Dietary heterocyclic amines and microsatellite instability in colon adenocarcinomas
Anna H. Wu,2,
Darryl Shibata1,
Mimi C. Yu,
Mei-Ying Lai and
Ronald K. Ross
Department of Preventive Medicine and
1 Department of Pathology, University of Southern California, Keck School of Medicine, Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Los Angeles, CA 90089-0251, USA
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Abstract
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Microsatellite instability (MSI) is now an accepted and important pathway in colon tumorigenesis, occurring in 1015% of sporadic colon cancers and almost all hereditary nonpolyposis colon cancers. Little is known about possible environmental influences on MSI status in colon cancer. We conducted an epidemiological study of 276 colon cancer cases in Los Angeles County that was designed to determine the population prevalence of MSI+ colon cancer and to identify environmental influences in the development of MSI+ tumors. Our results support the cigarette smoking and MSI+ association recently reported [Slattery,M.L., Curtin,K., Anderson,K. et al. (2000) J. Natl Cancer Inst., 92, 18311836]. Risk of MSI+ colon cancers increased with increasing dose (number of cigarettes per day) and increasing duration (years of smoking) of smoking. Compared with never-smokers, those who smoked 120 pack-years and >20 pack-years showed odds ratios of 1.39 and 1.64, respectively (P for trend = 0.03). In addition, our results show, for the first time, that after adjustment for cigarette smoking habits and red meat intake, patients with MSI+ colon cancers had significantly higher dietary exposure to heterocyclic aromatic amines (HAA) as determined by two surrogates of high dietary HAA exposure: preference for well-done red meat and high frequencies of certain cooking practices (frying, barbecuing, broiling and using meat drippings). The risk of MSI+ colon cancer was increased 3-fold (smoking/red meat intake adjusted OR = 3.03, 95% CI = 1.06, 8.63) among patients who preferred to eat red meat that was very well-done and was increased >2-fold (smoking/red meat adjusted OR = 2.33, 95% CI = 1.00, 5.25) among those who frequently fried/barbecued/broiled their meats or used meat drippings. The risk of MSI+ colon cancer associated with cigarette smoking remained statistically significant after adjustment for high dietary HAA exposure. The significant association between cigarette smoking and dietary HAA with a specific subset of colon cancers may explain, at least in part, inconsistencies in results linking these two exposures to colon cancers. These results provide a potential mechanism of linking HAA exposure and cigarette smoking to a specific subset of colon cancers.
Abbreviations: HAA, heterocyclic aromatic amines; HNPCC, hereditary nonpolyposis colorectal cancer; MMR, mismatch repair; MSI, microsatellite instability.
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Introduction
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Mismatch repair (MMR) deficiency and associated microsatellite instability (MSI) are hallmarks of hereditary nonpolyposis colorectal cancer (HNPCC) (1) and a subset of sporadic colorectal cancers (2,3). There has been much recent interest in identifying environmental influences that may be associated with loss of MMR. A recent article by Slattery et al. (3) demonstrated that cigarette smoking may be significantly associated with MSI in patients with adenocarcinoma of the colon. This observation suggests that certain environmental exposures are not only associated with higher risks of cancer but with specific subtypes of cancers.
Heterocyclic aromatic amines (HAA), a potent class of animal carcinogens, are common pyrolysis products produced by high temperature cooking of meats including beef, poultry, fish and pork (4,5). Dietary exposures to HAA have been associated with an increased risk of colon cancer and colon adenoma in some studies (610). However, the association between colon cancer and HAA exposure is inconsistent (1113). One of the challenges in studying the role of HAA in colon and other cancers is the well-recognized difficulty in assessing intake frequencies and amounts of specific food items in the diet. In addition, the formation of HAA in cooked foods varies depending on the type of meat and cooking conditions, including the method, temperature and duration of cooking. Cooking methods such as frying, grilling and broiling, and other high temperature cooking are known to yield high concentrations of HAA (4,5,9). Surrogates of high HAA exposure, including selected methods of cooking (such as frying, grilling and broiling), meat doneness level (well-done to very well-done), surface browning (charred) and intake of gravy (gravy with meat drippings), have been used in previous studies to investigate the association between HAA exposure and colon cancer risk (69,12,13).
Investigations that can link specific lifestyle exposures (such as dietary HAA) with molecular alterations are extremely powerful because (i) they provide independent and unbiased confirmation of the specific exposure implicated and (ii) they also offer insights regarding the possible mechanisms leading to the malignant phenotype. The present study investigated the possible association between cigarette smoking (of which HAA is a constituent), dietary HAA and microsatellite instability in colon carcinogenesis, based on a population-based sample of 276 histologically confirmed colon cancer cases in Los Angeles County, California.
