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Carcinogenesis Advance Access originally published online on August 8, 2007
Carcinogenesis 2007 28(9):2019-2027; doi:10.1093/carcin/bgm179
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Meat intake, preparation methods, mutagens and colorectal adenoma recurrence

María Elena Martínez1,2,3,*, Elizabeth T. Jacobs1,2, Erin L. Ashbeck1, Rashmi Sinha4, Peter Lance1,2,5, David S. Alberts1,2,5 and Patricia A. Thompson1,3,6

1 Arizona Cancer Center
2 Mel and Enid Zuckerman Arizona College of Public Health
3 Department of Nutritional Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ, USA
4 Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD 20852, USA
5 Department of Medicine
6 Department of Pathology, University of Arizona, Tucson, AZ 85724, USA

* To whom correspondence should be addressed. Tel: +1 520 626 8130; Fax: +1 520 626 9275; Email: emartinez{at}azcc.arizona.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Red meat intake has been shown to be associated with higher risk of colorectal cancer. Though the exact mechanisms responsible for this association remain unknown, several tumorigenic properties of meat have been proposed. One well-supported biologic mechanism is elevated exposure to the genotoxic formation of heterocyclic amines (HCAs), which occur when meat is cooked at high temperatures for a long period of time. We prospectively assessed the relation between type of meat, meat preparation method, doneness, a metric of HCAs and other mutagens and colorectal adenoma recurrence among 869 participants in a chemoprevention trial of ursodeoxycholic acid. Unconditional logistic regression analyses were used to estimate odds ratios (ORs) and associated 95% confidence intervals (CIs). Most meat variables assessed were positively but weakly associated with recurrence of any adenoma. In contrast, recurrence of advanced or multiple adenomas was more strongly associated with a number of the meat exposure variables evaluated. For recurrence of advanced lesions, significant associations were detected among individuals in the highest when compared with the lowest tertile of intake for pan-fried red meat (OR = 1.85; 95% CI = 1.10–3.13) and well/very well done red meat (OR = 1.71; 95% CI = 1.02–2.86). Significant positive associations were shown for recurrence of multiple adenomas and the following variables: processed meat (OR = 1.83; 95% CI = 1.10–3.04), pan-fried red meat (OR = 1.63; 95% CI = 1.01–2.61), well/very well done red meat (OR = 1.68; 95% CI = 1.03–2.74), 2-amino-3,4,8-trimethylimidazo[4,5,-f]quinoxaline (OR = 1.74; 95% CI = 1.07–2.82) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (OR = 1.68; 95% CI = 1.03–2.75). Our results support a meat mutagen exposure hypothesis as a potential mechanism for recurrence of clinically significant adenomatous polyps.

Abbreviations: BaP, benzo(a)pyrene; CHARRED, computerized heterocyclic amines resource for research in epidemiology of disease; CI, confidence interval; DiMeIQx, 2-amino-3,4,8-trimethylimidazo[4,5,-f]quinoxaline; HCA, heterocyclic amine; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NOC, N-nitroso-compound; OR, odds ratio; PAH, polycyclic aromatic hydrocarbon; PhIP, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine; UDCA, ursodeoxycholic acid


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Correlational studies show strong positive associations between national per capita animal fat and meat intake and colorectal cancer (1,2). Results of an earlier meta-analysis showed significant positive associations between red meat consumption and colorectal cancer (3), and a more recent summary of prospective studies also found significantly increased risk of colorectal cancer associated with higher red meat intake (4). Higher intake of beef was also shown to be positively associated with colorectal adenomas based on a systematic review of published data, but this was not statistically significant (5).

