| RESEARCH SUMMARY: This laboratory has focused its research efforts on understanding the pathways involved in initiation of aromatic and heterocyclic amine carcinogenesis. We identify potentially important pathways in vitro. Once the mechanism of metabolism and specific products of metabolism are identified, this information is applied to assess the significance of the pathway using in vivo models. Metabolites are separated in pure form by various chromatographic techniques including HPLC and identified by NMR and mass spectrometry. An important part of these studies has been the ability to translate our in vitro observations to in vivo studies with occupationally exposed individuals. We have assessed urine samples from workers manufacturing benzidine collected as part of a joint collaborative study between the U.S. National Cancer Institute's Environmental Epidemiology Branch and the Indian National Institute of Occupational Health. Results are consistent with benzidine causing bladder cancer in humans following N-glucuronidation in liver of either benzidine or its N-acetylated product, transport of N-glucuronides to the kidney, and excretion and accumulation in urine where acidic conditions hydrolyze glucuronides to their free amines in the bladder lumen. Activation of amines to bind DNA is thought to occur in the bladder. The major DNA adduct detected in urothelial and peripheral white blood cells from exposed-workers is the N-acetylbenzidine C-8 guanosine adduct. This adduct causes frameshift mutations, point mutations, and sister chromatid exchanges in in vitro systems, and mutations in ras protooncogenes in tumors. The adduct can be produced by peroxidatic, cytochrome P-450, or HOCl activation of N-acetylbenzidine. Thus, initiation of aromatic amine bladder carcinogenesis involves multiple pathways and organ systems.
There is a close association between cancers (bladder and colon) initiated by aromatic and heterocyclic amines and infection/inflammation and injury. During the inflammatory response, upregulation of inducible nitric oxide synthase produces high levels of nitric oxide (NO). NO interacts with either oxygen or superoxide to produce reactive nitrogen oxygen species (RNOS). RNOS are bactericidal and tumoricidal and contribute to the deleterious effects attributed to inflammation on normal tissues. Therefore, we propose that RNOS play a role in the initiation of aromatic and heterocyclic amine carcinogenesis (see scheme below). We have shown that RNOS transform both aromatic and heterocyclic amines to products, which can be activated to form DNA adducts. In particular, the heterocyclic amine cooked meat carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) form N-nitroso derivatives, N-NO-IQ and N-NO-MeIQx. These nitrosamines are mutagenic and can be activated to form DNA adducts identical to those formed by P450 activation of IQ or MeIQx. Using a colitis-induced colon cancer model with ApcMin/+ mice, we have demonstrated that mice treated with dextran sulfate sodium to induce colitis and given an oral dose of 40 mg/kg IQ had a 100% incidence of invasive colorectal tumors. By contrast, mice not treated with DSS, but given IQ had no tumors. In addition, a specific inhibitor of iNOS, PBIT, significantly reduced the incidence of colon cancer. Studies are continuing by identifying biological and genetic changes in the tumors and N-nitrosamine products in urine. This information is being used to provide practical benefits for cancer prevention, biomarkers, and risk assessment. For example, dietary recommendations for ulcerative colitis patients should include the avoidance of high temperature cooked red meat.
This model depicts the relationship between chronic inflammation/infection and injury, well-done red meat in the diet, and colon cancer. Mediators of inflammation (endotoxin and cytokines), oxidant stress, and injury induce the synthesis of inducible nitric oxide synthase (iNOS), increasing NO synthesis from L-arginine (L-Arg). Well-done red meat provides heme peroxidase and heterocyclic amines (MeIQx or IQ), while the inflammatory process provides NO, MPO, H2O2, and HOCl. Together these pathways generate RNOS, which produce N-nitroso heterocyclic amines (N-NO-MeIQx) capable of initiating colon cancer. Our research is designed to test this model of colon carcinogenesis. |
