Selenium-rich royal jelly inhibits hepatocellular carcinoma through PI3K/AKT and VEGF pathways in H22 tumor-bearing mice.

Royal jelly (RJ) and selenium (Se)-rich foods have well-known health benefits that are attributable to a broad range of pharmacological effects including antioxidant, anti-tumor, and immunoregulatory activities. However, the physiological effects of Se-rich RJ, which is produced by feeding Apis mellifera (Hymenoptera: Apidae) sodium selenite sucrose solution, are not well understood. The anti-hepatoma activity and mechanism of Se-rich RJ in H22 tumor-bearing mice were investigated in the current study. The findings showed that the content of organic and inorganic Se in Se-rich RJ was significantly higher than that in RJ. Furthermore, interleukin-2 (IL-2) levels and tumor necrosis factor-α (TNF-α) production in serum were increased and the malondialdehyde (MDA) content in liver was decreased in mice fed RJ and Se-rich RJ. 16SrRNA sequencing and serum untargeted metabolomics showed that RJ and Se-rich RJ could modulate the gut microbiota, and fisetin and L-glutathione oxidized were the main anti-tumor components in RJ and Se-rich RJ. Further analysis showed 11-deoxy prostaglandin F1ß was the specific anti-tumor metabolite in mice treated with Se-rich RJ compared with RJ. The results indicated that RJ and Se-rich RJ could inhibit the expression of PI3K and phosphorylation of AKT, induce cell apoptosis through the activation of caspase-9 and caspase-3, and regulate Bcl-2/Bax expression. RJ and Se-rich RJ also inhibited the expression of COX-2 and VEGF. To summarize, the findings clearly demonstrate that Se-rich RJ could inhibit tumor growth by inducing apoptosis and inhibiting angiogenesis as well as exhibit anti-tumor effects by improving immune function and antioxidant activities. The results indicated that Se-rich RJ could be a potential functional food for the management and prevention of cancer.

Microcystis toxin-mediated tumor promotion and toxicity lead to shifts in mouse gut microbiome.

Microcystins (MCs) are the most prevalent cyanotoxins reported in freshwater. While numerous studies have examined the toxicological impacts of MCs on mammalian systems, very few have examined the chronic impacts of MCs on the gut microbiome of exposed organisms. Our understanding of the relationship of MCs, especially lysed toxic cyanobacteria, and the gut microbiota is very limited. The objective of this study was to determine the impacts of MC-LR and Microcystis lysate ingestion on the gut microbiome in a hepatocellular carcinoma mouse model, simulating a high-risk population and exposure at an environmentally relevant MC level. Mice were assigned to 4 groups (MC-LR; Microcystis lysate; Negative control; Positive (liver carcinogen) control). Fecal samples were collected every 8 weeks. Bacterial community and colony counts were analyzed. The abundance of Firmicutes in the positive control and lysate group was higher than the negative control and MC group. Exposure to MC-LR or lysate was associated with significantly decreased bacterial diversity. A distinct separation of the three groups (MC-LR/lysate/carcinogen) from the negative was much more apparent in their gut microbiome as the exposure time increased. The MC-LR and lysate groups showed gut microbiome structure responding to lipid metabolism disturbance and high stress. Bacterial colony count was significantly lower in all the treated groups than the negative control. Our study highlights that chronic exposure to MC-LR and Microcystis lysate negatively impacts gut microbiome succession and altered the bacterial community structure into the one similar to the carcinogen group, which may indicate that the change favors progression of hepatocellular carcinoma. In a future study, more in-depth investigation is warranted to better understand the liver-gut nexus in promoting liver cancer among those exposed to MC and toxic cyanobacteria.

Iron-Oxide Nanocluster Labeling of Clostridium novyi-NT Spores for MR Imaging-Monitored Locoregional Delivery to Liver Tumors in Rat and Rabbit Models.

