other Helicobacter species. To check for motility and morphology, the organism was harvested, suspended in brain heart infusion broth with horse serum and yeast extract (Remel Co.), and visualized by phase-contrast microscopy. For the in vivo studies, the organism was harvested and suspended in sterile phosphate- buffered saline (PBS), and the concentration was adjusted to a McFarland equivalence tur- bidity standard of 1.0 (3 × 108 bacteria/mL). We tested viability of the suspended organisms by phase-contrast microscopy to determine motility and by subculturing onto Brucella blood agar plates with TVP as described above, both before and after animal inoculations. All mice were obtained from the Animal Production Area at NCI Frederick. The National Cancer Institute (NCI)-Frederick is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and follows the U.S. Public Health Service policy for the care and use of laboratory animals. We provided animal care in accordance with the procedures as outlined by the Institute of Laboratory Animal Resources (1996). Animals were treated humanely and with regard for alleviation of suffering. Animal treatments consisted of a single dose of 1.5 × 108 bacteria in 0.5 mL PBS. Treated mice were maintained in semi-rigid isolators (Charles River, Boston, MA), and con- trols were housed in rooms free of the bacteria. Mice were kept in polycarbonate cages with cel- lulose ﬁber chips as bedding. We assayed feces samples for the presence of Helicobacter spp. by passing the samples through a 0.45-µm ﬁlter and then streaking them onto Brucella agar with horse blood and TVP at 37°C under microaerophilic conditions. We compared appearance of growth of these cultures with that of starting cultures of H. hepaticus. Perinatal exposure by maternal injection with H. hepaticus. We used uninfected male and female A/JCr (A/J) mice for this study. We injected 4-week-old females ip or ig with 0.5 mL bacterial suspension. Feces samples from these mice were checked for presence of the bacteria after 3 weeks, and females positive for fecal bacteria were bred with uninfected males. We allowed some females to produce more than one litter. Male offspring were weaned and euthanized when moribund or at 18 or 24 months of age. Mothers were kept until euthanized because of morbidity. Female offspring were not retained because H. hepati- cus–associated liver tumorigenesis has been observed mainly in male mice. Perinatal exposure by maternal and neona- tal exposure to H. hepaticus through feces. Six cages containing 2-year-old A/J males with feces positive for H. hepaticus were available (one to three per cage). At the start, we removed the males from a cage that they had occupied for 4 days and transferred the soiled bedding to cages housing pregnant females (one or two such cages for each cage of males). Litters were born an average of 7 days after initial exposure to soiled bedding. This H. hepaticus exposure procedure was repeated daily, 5 days/week, until pups were weaned. We weaned 32 male offspring from nine litters and maintained them until moribund; remaining mice were eutha- nized at 24 months of age. We monitored feces from their cages periodically for the presence of H. hepaticus, collecting a total of eight samples over time from each cage. Six of the male offspring from cages with positive tests for fecal Helicobacter were bred to uninfected females in order to test their capa- bility for infection of subsequent generations. We obtained a total of 18 male F2 offspring. Experimental infection of weanling mice. Uninfected A/J males were treated at 3–4 weeks of age with H. hepaticus suspension ig or with PBS only. Mice exposed to the bacteria were maintained in isolators. At 2, 4, 12, 24, and 54 weeks after treatment, fecal samples were cultured to determine the presence of H. hepaticus, and mice from each group were euthanized as indicated in Table 1. Mice that died naturally or were moribund during the course of the study