The mechanisms responsible for the tumorigenic effect of red meat in the colorectum in humans are uncertain, but have been linked in animal and epidemiologic studies to the role of meat in the diet as a source of total or saturated fat, carcinogens or iron (reviewed in ref. 6). Results of several studies indicate that regular consumption of well done or pan-fried meat is associated with increased risk for colorectal or rectal cancer (7–10). Additionally, published reports suggest that risk of colorectal cancer or adenoma may be increased among individuals who consume meat with a heavily browned surface, but not among those who consume meat with a medium or lightly browned surface (8,9,11,12). Studies have also demonstrated that when meat is cooked at high temperatures for a long period of time, mutagenic heterocyclic amines (HCAs) are formed (13,14). Further, the process of grilling certain meats over a direct flame has been associated with greater production of the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (BaP) (15). To date, null or weak positive associations have been shown for individual measures of HCA, PAH or mutagenicity exposure and colorectal cancer (9,10,16). Available data on meat intake and meat mutagens in relation to adenoma end points (11,17–19), including adenoma recurrence (20,21), show weak positive associations or null effects.

Individuals who undergo colonoscopy and polypectomy provide a unique study population for the prospective analysis of metachronous lesions, as is the case for participants in adenoma recurrence studies. Furthermore, depending on the study size, it is possible to assess recurrence of more clinically important lesions, such as large, advanced and/or multiple adenomas. In all, 20–50% of individuals with adenomas recur within a 3- to 5-year period, of whom 10–20% will probably recur with advanced adenomas, defined as adenomas of diameter ≥10 mm, villous histology and/or high-grade dysplasia (22). The clinical relevance of these end points are underscored in the colorectal cancer surveillance guidelines, where individuals who are found to have large adenomas, adenomas with high-grade dysplasia or any villous component, or those with multiple lesions are advised to undergo a follow-up exam sooner (within 3 years) than those presenting with single, small tubular adenomas with low-grade dysplasia (5–10 years) (23).

We assessed the relation between meat types, meat preparation methods, meat doneness, meat-derived HCAs, BaP, mutagenicity exposure indices and colorectal adenoma recurrence. In addition, we examined these associations for different adenoma end points, including advanced and multiple recurrences. We hypothesized that individuals who consume higher quantities of meats enriched for HCAs and other mutagens were more likely to recur with new adenomas than those who consumed lower quantities of these compounds.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Study population
Analyses were conducted in a subset of participants from the ursodeoxycholic acid (UDCA) trial, the details of which have been reported elsewhere (24). Briefly, the UDCA trial was a double-blind controlled trial conducted to compare the effect of UDCA versus placebo on adenoma recurrence among individuals who had undergone colonoscopy and had one or more adenomas removed. Participants were randomized between 1996 and 2000 and were followed for an average of 31.0 months. We excluded individuals with a personal history of inflammatory bowel disease or hereditary colon cancer syndromes. A total of 1285 participants were randomized to either the treatment or placebo group. Of these, 1192 participants (92.8%) completed the study by undergoing one or more follow-up colonoscopies 6 months or more after qualifying exam. The UDCA intervention had no significant effect on adenoma recurrence (24). Of the 1192 study participants, 869 (73%) individuals filled out an Arizona Cancer Center Meat Preparation Questionnaire at baseline and comprise the current study population. The remaining 323 participants did not have the opportunity to provide this information because their follow-up had already initiated when the present substudy began. There were no significant differences with regard to risk factors of interest (i.e. age, gender, etc.) between the 1192 participants and those that made up the substudy population for the present analyses (data not shown). Written informed consent was obtained from each subject. The study was approved by the University of Arizona Human Subjects Committee and the Institutional Review Board.

Study end points
Adenoma recurrence was defined as detection of one or more colorectal adenomass or a colorectal cancer occurrence 6 months or more after randomization. We defined advanced recurrences as adenomas that were 1 cm in size or larger and/or those with a tubulovillous or villous histology; this group also includes colorectal cancers that occurred during the follow-up period. Recurrence of more than a single adenoma in the same patient during follow-up examinations was defined as multiple. We categorized the location of the adenoma recurrence into proximal, distal and both locations. Distal recurrence was defined as anatomical location in the distal colorectum that extends from the rectum through the splenic flexure, whereas proximal location was defined as that proximal to the splenic flexure.