PURPOSE: To label Clostridium novyi-NT spores (C. novyi-NT) with iron oxide nanoclusters and track distribution of bacteria during magnetic resonance (MR) imaging-monitored locoregional delivery to liver tumors using intratumoral injection or intra-arterial transcatheter infusion. MATERIALS AND METHODS: Vegetative state C. novyi-NT were labeled with iron oxide particles followed by induction of sporulation. Labeling was confirmed with fluorescence microscopy and transmission electron microscopy (TEM). T2 and T2* relaxation times for magnetic clusters and magnetic microspheres were determined using 7T and 1.5T MR imaging scanners. In vitro assays compared labeled bacteria viability and oncolytic potential to unlabeled controls. Labeled spores were either directly injected into N1-S1 rodent liver tumors (n = 24) or selectively infused via the hepatic artery in rabbits with VX2 liver tumors (n = 3). Hematoxylin-eosin, Prussian blue, and gram staining were performed. Statistical comparison methods included paired t-test and ANOVA. RESULTS: Both fluorescence microscopy and TEM studies confirmed presence of iron oxide labels within the bacterial spores. Phantom studies demonstrated that the synthesized nanoclusters produce R2 relaxivities comparable to clinical agents. Labeling had no significant impact on overall growth or oncolytic properties (P >.05). Tumor signal-to-noise ratio (SNR) decreased significantly following intratumoral injection and intra-arterial infusion of labeled spores (P <.05). Prussian blue and gram staining confirmed spore delivery. CONCLUSIONS: C. novyi-NT spores can be internally labeled with iron oxide nanoparticles to visualize distribution with MR imaging during locoregional bacteriolytic therapy involving direct injection or intra-arterial transcatheter infusion.

Dysregulated hepatic bile acids collaboratively promote liver carcinogenesis.

Dysregulated bile acids (BAs) are closely associated with liver diseases and attributed to altered gut microbiota. Here, we show that the intrahepatic retention of hydrophobic BAs including deoxycholate (DCA), taurocholate (TCA), taurochenodeoxycholate (TCDCA), and taurolithocholate (TLCA) were substantially increased in a streptozotocin and high fat diet (HFD) induced nonalcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC) mouse model. Additionally chronic HFD-fed mice spontaneously developed liver tumors with significantly increased hepatic BA levels. Enhancing intestinal excretion of hydrophobic BAs in the NASH-HCC model mice by a 2% cholestyramine feeding significantly prevented HCC development. The gut microbiota alterations were closely correlated with altered BA levels in liver and feces. HFD-induced inflammation inhibited key BA transporters, resulting in sustained increases in intrahepatic BA concentrations. Our study also showed a significantly increased cell proliferation in BA treated normal human hepatic cell lines and a down-regulated expression of tumor suppressor gene CEBPα in TCDCA treated HepG2 cell line, suggesting that several hydrophobic BAs may collaboratively promote liver carcinogenesis.

Profound impact of gut homeostasis on chemically-induced pro-tumorigenic inflammation and hepatocarcinogenesis in rats.

BACKGROUND & AIMS: Due to its anatomic connection, the liver is constantly exposed to gut-derived bacterial products or metabolites. Disruption of gut homeostasis is associated with many human diseases. The aim of this study was to determine the role of gut homeostasis in initiation and progression of hepatocellular carcinoma (HCC). METHODS: Disruption of intestinal homeostasis by penicillin or dextran sulfate sodium (DSS) and its restoration by probiotics were applied in a diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. RESULTS: Patients with liver cirrhosis and HCC had significantly increased serum endotoxin levels. Chronic DEN treatment of rats was associated with an imbalance of subpopulations of the gut microflora including a significant suppression of Lactobacillus species, Bifidobacterium species and Enterococcus species as well as intestinal inflammation. Induction of enteric dysbacteriosis or intestinal inflammation by penicillin or DSS, respectively, significantly promoted tumor formation. Administration of probiotics dramatically mitigated enteric dysbacteriosis, ameliorated intestinal inflammation, and most importantly, decreased liver tumor growth and multiplicity. Interestingly, probiotics not only inhibited the translocation of endotoxin, which bears pathogen-associated molecular patterns (PAMPs) but also the activation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1). As a result, the production of pro- and anti-inflammatory cytokines was skewed in favor of a reduced tumorigenic inflammation in the liver. CONCLUSIONS: The data highlights the importance of gut homeostasis in the pathogenesis of HCC. Modulation of the gut microbiota by probiotics may represent a new avenue for therapeutic intervention to treat or prevent HCC development.

Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4.

Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation, and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and TLR4 activation in non-bone-marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC, suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.

Helicobacter hepaticus--induced liver tumor promotion is associated with increased serum bile acid and a persistent microbial-induced immune response.

Chronic microbial infection influences cancer progression, but the mechanisms that link them remain unclear. Constitutive androstane receptor (CAR) is a nuclear receptor that regulates enzymes involved in endobiotic and xenobiotic metabolism. CAR activation is a mechanism of xenobiotic tumor promotion; however, the effects of chronic microbial infection on tumor promotion have not been studied in the context of CAR function. Here, we report that CAR limits the effects of chronic infection-associated progression of liver cancer. CAR knockout (KO) and wild-type (WT) male mice were treated with or without the tumor initiator diethylnitrosamine (DEN) at 5 weeks of age and then orally inoculated with Helicobacter hepaticus (Hh) or sterile media at 8 weeks of age. At approximately 50 weeks postinoculation, mice were euthanized for histopathologic, microbiological, molecular, and metabolomic analyses. Hh infection induced comparable hepatitis in WT and KO mice with or without DEN that correlated with significant upregulation of Tnfα and toll receptor Tlr2. Notably, DEN-treated Hh-infected KO mice exhibited increased numbers of liver lobes with dysplasia and neoplasia and increased multiplicity of neoplasia, relative to similarly treated WT mice. Enhanced tumor promotion was associated with decreased hepatic expression of P450 enzymes Cyp2b10 and Cyp3a11, increased expression of Camp, and increased serum concentrations of chenodeoxycholic acid. Together, our findings suggest that liver tumor promotion is enhanced by an impaired metabolic detoxification of endobiotics and a persistent microbial-induced immune response.

Gut microbes define liver cancer risk in mice exposed to chemical and viral transgenic hepatocarcinogens.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. METHODS: Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. RESULTS: Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-kappaB (NF-kappaB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/beta-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. CONCLUSIONS: Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention.

Candida albicans enhances experimental hepatic melanoma metastasis.

Candida albicans infections are very frequent in cancer patients, whose immune system is often compromised, but whether this fungal pathogen affects cancer progression is unknown. C. albicans infection involves endogenous production of inflammatory cytokines such as tumour necrosis factor alpha (TNF-alpha) and interleukin-18 (IL-18). Increased levels of these cytokines have already been correlated with metastasis of most common cancer types. In this study, a well-established model of IL-18-dependent hepatic melanoma metastasis was used to study whether C. albicans can alter the ability of murine B16 melanoma (B16M) cells to colonize the liver. First, we determined the ability of intrasplenically (IS) injected B16M cells to metastasize into the liver of mice challenged with 5 x 10(4) C. albicans cells by three different routes (intravenous, IV; intrasplenic, IS; or intraperitoneal, IP) 12 h prior to injection of B16M cells. We demonstrated that C. albicans significantly increased metastasis of B16M cells with all three fungal injection routes. Pro-metastatic effects occurred when hepatic colonization with B16M cells place after the peak of TNF-alpha and IL-18 levels had been reached in the hepatic blood of fungal challenged mice. In a second set of experiments, mice were fungal challenged 4 days after injection of B16M cells. In these mice, C. albicans also potentiated the growth of established micro-metastases. Significantly, the fungal challenge had pro-metastatic effects without the C. albicans being able to reach the liver, suggesting that soluble factors can promote metastasis in remote sites. Mouse treatment with antifungal ketoconazol abrogated hepatic TNF-alpha stimulation by C. albicans and prevented the enhancement of hepatic metastasis in fungal challenged-mice. Therefore, the pro-inflammatory microenvironment generated by the host's systemic response to C. albicans stimulates circulating cancer cells to metastasize in the liver.

Chronic Mycobacterium marinum infection acts as a tumor promoter in Japanese Medaka (Oryzias latipes).

