.67 8-oxo-dG residues per 105 dG, whereas one with minimal hepatic change had 1.74 residues/105 dG. However, because of the focal nature of the hepatitis, lack of corre- spondence with 8-oxo-dG levels cannot be concluded with certainty. Fecal samples taken from cages of experi- mentally infected mice at 2–24 weeks after exposure were positive for H. hepaticus. No samples were taken at 36 weeks. For the three cages of mice at 54 weeks after exposure, two Table 2. Histopathologic ﬁndings in A/J mice exposed to H. hepaticus perinatally and in controls [n (%)]. Characteristic No. Average age (days) Helicobacter hepatitis positive Average grade Average age (days) Hepatocellular tumors present Multiple hepatocellular tumorsb Hepatocellular carcinoma Typhlitis Hyperplasia of GALT Mothers exposed ip 27 674 ± 18 11 (41) 3.0 ± 0.4* 669 ± 20 9a (33)**, # 7 (26)##,† 5 (18) 1 (4) 1 (4) Mothers exposed ig 26 674 ± 28 9 (35) 2.0 ± 0.3* 713 ± 18 2 (8)** 0## 1 (4) 3 (12) 3 (12) Values shown are mean ± SE or number (%) except where indicated. aEight had hepatocellular tumors (three with adenomas only, ﬁve with adenoma and carcinoma, one each with hepato- cellular adenoma and carcinoma and either cholangioma or cholangiocarcinoma), and one had cholangioma only. The liver with hepatocholangiolar adenoma was the only liver with tumor that did not have Helicobacter hepatitis.bMultiplicity could not be quantified because of extensive tumor involvement throughout many of the livers. Significance of differ- ences between values with matched superscripts: *p = 0.07, **p = 0.039, #p = 0.0035, ##p = 0.01, †p = 0.0026. 1354 VOLUME 116 | NUMBER 10 | October 2008 • Environmental Health Perspectives