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Materials and methods
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We conducted an epidemiological study of colon cancer in Los Angeles County that was designed to determine the population prevalence of MSI+ colon cancer and to identify possible environmental influences in the development of MSI+ tumors. In brief, we randomly sampled ~5% of subjects aged 7079 years, 10% of those aged 6069 years, and all who were <60 years and were diagnosed with an incident, histologically confirmed adenocarcinoma of the colon. Subjects were identified by the Los Angeles County Cancer Surveillance Program (CSP), the population-based tumor registry for the study area during a 12 month period of case ascertainment between 1995 and 1996. 448 colon cancer patients were recruited. The race and gender distributions of those included in the sampling scheme of the parent cross-sectional study are representative of all colon cancer patients identified by the CSP.
English-speaking, eligible subjects participated in an in-person interview that asked about family history of cancer and lifestyle factors, including lifetime smoking habits. To assess methods of cooking meats, subjects were asked the frequencies of consuming meat gravies made with meat drippings; frequencies of eating red meat, chicken or fish that had been prepared by microwave (excluding simply warming the food), boiled, roasted/baked, fried/stir-fried, barbecued/grilled/charbroiled or oven-broiled; and their preference for `doneness' of red meat (i.e. rare, medium rare, medium well, well done, very well done). Ninety-four percent of interviewed patients signed a consent allowing release of tumor materials.
The current analysis is based on the first 276 colon cancer patients with completed interviews and for whom we were able to secure tumor blocks for MSI analysis. Comparable age, gender and race distributions were found between subjects tested for MSI (n = 276) and those not tested (n = 172). The study was approved by the Institutional Review Board of the Keck School of Medicine of the University of Southern California, and written informed consent was obtained from each case before interview.
Using available paraffin sections, cancerous tissue was identified and microdissected along with adjacent, histologically normal tissue. DNA extracted from these paraffin sections was screened for MSI at four CA-dinucleotide MS loci (14) and two poly-A repeats (BAT25, BAT26) (15). The definition of MSI+ cancers conformed to the criteria of MSI-H suggested by a recent US National Institutes of Health (NIH) workshop as they showed microsatellite instability in at least two of the loci tested (1).
Our statistical analyses compared selected demographic and lifestyle characteristics between MSI+ and MSI colon cancer patients. We constructed a cooking/HAA score by summing four individual scores assigned to frequencies of each of the four cooking practices that are known to be associated with high levels of HAA; namely, frying, barbecuing, broiling and using meat drippings. Frequencies of 2+/week or higher were assigned a score of 1; lower frequencies were scored as zero. Unconditional logistic regression with adjustment for age, race and gender was conducted to calculate odds ratios (ORs) and corresponding 95% confidence intervals (CIs), treating MSI+ subjects as `cases' and MSI subjects as `controls'. All P values quoted are two-sided. Values of P < 0.05 were considered statistically significant. Statistical analyses were performed using the software package EPILOG (16).
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Results
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The prevalence of MSI+ colon cancers was 12.7% (35 of 276). The proportion of MSI+ tumors was highest in the under 50 age group (13 of 65; 20.0%), intermediate in those 70 years of age or older (7 of 47; 14.9%) and lowest among those between ages 5069 (15 of 164; 9.1%). Males (18 of 146; 12.3%) versus females (17 of 130; 13.1%) and whites (20 of 152; 13.2%) versus non-whites (15 of 124; 12.1%) showed comparable proportions of MSI+ tumors. The prevalence of MSI+ tumors was significantly higher in right-sided (proximal) (32 of 115; 27.8%) than in non-right-sided (distal) colon cancers (3 of 161; 1.9%).
The risk of MSI+ tumors increased with both the amount and duration of cigarette smoking (Table I
). Early age when started to smoke was associated with an increased risk of MSI+ colon cancers (P for trend = 0.08). Cumulative lifetime pack-years of smoking (packs of cigarettes smoked per day multiplied by total years of smoking; thus, an index of cumulative lifetime exposure to cigarette smoking) was associated with a significantly increased risk of MSI+ tumors (P for trend = 0.03).
Intake of red meat was not statistically significantly related to risk of MSI and colon cancer. Compared with individuals with the lowest tertile of red meat intake, the smoking adjusted ORs for MSI+ tumors in association with the upper two tertiles of red meat intake were 1.12 and 1.21, respectively. However, the risk of MSI+ colon tumors was increased >2-fold among patients who preferred to eat red meats that were well-done (P for trend = 0.09) (Table II
). This association became stronger and was statistically significant after adjustment for cigarette smoking and red meat consumption (P for trend = 0.04; Table II
). In addition, patients who preferred to cook their meats by frying, barbecuing or broiling, or who used meat drippings in the gravies at least two or more times per week showed increased risks of MSI+ tumors; the ORs ranged from 1.4 to 1.9. In contrast, weekly cooking with microwave, boiling and roasting (methods associated with low yields of HAA) was associated with lowered risks of MSI+ tumors. The ORs were 0.35 (P = 0.08), 0.38 (P = 0.16) and 0.94 (P = 0.91), respectively, in association with cooking meat using microwave, boiling or roasting. Since frying, barbecuing, broiling and using meat drippings are associated with higher levels of dietary HAA, we constructed a `cooking/HAA' index based on the summation of four individual scores assigned to each of the four cooking practices (1 for frequencies of 2+/week or higher, 0 otherwise). There was a 2.5-fold increase in risk of MSI+ tumors in association with high dietary cooking/HAA score (1+ versus zero; P = 0.03). This association remained statistically significant after adjustment for cigarette smoking and red meat intake (adjusted OR = 2.33, P = 0.05).