Assessment of meat variables
We assessed intake of total meat at baseline using the Arizona Food Frequency Questionnaire, a self-administered, semi-quantitative food frequency questionnaire that asks about usual dietary intake in the previous year. Intake of red meat includes beef, pork, hot dogs, ham, bacon, sausage, veal, venison, hamburgers, cheeseburgers and meat loaf. Intake of poultry and fish includes chicken, turkey and fish. Processed meat intake includes bacon, sausage, hot dogs, ham, bologna, salami and lunch meat. Estimates of total meat, red meat, poultry, fish and processed meat in this analysis are calculated from Arizona Food Frequency Questionnaire responses.

The Arizona Cancer Center Meat Preparation Questionnaire was used to assess consumption of red meat and poultry with respect to food preparation methods and degree of doneness over the previous year. This instrument was developed to be used as a self-administered questionnaire using the National Cancer Institute's interview-administered questionnaire (25). The questionnaire includes 10 meat, poultry and fish items and asks about frequency of consumption and portion size of each in the previous year. In addition, for each meat item the cooking method was reported as follows: pan-fried, grilled/barbecued and oven broiled for red meat; baked/roasted, stewed, oven broiled and grilled/barbecued for chicken or turkey; and deep fat fried and pan-fried for chicken. Participants were provided with color photographs of steak, hamburger, chicken, pork chops and bacon cooked to different levels of doneness that included rare, medium, well done and very well done. We derived individual daily estimated intakes of HCAs, PAHs and mutagenicity from meat using the National Cancer Institute's database known as the computerized heterocyclic amines resource for research in epidemiology of disease (CHARRED) (http://www.charred.cancer.gov/). Using consumption frequency and portion size, we estimated gram intake of each meat item (steak, hamburger patty, pork chops, bacon, etc.). We further computed daily intake (nanogram) of individual HCAs, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5,-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b] pyridine (PhIP) as well as BaP and mutagenicity (revertant colonies/day), by taking into account the type of meat, cooking technique and doneness level (written response and photographs). As previously reported (26,27), estimates of exposure levels of HCAs in cooked meats in the CHARRED database are based on direct measurements of HCA compounds present in meat samples prepared under different cooking practices and temperatures using solid-phase extracts analyzed by high performance liquid chromatography. BaP was estimated from direct measures of meat preparations using thin-layer chromatography (15). The Salmonella typhimurium strain TA98 standard plate incorporation assay was used to derive the mutagenic activity of meat sample extracts (28). The presence and relative amounts of many of the HCAs measured by Sinha et al. (26,27) and used in the construction of the CHARRED database are consistent with the findings for meats cooked in a similar manner by other researchers (29).

Risk factor and covariate data
Self-administered questionnaires were used to obtain data on sociodemographic variables, family history of colorectal cancer in first-degree relatives, history of polyps prior to the baseline examination, aspirin use and cigarette smoking. In addition, we considered baseline adenoma characteristics as potential confounding variables since these have been shown to be significant predictors of adenoma recurrence in this study population (22). Data on adenoma characteristics (i.e. number, size, location and histology) were obtained from the medical record and the pathology report, as previously reported (22).

Statistical analysis
We calculated the mean intake of each HCA, BaP and mutagenicity contributed from various commonly consumed meats and cooking methods and assessed the percent contribution. We assessed the main effects of meat consumption, red meat cooking methods, red meat doneness, HCAs and other meat mutagens on adenoma recurrence by unconditional logistic regression (30). We categorized the continuous variables into tertiles based on the distribution in the total population. For broiled, baked and microwaved red meat, the percentage of people reporting 0 g was considerably >50%; therefore, for each of these variables the first category includes all participants reporting 0 g, and the median consumption for the remaining participants defined the cut point between the second and third categories. Modeling of adenoma recurrence end points (i.e. advanced versus non-advanced and multiple versus single) was conducted by polytomous logistic regression using maximum-likelihood multinomial logit models.

