During this year we have concentrated this project's resources on an expanding effort to understand the mechanisms by which the liver pathogen Helicobacter hepaticus leads to the development of liver cancer. This organism, which first appeared and was identified as a new species in our mice, provides the first animal model for the causation of cancer by a microorganism. As such, it has high relevance to the established association of stomach cancer with infection by H. pylori, and the putative involvement of infections with cancers of esophagus, intestine, urinary bladder, cervix, and penis. In our mouse colony, the presence of H. hepaticus infection was first indicated by the development of hepatitis, and later liver adenomas and carcinomas in high incidence, in a strain (A/JNCr) that normally is resistant to hepatocarcinogenesis. As this is the first model of its type, our initial efforts to establish fruitful lines of inquiry into mechanism have included a survey of possible changes, that have been implicated in liver carcinogenesis in other models. The tumor suppressor gene, p53, and the H-, K-, and N-ras oncogenes were examined for mutations, with none found; this is suggestive of a tumor promotion type mechanism. Search for formation of alkyl DNA adducts, which could result from nitric oxide release by inflammatory cells and in vivo nitrosation, has likewise been unfruitful thus far. Neither bacteria from agar cultures nor liver homogenates were mutagenic in the Ames test, results which again point to a nongenotoxic mechanism. Positive results, however, were obtained when levels of 8-hydroxydeoxyguanosine, from oxidative DNA damage, were measured. These increased significantly, and from a very early stage in the infection, before frank hepatitis was evident. These findings indicate that release of reactive oxygen species in the infected livers may be part of the mechanism of carcinogenesis. Reactive oxygen has been implicated in tumor promotion in liver and other models. We have succeeded in transmitting the disease by intubation of pure cultures, reproducibly and with short latency; this will permit controlled experimentation, which for the immediate future will be focused on reactive oxygen. A polymerase chain reaction (PCR) method has been developed which distinguishes hepaticus from other Helicobacter species, and one hepaticus strain from another, and progress has been made in a search for a putative cytotoxin gene. With these methods in hand, rapid progress with investigation of the H. hepaticus liver tumorigenesis phenomenon is anticipated.