An accumulating body of research indicates there is an increased cancer risk associated with chronic infections. The genus Mycobacterium contains a number of species, including M. tuberculosis, which mount chronic infections and have been implicated in higher cancer risk. Several non-tuberculosis mycobacterial species, including M. marinum, are known to cause chronic infections in fish and like human tuberculosis, often go undetected. The elevated carcinogenic potential for fish colonies infected with Mycobacterium spp. could have far reaching implications because fish models are widely used to study human diseases. Japanese medaka (Oryzias latipes) is an established laboratory fish model for toxicology, mutagenesis, and carcinogenesis; and produces a chronic tuberculosis-like disease when infected by M. marinum. We examined the role that chronic mycobacterial infections play in cancer risk for medaka. Experimental M. marinum infections of medaka alone did not increase the mutational loads or proliferative lesion incidence in all tissues examined. However, we showed that chronic M. marinum infections increased hepatocellular proliferative lesions in fish also exposed to low doses of the mutagen benzo[a]pyrene. These results indicate that chronic mycobacterial infections of medaka are acting as tumor promoters and thereby suggest increased human risks for cancer promotion in human populations burdened with chronic tuberculosis infections.

Marked liver tumorigenesis by Helicobacter hepaticus requires perinatal exposure.

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 injection 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 x 10(8) 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 bacterium or its toxins. Mechanisms of human perinatal sensitivity to such phenomena can be studied with this model.

Bifidobacterium longum as an oral delivery system of endostatin for gene therapy on solid liver cancer.

To overcome difficulties that hampered widespread application of a specific delivery system in cancer gene therapy and to inhibit the growth of solid liver cancer, we utilized a strain of Bifidobacterium longum as a delivery system to transport an endostatin gene that can inhibit growth of tumor. The B. longum strain with the endostatin gene (B. longum-En) was taken orally by tumor-bearing nude mice through drencher preparation. The results showed that B. longum-En could strongly inhibit the growth of solid liver tumor in nude mice and prolong the survival time of tumor-bearing nude mice. Furthermore, tumor growth was inhibited more efficiently when the B. longum-En treatment included selenium. Enriching the B. longum-En treatment with selenium improves the activity of NK and T cells and stimulates the activity of IL-2 and TNF-alpha in BALB/c mice. These results suggest that B. longum may be a highly specific and efficient vector for transporting anticancer genes in cancer gene therapy.

Bifidobacterium adolescentis as a delivery system of endostatin for cancer gene therapy: selective inhibitor of angiogenesis and hypoxic tumor growth.

In order to overcome difficulties that hampered widespread application of antiangiogenesis in cancer therapy, a highly specific delivery system may be engaged in vivo to deliver and express antiangiogenic genes. We selected a strain of Bifidobacterium adolescentis (B. adolescentis) as the delivery system to transport endostatin gene to solid tumors. B. adolescentis with endostatin gene were injected into tumor-bearing mice through the tail vein. After the mice were sacrificed, the tumor and some normal tissues of the mice were examined. B. adolescentis were only found in the tumors and no bacilli were found in other normal tissues. Also, a strong inhibition of angiogenesis had been shown to inhibit local tumor growth in the administrated group. These results suggested that B. adolescentis only germinated and proliferated in solid tumors and might be a highly specific and efficient vector for transporting anticancer genes into target tumor in cancer gene therapy.

Impacts of chemicals on liver cancer risk.

Primary liver cancer (PLC) is of multifactorial etiology. Chronic infections by hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for most PLC cases worldwide, although mechanisms through which the infections cause PLC are still unknown. Epidemiologic and experimental evidence indicates that exposure to certain chemicals can also contribute significantly to PLC development, some of which have been designated as human liver carcinogens (Group 1) by the International Agency for Research on Cancer. These include aflatoxins and chronic consumption of alcoholic beverages. Many naturally occurring and synthetic chemicals have been shown to induce liver cancer in experimental animals. Humans are exposed to these carcinogens via accidental contamination of food or water; usually at levels far lower than those that are carcinogenic to experimental animals. Consequently, assessment of possible human PLC risk associated with such exposures is complex and uncertain. Evidence regarding aflatoxin as a human carcinogen has been extensively documented and is reviewed as an example of the usefulness of parallel experimental and epidemiological investigations in cancer risk assessment. Aflatoxins are toxic metabolites of certain spoilage molds that are potent liver carcinogens in experimental animals and frequently contaminate human diets. Collectively, epidemiologic data together with evidence from many types of experimental models defines the role of aflatoxin exposure in PLC causation. Molecular epidemiology involving the use of biomarkers of exposure has been particularly effective in linking aflatoxin exposure to PLC. Biomarkers of aflatoxin exposure have been validated with particular thoroughness. Dose-response relationships between biomarker levels and liver tumor incidence were first established in experimental animals. The biomarkers were then employed in pilot studies of limited scale in humans to define sensitivity, specificity, accuracy, and reliability parameters. Further validation in transitional epidemiological studies assessed intra- and interindividual variability, background levels, external dose-marker relationship, and feasibility for use in larger population-based studies. Finally, prospective epidemiological studies were conducted to evaluate biomarker effectiveness in identifying PLC risk.