A priori, we considered potential confounding variables including age, sex, history of previous polyps and number of colonscopies performed during the trial. We then evaluated additional covariates, including dietary, non-dietary and baseline adenoma characteristics shown in Table I. None of these additional covariates generated a substantial change in the main effects [i.e. >10% difference in the odds ratio (OR)] in multivariate models, and thus were not included in our final models. In addition, although the trial intervention had no effect on overall adenoma recurrence, inclusion of a covariate for treatment effect made no difference in the results of our final multivariate models. Tests for trend were conducted by modeling tertile median exposure values. All analyses were done using the Stata Version 9 statistical package (College Station, TX).


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  		Table I. Baseline characteristics of study population according to tertile of total meat intake

 

   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Table I presents age- and gender-adjusted study population characteristics in relation to tertiles of total meat intake. Participants in the upper tertile of meat consumption were younger and more likely to be male as compared with those in the lower tertile. All selected dietary variables and alcohol consumption were positively correlated with total meat intake. Individuals who consumed more meat were less likely to be current smokers and more likely to have a higher body mass index than those who consumed less meat.

We calculated mean mutagenicity, DiMeIQx, MeIQx, PhIP and BaP contributed from various commonly consumed meats and cooking methods, as described in the Materials and Methods (Table II). For mutagenicity, several pan-fried and grilled meats contributed to its intake but the highest contributor was grilled chicken. Grilled hamburgers were among the highest contributors to DiMeIQx and MeIQx, although several additional meats had high content of these HCAs. Consumption of grilled steak, pan-fried and grilled chicken accounted for almost 75% of the estimated PhIP intake in this group of study participants. As well, grilled steak and chicken almost entirely constituted the estimated total BaP dietary intake from these meat sources.


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  		Table II. Intake of mutagens and percent contribution (%) from meats and cooking methods

 
Table III shows the relation between meat consumption and adenoma recurrence, including advanced and multiple recurrences. Although no association was shown for total meat, red meat or poultry and fish intake, a positive non-significant association was shown for individuals in the upper versus the lower tertile of processed meat and adenoma recurrence. When we assessed the relation of meat intake by advanced and non-advanced recurrences, no associations were shown for any of the meat characteristics and non-advanced recurrence. For advanced recurrence, all associations were slightly stronger than those for non-advanced lesions, but none was statistically significant. With the exception of poultry and fish intake, stronger associations were observed for recurrence with multiple compared with single lesions. Furthermore, participants in the upper tertile of processed meat intake had significantly higher odds of recurring with multiple adenomas than those in the lower tertile [OR = 1.83; 95% confidence interval (CI) = 1.10–3.04].


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  		Table III. Association between meat consumption and adenoma recurrence (N = 869)

 
For the association between red meat cooking method and adenoma recurrence, positive but not significant associations were shown for overall recurrence and pan-fried (OR = 1.33; 95% CI = 0.93–1.89) as well as broiled meat (OR = 1.40; 95% CI = 0.91–2.16) (Table IV). The relation between pan-fried meat and advanced recurrence was stronger than that for non-advanced adenomas and was statistically significant (OR = 1.85; 95% CI = 1.10–3.13). A significant higher odds of recurrence with multiple lesions was also shown for higher intake of pan-fried meat (OR = 1.63; 95% CI = 1.01–2.61), whereas no association was observed for recurrence with only a single lesion. Weakly positive, but non-significant, associations with overall recurrence were shown for higher versus lower tertile intake of rare/medium and well/very well done meat. However, stronger and significant associations were shown for well/very well done red meat and advanced adenoma recurrence (OR = 1.71; 95% CI = 1.02–2.86) as well as recurrence with multiple adenomas (OR = 1.68; 95% CI = 1.03–2.74).


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  		Table IV. Association between red meat cooking methods, meat doneness and adenoma recurrence (N = 869)

 
We next assessed intake of HCAs, mutagenic activity and BaP and adenoma recurrence based on estimated values using the CHARRED database (Table V). Non-significant positive associations were shown for mutagenic activity, DiMeIQx and MeIQx and overall recurrence; no positive associations were observed for PhIP and BaP. For advanced recurrence, the associations were stronger for DiMeIQx (OR = 1.59; 95% CI = 0.95–2.67) and MeIQx (OR = 1.60; 95% CI = 0.95–2.69). Recurrence of multiple adenomas was significantly associated with higher intake of DiMeIQx (OR = 1.74; 95% CI = 1.07–2.82) and MeIQx (OR = 1.68; 95% CI = 1.03–2.75). When we conducted the multivariate models including red meat as a covariate, we observed slight attenuations in the point estimates but the overall trend of the associations remained unchanged (data not shown).


