Hepatocarcinogenesis chemical models Introduction Earliest observations that human exposure to certain chemicals is related to an increased incidence of cancer John Hill 1761 Nasal cancer in snuff users Sir Percival Pott 1775 Scrotal cancer in chimney sweeps Soot and coal tar Experimental chemical carcinogenesis Yamagiwa and Ichikawa 1918 Multiple applications of coal tar to rabbit ears produced skin carcinomas First demonstration that a chemical could produce cancer in an animal Confirmed Pott s initial observation and linked human epidemiology and animal carcinogenicity Somatic mutation theory Theodor Boveri 1914 Concept that cancer involves an alteration in the genetic material of the somatic cell Chromosome abnormalities Furth and Kahn 1934 Isolated single cell clones from a tumor and showed that injection into a healthy host could reproduce disease Cancer stable heritable cellular alteration Chemical carcinogenesis James and Elizabeth Miller 1950s Observed that a wide variety of structurally diverse chemicals could produce cancer in animals Proposed that all of these chemicals require metabolic activation to electrophilic reactive intermediates Covalently bind to nucleophilic centers on proteins RNA or DNA Electrophilic theory of chemical carcinogenesis Evidence for genetic mechanisms 1 Cancer is a heritable stable change 2 Tumors are generally clonal in nature 3 Many carcinogens are metabolized to electrophilic intermediates that covalently bind to DNA 4 Many carcinogens are also mutagens 5 Many cancers display chromosomal abnormalities 6 Transformed phenotype can be transferred from a tumor cell to a non tumor cell by DNA transfection Genotoxic agents Direct acting carcinogens N methyl N nitrosourea MNU N methyl N nitro N nitrosoguanidine MNNG Indirect acting carcinogens Dimethylnitrosamine DMN Benzo a pyrene Radiation Inorganic agents Image removed due to copyright reasons Epigenetic agents Immunosuppresive xenobiotics Asbestos Hormones Promoters 12 O tetradecanoylphorbol 13 acetate Phenobarbital Evidence for epigenetic mechanisms 1 Cancer is associated with altered differentiation and proliferation 2 The cancerous state of tumors is sometimes reversible 3 Carcinogenesis is induced by nonmutagenic substances 4 Not all carcinogens are mutagens 5 Carcinogenesis is associated with changes in DNA methylation Multistage carcinogenesis Initiation Genotoxic event Promotion Clonal expansion of an initiated cell Progression Development of a malignant tumor Initiation promotion model 12 O tetradecanoylphorbol 13 acetate TPA belongs to a family of compounds called phorbol esters that are isolated from croton oil and are active almost exclusively on mouse skin TPA is also known as phorbol 12 myristate 13 acetate PMA Phenobarbital DDT chlordane and TCDD are hepatic tumor promoters Figure removed for copyright reasons Figure removed for copyright reasons See http www plantpictures de Features of tumor promoters 1 Following a sub threshold dose of an initiating carcinogen chronic treatment with a tumor promoter will produce many tumors 2 Initiation at a sub threshold does alone will produce very few if any tumors 3 Chronic treatment with a tumor promoter in the absence of initiation will produce very few if any tumors 4 The order of treatment is critical initiation must precede promotion Mouse skin model Berenblum 1941 Alternating doses of croton oil and benzo a pyrene Mottram et al Single sub effective dose of benzo a pyrene followed by repetitive croton oil treatments 1 SW mice 200 nmol DMBA 2 1 week later 2 5 nmol TPA twice a week for 20 weeks 3 After 15 weeks 12 14 benign papillomas Mechanisms of tumor promotion Clonal expansion of initiated cells by providing a selective growth advantage or by repressing normal cell growth or both The specific phorbol ester is protein kinase C PKC Serine and threonine kinase and a Ca2 and phospholipid dependent enzyme Diacylglycerol is also a potent tumor promoter in mouse skin Rodent models of liver cancer Most rat strains have 5 lifetime incidence of primary hepatocellular tumors In contrast outbred Swiss Webster mice have 35 incidence in males and 5 incidence in females In the B6C3F1 National Toxicology Program NTP mouse the range is 25 40 for males and 4 6 9 7 for females In bioassays for carcinogenicity the liver is the most commonly affected site Hepatic carcinogenesis 2 major pathways have been described Oval cell proliferation leading to lesions composed of extensive connective tissue matrix investing a metaplastic ductal system cholangiofibrosis or adenofibrosis Altered hepatic foci hepatic nodules and hepatocellular carcinoma HCC Much of our current understanding comes from nitrosamine or aflatoxin studies in rats relatively non toxic at carcinogenic doses Altered hepatic foci Hepatocellular tumors develop from foci of altered hepatocytes Increased eosinophilia or basophilia or because of rearrangement of RER may be striped or tigroid in appearance In the rat many foci express fetal enzymes such as gamma glutamyl transferase GGT and the placental form of GSH S transferase Image removed due to copyright reasons Some aspects poorly understood The changes are not seen in all foci Foci in mice do not have GGT or placental GSH S transferase Whether all foci develop into tumors is not known The origin of the foci is also not known As they grow the foci become nodules 2 step hepatocarcinogenesis Initiation followed by promotion Rodents appear to have no absolute requirement for deliberate exposure to genotoxic carcinogens for neoplasia to develop Spontaneously initiated cells in the liver Low level environmental exposure to genotoxic carcinogens or inherent metabolic processes leading to oxidative stress Genotoxic hepatocarcinogens Metabolic activation of dimethylnitrosamine DMN or diethylnitrosamine DEN Ultimate carcinogen is methyl diazonium ion Methyl carbonium ion forms pre mutagenic O6guanine and O4 thymidine Epigenetic hepatocarcinogens 2 classes have been widely investigated Phenobarbital PB Peroxisome proliferators PB causes induction of mixed function oxidase enzymes Causes liver enlargement as well as CYP enzyme induction Hyperplasia hypertrophy of cells in centrilobular region due to proliferation of SER PB promotion If PB is given to rats for 18 months there may be a small increase in the number of hepatic tumors If treatment is preceded by short exposure to genotoxic carcinogen such as DEN administration of PB results in