Impact of Helicobacter hepaticus infection in B6C3F1 mice from twelve National Toxicology Program two-year carcinogenesis studies.

Male and female B6C3F1 mice from 12 National Toxicology Program (NTP) 2-yr carcinogenesis studies were found to be infected with Helicobacter hepaticus. Many of the male mice from 9 of these studies had an associated hepatitis (affected studies). Helicobacter hepaticus has been reported to be associated with an increased incidence of hepatitis and hepatocellular neoplasms in the A/JCr male mouse. We attempted to determine if the data from the Helicobacter-affected NTP B6C3F1 mouse studies were compromised and unsuitable for cancer hazard identification. The incidences of neoplasms of the liver (both hepatocellular and hemangiosarcoma) but not of other organs in control male B6C3F1 mice were increased in affected studies as compared with control males from unaffected studies. The increased incidence of hepatocellular neoplasms was observed in those males exhibiting H. hepaticus-associated hepatitis. Other observations further differentiated control male mice from affected and unaffected studies. H-ras codon 61 CAA to AAA mutations were less common in liver neoplasms from males from affected studies as compared with historical and study controls. In addition, increases in cell proliferation rates and apoptosis were observed in the livers of male mice with H. hepaticus-associated hepatitis. These data support the hypothesis that the increased incidence of liver neoplasms is associated with H. hepaticus and that hepatitis may be important in the pathogenesis. Therefore, interpretation of carcinogenic effects in the liver of B6C3F1 mice may be confounded if there is H. hepaticus-associated hepatitis.

Epidermal growth factor and transforming growth factor-alpha-associated overexpression of cyclin D1, Cdk4, and c-Myc during hepatocarcinogenesis in Helicobacter hepaticus-infected A/JCr mice.

Helicobacter hepaticus is a new bacterial species that is homologous to Helicobacter pylori, a human gastric carcinogen. H. hepaticus causes chronic active hepatitis, with progression to hepatocellular tumors. We hypothesized that chronic up-regulation of epidermal growth factor (EGF), transforming growth factor-alpha, and nuclear oncogenes (cyclin D1 and c-Myc), all known to transform by overexpression, might contribute to tumorigenesis. Livers from mice that were 6-18 months old were analyzed, including nonneoplastic and preneoplastic tissues and tumors, along with age-matched controls, by immunohistochemistry and immunoblotting. EGF and transforming growth factor-alpha were increased at the earliest stage, with a further increase in EGF in tumors. Cyclin D1, cyclin-dependent kinase 4, and c-Myc were strongly increased in all infected livers, with even greater increases in tumors. An increase in cyclin D1/cyclin-dependent kinase 4 complex was also demonstrated in tumors, and its functionality was confirmed by an increase in the hyperphosphorylated:hypophosphorylated retinoblastoma protein ratio. Our findings suggest a possible cooperation of growth factors, cell cycle proteins, and transcription factors during the development of H. hepaticus-associated liver tumors and may have relevance to human cancers associated with bacterial, viral, or parasitic infections.

Lack of p53 and ras mutations in Helicobacter hepaticus-induced liver tumors in A/JCr mice.

Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas. This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection. A possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes. Since mutations in ras oncogenes have been widely detected in a variety of chemically induced and spontaneous mouse liver tumors and specific mutations in the p53 tumor suppressor gene have been associated with human bladder cancers attributed to chronic schistosomal infection, we studied exons 1 and 2 of the N-, K- and H-ras genes and exons 5-8 of the p53 gene for the presence of point mutations in 25 liver tumors from 10 naturally infected A/JCr mice, ranging in age from 16 to 24 months. The 20 adenomas and five carcinomas varied in size from 0.1 to 2.3 cm and arose in livers characterized by a wide assortment of pathological profiles, including hepatitis, inflammation, hyperplasia, hypertrophy, leukocyte infiltration, necrosis and focal phenotypic alteration. DNA samples extracted from formalin-fixed paraffin-embedded tissues were screened by PCR/SSCP analysis and showed no mutations in the analyzed genes. Complete absence of mutations in ras genes in 25 mouse liver tumors is unusual. Other genes may be targeted or H. hepaticus infection causes liver cancer through other pathways than direct damage to DNA.

Alteration in cell kinetics in control B6C3F1 mice infected with Helicobacter hepaticus.

The discovery of Helicobacter hepaticus infection, H. hepaticus hepatitis, and increased incidence of liver tumors in control males from several recent National Toxicology Program B6C3F1 mouse carcinogenicity bioassays raised questions regarding the suitability of these bioassays for hazard identification. The purpose of this study was to determine if changes in cell proliferation and death at terminal sacrifice might be linked to the increased liver tumor incidences among control males. In control males, enhanced rates of hepatocyte proliferation, as assessed by immunostaining for proliferating cell nuclear antigen (PCNA), and apoptosis, as assessed from hematoxylin and eosin- and TUNEL-stained preparations, were seen in 3 bioassays with H. hepaticus hepatitis. One bioassay with H. hepaticus infection without attendant hepatitis and one bioassay without H. hepaticus or hepatitis did not have elevated rates of hepatocyte proliferation or apoptosis. There was no significant effect on PCNA cell proliferation indices or apoptosis in females. The present findings are indicative of a clear association between the presence of H. hepaticus infection with attendant hepatitis, increased cell proliferation and apoptosis, and increased incidences of hepatocellular neoplasia in males but not in females. Thus, the interpretation of liver tumor responses in H. hepaticus-infected studies is considered to be confounded in male mice. The lack of enhanced cell proliferation or hepatocellular neoplasia in control females suggests that bioassay results from females are valid for hazard identification. Furthermore, the absence of enhanced cell proliferation in lungs and kidneys of male and females suggests that neoplastic effects at these sites are not exacerbated by H. hepaticus infection.

Promotion by Helicobacter hepaticus-induced hepatitis of hepatic tumors initiated by N-nitrosodimethylamine in male A/JCr mice.

A new murine Helicobacter species, Helicobacter hepaticus, infects the livers of mice, causing a progressive chronic active hepatitis culminating in hepatocellular tumors. To examine the role of chronic H. hepaticus infection in carcinogenesis, H. hepaticus-infected male infant mice of A/JCr strain were given a single i.p. dose of N-nitrosodimethylamine (NDMA). Noninfected A/J mice similarly treated with NDMA served as controls. The effect of hepatitis induced by H. hepaticus was studied for 64 wk. At 31-36 wk, the incidence of hepatocellular adenomas in infected mice was significantly higher than in noninfected mice (82 vs 52%; p = 0.05). The multiplicity of hepatocellular tumors was also significantly higher in infected mice compared to noninfected mice (3.2 +/- 0.09 vs 0.09 +/- 0.2; p = 0.03). At 51-64 wk, many (10/18) infected mice developed hepatocellular carcinomas while only 2 of 19 control mice developed such tumors (p = 0.005). Overexpression of cyclin D was observed in hepatocytes as well as adenomas induced by NDMA in H. hepaticus-infected mice, suggesting its role in inflammation, abnormal cell growth, and early neoplasia. High molecular weight keratins were highly expressed in hyperplastic oval cells in hepatitis and in liver tumors in mice with hepatitis, establishing a reliable marker for oval cells in formalin-fixed, paraffin-embedded tissue. Thus, chronic H. hepaticus infection significantly stimulated cyclin D expression, accelerated the development of liver tumors, increased the multiplicity of such lesions, and enhanced the progression of benign to malignant tumors.