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  		Table V. Association between mutagenicity, heterocyclic amines, BaP and adenoma recurrence (N = 869)

 
In secondary analyses, we assessed the relation between high-risk combinations of the variables that included the specific cooking method, preparation and well/very well done red meat. Results of these analyses show only slightly stronger associations for the various combinations tested than those observed for the single variables shown in the tables, with the exception of consumption of the combination of pan-fried and broiled red meat, where the ORs (95% CI) for recurrence of advanced lesions were 1.36 (0.79–2.35) and 2.43 (1.44–4.11) for the second and third tertiles, respectively, compared with the first tertile. Furthermore, given that anatomic site-specific differences in relation to meat intake have been proposed, we were interested in assessing the effect of the selected meat variables in relation to the location (proximal or distal) of the recurrent lesion within the colorectum. Although not entirely consistent across all characteristics, the observed stronger positive associations between red meat, processed meat and grilled meat and adenoma recurrence appeared to be restricted to the distal and not the proximal colon (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
In our analyses of colorectal adenoma recurrence, most meat variables assessed were positively associated with recurrence of any lesion, albeit these associations were weak and imprecise. However, when we assessed these variables in relation to recurrence of advanced and/or multiple adenomas, stronger and significant associations emerged for intake of processed meat, pan-fried red meat, well/very well done red meat, as well as intake of the HCAs, MeIQx and DiMeIQx.

Intake of red meat has been shown to be positively associated with risk of colorectal cancer and adenoma but the effect is modest. Although our results lack precision, the point estimates for advanced and multiple recurrence are consistent with those reported in previous meta-analyses of red meat consumption (3,4). We also observed a significant positive association between higher intake of processed meat and recurrence of multiple adenomas. These findings are in agreement with summary estimates from 22 case–control and seven cohort studies, which show a significantly higher risk of colorectal cancer associated with higher consumption of processed meat (OR = 1.29; 95% CI = 1.09–1.52 for case–control studies and Relative Risk = 1.39; 95% CI = 1.09–1.76 for cohort studies) (3). Few studies of the effect of processed meat on adenoma have been published and these show mixed results (11,18); however, one study showed a significant higher risk of advanced adenoma recurrence associated with higher processed meat consumption (20).

Several mechanisms for the increased risk of colorectal neoplasia associated with higher meat intake have been proposed; however, sound evidence in support of these is limited in human studies. Cooking method and doneness of meat affect mutagen and carcinogen levels (31,32). As noted earlier, risk of colorectal cancer or adenoma has been shown to be increased among individuals who consume meat with a heavily browned surface (8,9,11,12,33).

A prominent biological mechanism proposed for the adverse effect of higher meat consumption and specific cooking methods relates to the enhanced production of HCAs and other mutagens during certain cooking processes such as pan frying and grilling. Three decades ago, Sugimura et al. (34) demonstrated that charred parts of broiled meat contained highly mutagenic activity. Numerous animal and cell culture studies have confirmed this early work (6) with a number of studies showing that when meat is cooked at higher temperatures for a long period of time, genotoxic HCAs are formed from creatine or creatinine via the Maillard reaction (13,14). The formation of these reactive species results in covalent modification of the DNA or DNA adduct formation at guanine bases, which if unrepaired promote mutation. Our results are consistent with this line of evidence and show that consumption of well or very well done red meat is associated with a higher likelihood of recurrence of advanced and multiple adenomas, which is consistent with findings from a recently published study of adenoma prevalence (19). Although this positive association between adenoma risk and high-temperature cooked meats appears consistent, it remains unclear if the risk can be directly attributed to specific HCAs as these indirect measures cannot provide definitive evidence. Additional studies have reported a higher risk of colorectal or rectal cancer (7–10,35) or adenoma (11) associated with consumption of well done or fried meats, although there are inconsistencies (36,37). Our results also show significantly higher odds of recurrence of advanced and multiple lesions associated with higher intake of pan-fried meat, as is the case for several (810) but not all published studies (1238). It has been proposed (10) that frying temperature may account for the differences in findings across studies for pan-fried meat. Our results do not support a positive association between intake of grilled red meat, a method that often results in high content of HCAs (12,26) and BaP (6), and adenoma recurrence. However, in our study we collapsed well done and very well done red meat due to sample size limitations; this along with excluding information on meat cut type may have limited our ability to detect an effect, given the importance of temperature during cooking and meat cut type in the formation of HCAs and BaP (26). Nevertheless, we did detect a modest positive association for broiled red meat and total and advanced recurrence, although the results were imprecise.