were included for analysis with the end point group closest in time. A small number of mice were allowed to live until natural morbidity; their average ages are given in Table 1. We collected livers from all animals up to 54 weeks and divided them into lobes; we either froze portions of each lobe in liquid nitrogen or ﬁxed them in 10% neutral buffered formalin for histopathologic analysis. We ana- lyzed two lobes each from randomly selected livers, representing each time point and group, to determine levels of 8-hydroxydeoxyguano- sine (8-oxo-dG), a marker for oxidative DNA damage, in liver DNA as previously described (Sipowicz et al. 1997). Results for mice at 2, 4, and 12 weeks after exposure have been pub- lished previously (Sipowicz et al. 1997). Perinatal H. hepaticus and liver tumors To test the effect of different genetic back- grounds, we performed a second experiment with male weanling mice of the BALB/cAnNCr (BALB/c), C57BL/6NCr (C57BL/6), and A/J strains. Small groups of mice were euthanized at 3, 6, and 12 months of age, and the remainder were maintained until morbidity or natural death. Fecal samples were monitored periodi- cally for Helicobacter. Histopathology. We subjected all mice to complete necropsy and fixed tissues in 10% neutral formalin. Liver, stomach, and ceca, as well as any abnormal masses or lesions, were examined for each mouse. We evaluated sever- ity of pathologic changes in livers semi- quantitatively on a scale of 1 to 4 (minimal, mild, moderate, or severe), and diagnosed typ- ical H. hepaticus–associated hepatitis (chronic active hepatitis) on the basis of simultaneous presence of the following lesions: mild to severe multifocal necrosis, subacute inﬂamma- tion, inﬁltration of peribiliary leukocytes, bile ductule hyperplasia, oval cell hyperplasia, hepatocytomegaly, and lymphocytic infiltra- tion (Rice 1995; Ward et al. 1994a, 1994b). We also noted minimal foci of subacute inflammation and lymphocytic infiltrates, which are common liver lesions in normal mice of many strains, but they were not asso- ciated with Helicobacter infection and are thus not included here. Steiner’s silver stain was used for histologic confirmation of the pres- ence of helical bacteria in liver sections. Statistical analysis. We analyzed data using GraphPad InStat, version 3.00 (GraphPad Software, San Diego, CA). Tests included a t-test for parametric data and Mann-Whitney or Kruskal-Wallis tests for nonparametric data as appropriate. Results Liver tumorigenesis in male A/J offspring after maternal exposure to H. hepaticus. Females treated with H. hepaticus suspensions ip and Table 1. Hepatic and cecal lesions and hepatic oxidative DNA damage in A/J mice exposed ig as wean- lings to H. hepaticus and in controls. Weeks after exposure Group No. Tumors [n (%)] Chronic active hepatitis [n (%)] Oxidative DNA damagea No.b 8-Oxo-dG/105 dG ± SE Cecal proliferative typhlitis [No. (%), severity scorec] 2 4 12 24 36 54 > 54d Exp Con Exp Con Exp Con Exp Con Exp Con Exp Con Exp Con 15 15 15 16 15 16 10 10 15 16 5 5 6 4 0 0 0 0 0 0 0 0 0 0 0 0 1 (17) 1 (25) 0 0 0 0 2 (13) 0 1 (10) 0 0 0 1 (20) 0 0 0 3 (6) 3 (4) 3 (6) 2 (4) 3 (6) 3 (5) 5 (9) 5 (10) 4 (7) 4 (8) 5 (10) 5 (10) ND ND 1.3 ± 0.1* 0.8 ± 0.1 1.3 ± 0.1** 0.8 ± 0.03 2.1 ± 0.2** 1.2 ± 0.1 2.3 ± 0.1** 1.5 ± 0.1 2.1 ± 0.3 2.0 ± 0.2 2.3 ± 0.3 2.0 ± 0.1 1 (7), 1.0 0 2 (13), 1.0 ± 0 1 (7), 1.0 6 (40)*, 1.7 ± 0.2 1 (6), 1.0 7 (70)**, 1.6 ± 0.3 0 8 (53)**, 1.6 ± 0.2 1 (6), 1.0 2 (40), 2.0 ± 0 0 0 0 Abbreviations: Con, untreated controls; Exp, experimentally infected with H. hepaticus; ND, not done aResults for weeks 2–12 were reported previously by Sipowicz et al. (1997). bNumber of livers analyzed (total number of samples). cMean ± SE. dMean (± SE) ages of experimental and control mice were 743 ± 48 and 623 ± 68 days, respectively. *p < 0.05, and **p < 0.01, compared with control mice at the same time point. Environmental Health Perspectives • VOLUME 116 | NUMBER 10 | October 2008 1353