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Table II. Surrogates of dietary heterocyclic aromatic amine exposure and microsatellite instability in colon cancer
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We examined the relationship between cigarette smoking and risk of MSI+ tumors with adjustment for `dietary HAA' exposure. The odds ratio for pack-years of smoking remained statistically significant after controlling for doneness of meat and cooking/HAA score (
20 pack-years, adjusted OR = 1.45, 95% CI = 0.57, 3.68; >20 pack-years, adjusted OR = 1.59, 95% CI = 0.61, 4.14; P for trend = 0.03).
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Discussion
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Colorectal cancer is the third most common incident cancer in men and women in the US (17). Worldwide incidence is increasing, particularly in many Asian countries undergoing westernization (18). The environmental agents responsible for this increase in risk with westernization are unclear. There is accumulating evidence from both casecontrol and prospective studies that long-term cigarette smoking (1921) increases the risk of colon cancer. However, the findings are not totally consistent (22). The risk estimates associated with smoking are typically modest (between 1.5 and 2.0) and concerns of residual confounding by known or unmeasured risk factors remain. The collective results on dietary HAA and colon cancer, based largely on casecontrol studies, are also not in agreement (613). In addition to concerns of confounding by other dietary and non-dietary factors, differential recall bias is an inherent methodological limitation of casecontrol studies of dietary risk factors.
Given the importance of colon cancer as a major cause of cancer incidence and mortality worldwide, identification of modifiable lifestyle factors remains a priority. Cigarette smoking remains prevalent in the US (23) and rates are increasing in Asia and South America (24). Cooking practices associated with high HAA are common ways of preparing foods in the US and in many other western and eastern countries. Thus, seeking a definitive answer to the question of whether cigarette smoking and dietary HAA are etiologic factors for colon cancer is not only scientifically important but also has immense public health implications.
The present study, by combining epidemiology and molecular biology approaches, has examined these questions from a perspective that circumvents many of the methodological limitations of casecontrol studies. Our results support the MSIcigarette smoking link first reported by Slattery et al. (3). In addition, our study reports, for the first time, an association between MSI+ tumors and exposure to dietary HAA. Thus, the present study has considerably strengthened the notion that both cigarette smoking and dietary HAA exposure are causally linked to colon cancer development, by demonstrating that these risk factors are more pronounced for specific biologic subtypes. Cigarette smoke contains over 50 carcinogens, including several important HAAs, polycyclic aromatic hydrocarbons and tobacco-specific nitrosamines (25). 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), an HAA that is found in high concentrations in cooked meats also occurs in relative abundance in cigarette smoke (26). Thus, it is plausible that HAA from dietary sources as well as HAA in cigarette smoke are the common agents underlying our observations. Alternatively, other constituents in tobacco smoke in addition to HAA may also have a role in the development of MSI+ colon cancers.
Colon cancer can be divided into two broad subgroups based on genomic instability (13,27). The majority of colon cancers appear to exhibit chromosomal instability manifested by aneuploidy and losses of heterozygosity. A smaller subset (~1015%) exhibits loss of mismatch repair manifested by microsatellite instability. These two subsets of colon cancer differ considerably in location, prognosis and types of somatic mutations in oncogenes and tumor suppressor genes (13,27). Thus, different etiologic factors (either environmental and/or genetic) may be operating in the development of the two types of colon cancer. The significant association between cigarette smoking and dietary HAA with a specific subset of colon cancers may explain, at least in part, prior inconsistencies in results linking these two exposures to all colon cancers.
Recent studies have demonstrated that an epigenetic mechanism underlies the loss of mismatch repair in sporadic MSI+ colon cancers (2830). Specifically, the majority of sporadic MSI+ colon cancers exhibit loss of expression of the MLH1 gene that is associated with somatic hypermethylation of its normally unmethylated promoter (2830). The exact mechanisms responsible for silencing of genes by methylation are uncertain. The significant associations between MSI+ colon cancer and cigarette smoking/dietary HAA observed in the present study strongly implicate a possible influence of environmental factors on epigenetic fidelity. Recent studies have noted epigenetic changes in many different types of cancer cells (31). It is possible that environmental exposures contribute to the silencing of tumor suppressor genes via epigenetic mechanisms.
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Notes
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2 To whom correspondence should be addressedEmail: annawu{at}hsc.usc.edu 
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Acknowledgments
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This work was supported by contract no. N01-CN-67010 from the National Cancer Institute. Incident cancer cases for this study were collected by the USC Cancer Surveillance Program (CSP), which is supported under subcontract by the California Department of Health Services. The CSP is also part of the National Cancer Institute's Division of Cancer Prevention and Control Surveillance, Epidemiology and End Results Program, under contract number N01-CN-25403.
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Received April 24, 2001;
revised July 2, 2001;
accepted July 12, 2001.