Over the past decade, several HCAs have been isolated from cooked meat (14). Earlier epidemiological studies were unable to directly assess exposure to these carcinogens due to inadequate dietary questionnaires that lacked information on meat cooking methods or degree of doneness. Results of laboratory and animal studies have demonstrated the mutagenicity and carcinogenic potential of these products (14,39). To date, published epidemiological studies have yet to provide consistent support for the meat doneness carcinogen hypothesis; studies of colorectal cancer and adenoma show weak or null associations. It is difficult to compare across these observational studies, given the lack of uniformity in the list of meats and meat preparation methods included in the questionnaires.

Although we observed positive associations for total mutagenicity, DiMeIQx and MeIQx and overall recurrence, none was statistically significant. However, we found significant positive associations for higher intake of DiMeIQx and MeIQx and multiple recurrent adenomas and borderline significant associations for advanced recurrences. Positive associations between DiMeIQx and colorectal neoplasia have been reported (11,17,18) but the risk estimates have been weak and imprecise, with the exception of one large case–control study of colorectal cancer (10), where DiMeIQx had the strongest association of all the meat-derived compounds. Data on MeIQx are similar to those of DiMeIQx but no statistically significant associations have been reported (10,11,17,18). It is important to note that the CHARRED database estimates exposure levels for only three high-interest HCAs and one high-interest PAH. Thus, it is possible that in our studies DiMeIQx or MeIQx may not necessarily be the etiologic agents but rather act as proxies for other etiologically relevant compounds (40,41).

In addition to cooked red meats, consumption of high levels of processed meats have long been implicated in human colorectal carcinogenesis. Processed meats such as bacon, sausage and ham contain preformed N-nitroso-compounds (NOCs) as well as nitrites and nitrates, which can be converted to NOCs in vivo (42). The carcinogenicity of NOCs, at a variety of anatomical sites, is well established in animals (43) though direct evidence that NOCs in food products act as carcinogens in humans remains elusive (44). As noted earlier, higher intake of processed meats is associated with a modest 20–30% increase in colorectal cancer risk based on a summary of 14 prospective studies (3,4). Our results lend further support to a positive association between high consumption of processed meats and colorectal carcinogenesis, with higher intake of processed meats associated with higher odds of recurrence of multiple adenomas. Given the fairly consistent nature of the association between processed meat consumption and cancer risk in the colorectum, development and validation of dietary instruments to estimate NOC exposures, similar to or within the context of CHARRED, may be a useful next step to identify NOCs as contributors to risk of cancer.

