LINUS PAULING INSTITUTE RESEARCH REPORT

Food-borne Carcinogens and Hereditary Colorectal Cancer Risk

Andrew B. Buermeyer, Ph.D.
Assistant Professor of Environmental and Molecular Toxicology

Lynch Syndrome (also known as hereditary nonpolyposis colorectal cancer) is the most common form of genetically inherited predisposition for colorectal cancer, representing 2-5% of the total cases. Individuals with Lynch Syndrome face a lifetime risk of approximately 80% for colorectal or other internal cancer, especially endometrial, ovarian, and gastric cancers. The disease is caused by inherited defects in DNA mismatch repair (MMR) genes, which code for enzymes and proteins that can repair strands of DNA that are mismatched, i.e., bases on one strand are not properly aligned with bases on the other strand of the double helix. DNA consists of two strands of helical nucleotides, or bases, which are joined by bonds formed between adenine and thymine or guanine and cytosine (see illus.). Bases typically become mismatched as a result of DNA synthesis errors during replication in dividing cells. If mismatched bases are not corrected, they give rise to mutations, and normal gene activity can be disrupted. MMR is the most prominent mechanism to repair these errors in DNA. Mutations in two essential MMR genes, MLH1 or MSH2, account for most cases of Lynch Syndrome, although mutations also have been identified in other MMR genes. Lynch Syndrome patients inherit one copy of a defective MMR gene and typically develop cancer in their thirties or forties. Inheritance of two defective copies of MLH1 or MSH2 increases cancer risk and can result in severe childhood lymphoma or leukemia. Inactivation of MMR caused by decreased production of MLH1 also is associated with approximately 15% of more common colorectal cancer cases diagnosed when patients are in their sixties or seventies. Although much has been learned in recent years about the functions of MMR proteins, the precise etiology of cancer associated with MMR deficiency remains unclear. For example, the primary site of cancer in Lynch syndrome patients appears to have shifted from the stomach to the colon over the last century, suggesting that environmental factors discussed below might significantly influence disease initiation or progression.

Two prominent cellular functions of MMR contribute to protection against carcinogenesis. First, MMR correction of DNA replication errors suppresses mutation in dividing cells. Loss of proper MMR function elevates mutation rates by 10- to 100-fold and can be observed as an increased frequency of variations in the size of short DNA sequences called “microsatellites.” Such microsatellite instability is detected in many cancers. MMR also suppresses mutation and activates pathways leading to cell death (so-called apoptosis) in response to exposure to DNA damaging agents. Such cellular responses appear to involve specific recognition by MMR proteins of DNA damage, such as oxidative damage or alkylation—a chemical modification of DNA bases. The principal role for MMR in responding to DNA damage is not to repair the lesions, which is mainly the responsibility of other base and/or nucleotide excision repair mechanisms, but instead to recognize that the level of damage has exceeded the cell’s capacity for repair. This, in turn, leads to cell death. The increased spontaneous mutation in MMR-deficient cells certainly contributes significantly to increased cancer risk by increasing the likelihood of inactivating mutations in tumor suppressor genes and activating mutations in oncogenes. The extent to which loss of MMR-dependent cellular responses to DNA damage influences cancer risk is unknown. The heightened susceptibility of MMR-deficient mice to cancer induced by alkylating agents that damage DNA suggests that such effects may be of significant concern in humans.

Few studies have examined the impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. 2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) is a heterocyclic amine formed during the high-temperature cooking of meat and is known as a “cooked-meat mutagen.” It is mutagenic in bacteria, and mutagenic and carcinogenic in rodents. PhIP induces colon, mammary, and prostate tumors in rats, and T-cell and B-cell lymphoma in mice. PhIP also can induce intestinal tumors in genetically susceptible mice, as well as aberrant crypt foci—preneoplastic lesions thought to represent an early step in colorectal carcinogenesis—in both rats and mice. PhIP damages DNA through the formation of chemical adducts, or additions, predominately on guanine bases in DNA. Epidemiological studies suggest that colon cancer risk is increased for persons who consume high levels of cooked-meat mutagens, and PhIP has been classified by the National Toxicology Program as “reasonably anticipated to be a human carcinogen.” In cultured human cancer cells lacking MMR, treatment with PhIP produces higher levels of mutation than similar treatment of cells with adequate MMR, suggesting that PhIP DNA adducts may be recognized in vivo by MMR proteins and subject to damage surveillance. In the absence of MMR, exposure to PhIP would be predicted to increase risk of carcinogenesis since mutation rates are increased. However, the impact of cooked-meat mutagen exposure in individuals with MMR deficiencies is unknown.

With support from an LPI pilot project grant, we determined the effect of MMR status on mutagenesis and carcinogenesis induced by PhIP in mice genetically engineered to model Lynch Syndrome. Mice lacking the DNA mismatch repair gene, MLH1, spontaneously develop intestinal tumors, and, similar to Lynch Syndrome, these tumors appear to progress rapidly to carcinoma. These mice also develop lymphomas, potentially similar to the severe hematological malignancy seen in MMR-deficient humans. We measured mutations induced by PhIP in mice using a special technique in which mutated genes from intestinal cells were isolated and analyzed. PhIP exposure induced 3-fold more mutations in the colon and small intestine of MLH1-deficient mice compared to normal littermates. The increased levels of mutations in mice without MMR were due to the induction of types of mutations not typically associated with PhIP. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs of DNA bases. Our results suggest that MMR-deficiency would increase the likelihood of PhIPinduced carcinogenic mutations.

In related studies, PhIP exposure increased the incidence and frequency of colonic aberrant crypt foci, a proposed biomarker for colon carcinogenesis. In MMR-deficient mice PhIP increased the frequency of mutations in colon cells by 3-fold and the frequency of aberrant crypt foci by 4-fold. Thus, MMR suppression of PhIP-induced mutation may help prevent the development of aberrant crypt foci. PhIP exposure did not significantly increase the development of lymphoma or small intestinal adenoma in the mice, which may require exposure of younger animals to PhIP and/or more prolonged exposure than in our study. Due to a limited number of tumors detected in colon, effects on PhIP-induced colon tumorigenesis could not be assessed directly. However, the apparently heightened susceptibility to induction of aberrant crypt foci in MMR-deficient mice is consistent with the hypothesis that MMR-deficiency increases risk of colon carcinogenesis due to PhIP. Therefore, additional studies to determine cancer risk of MMR-deficient individuals who consume cooked-meat mutagens are warranted.

Since the discovery of inherited MMR-deficiency as the cause of Lynch Syndrome, there has been an explosion of research on the mechanism and functions of MMR and the etiology of cancer caused by defects in MMR. Despite such intensive efforts, very little is known about the environmental factors that modulate the risk of cancer in individuals with MMR deficiency. Our project provides the first direct evaluation of risk of intestinal tumorigenesis in this genetically susceptible animal model due to exposure to a common human carcinogen and suggests that exposure to this cooked-meat mutagen may be a significant concern for individuals at risk for MMR-deficiency. Such risk is not limited to only those with an inherited deficiency, such as Lynch Syndrome. MMR activity also can be inhibited by environmental factors like chronic oxidative stress or hypoxia. Exposure to PhIP and other cooked-meat mutagens is difficult to avoid but can be reduced through adjustment of cooking practices and diet, thereby reducing risk of carcinogenesis. The approach described here might also be useful for investigating pharmacological and dietary strategies to prevent the development of cancer in individuals with MMR-deficiency. As current management of Lynch Syndrome requires extensive and invasive screening procedures, surgery, and, often, chemotherapy, the identification of efficacious chemopreventive approaches is highly desirable.

Last updated November, 2005


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