Total mutagenic activity is a biological measure that integrates all classes of mutagens according to their mutagenic potential. Using total mutagenic activity is thought to be a superior measure compared with the sum of different HCAs (45) since the latter does not take into account different mutagenic potentials of each compound and is weighted by the HCA content that is most abundant in meat. However, although we observed positive associations for mutagenicity and all recurrence end points, the ORs lacked precision, similar to what has been reported in the literature (9–11,18). Methods based on approximating HCA and PAH tumor potential from bacterial-derived mutagenic potency have been suggested to inadequately reflect carcinogenic potency (46), as these fail to capture information on bioactivation, cell uptake and retention and other determinants of in vivo tissue-specific exposures (41,47,48). This is one potential limitation of the CHARRED database. However, the use of the CHARRED database is supported by the early work of Hatch et al. (49), who assessed the mutagenic and carcinogenic potencies of 10 HCAs and 24 aromatic amines. Results of these studies show that mutagenic activity in bacterial models correlate with carcinogenicity in rodent studies for a number of the HCAs with notable exceptions. For example, PhIP is among the most mutagenic of the HCAs in mammalian cell systems (41,50) and a strong colon carcinogen in rodents (51) but a weak acting mutagen in the Salmonella based assay (50). Thus, whereas the ability of mutagenic potency derived from short-term bacterial assays to approximate carcinogenic activity and hazard levels with exposures such as those assessed in the present study is accepted, the appropriate caveats associated with the use of mutagenic potency as a proxy for carcinogenic potency should be considered when interpreting these results.

Consistent with results of several published studies (10,17–19), we found no effect of PhIP on any of our adenoma end points. Although PhIP can be formed in abundant amounts in grilled and pan-fried meats, especially those that are very well done, there may be large variability in its formation especially at the extreme level of doneness. In addition, active metabolism and differential bioavailability (47) may influence individual exposure and susceptibility. Larger studies that allow analysis to be restricted to consumers of very well done meat along with studies that include information on genetic variation thought to influence PhIP carcinogenicity in humans may improve detection of suspected associations between meat preparation, carcinogen exposures and risk in epidemiologic studies (52).

In our study, we found no increased odds of recurrence in any of our adenoma end points associated with measures of BaP intake. BaP is a PAH, which is produced during the process of grilling meat over a direct flame. Few epidemiological studies have assessed BaP intake and colorectal adenoma or cancer and results are mixed (10,11,17,19); however, in the study by Butler et al. (10) a significant 2-fold increase in the risk of colorectal cancer was shown for African Americans.

We were intrigued by the relative consistency of our findings across the different adenoma end points, which show that a number of meat variables and meat mutagens have impact on recurrence of more clinically significant lesions, particularly multiplicity. These results suggest that certain meat mutagens and meat preparation methods increase the odds of recurring with specific types of lesions. However, our data do not agree with those of the large Prostate, Lung, Colon and Ovarian study, where stronger effects were shown for non-advanced single lesions, albeit the associations were not statistically significant (11). In a recent publication (19), only intake of well done meat was associated with higher risk of large but not small adenomas among the various meat or meat mutagen variables.

Strengths of our study include the prospective nature of our data, which allows the assessment of adenoma formation and recurrence of clinically important lesions. In addition, the use of a separate, comprehensive questionnaire that includes photographs to ascertain meat doneness is an important strength of our ability to estimate exposure levels. However, in spite of this, the questionnaire does not take into account all contributing factors to HCA and mutagen content (i.e. specific cut of meat, use of marinades, microwaving prior to cooking, frequency of flipping meat, etc.). Limitations related to studies of adenoma recurrence include a short length of follow-up and inability to assess the onset of first-time adenomas. In addition, it is well recognized that all observational studies of meat intake and cancer risk are challenged by the high correlation of the HCAs with other dietary factors and mutagenic agents, which is not always possible to disentangle in statistical models. Lastly, it remains widely believed that polymorphisms in key enzymes involved in the metabolism of aromatic and heterocyclic aromatic amines contribute to individual susceptibility to the HCAs and PAHs and may explain differential susceptibility to dietary and environmental HCAs (53). We will be pursuing these interactions in future studies utilizing the derived HCA metric from dietary intake and meat preparation to estimate exposure.

In conclusion, our results show no significant associations for meat intake and adenoma recurrence. Our data also show null to weak positive associations between mutagenicity and HCAs assessed in relation to overall colorectal adenoma recurrence. Importantly, our results show significantly higher odds of recurrence of advanced and/or multiple adenomas among individuals with higher intake of processed meat and pan-fried red meat. We found evidence in support of the meat doneness hypothesis that implicates meat mutagens as relevant human carcinogens, where we observed significant associations with recurrence of advanced and multiple lesions for well/very well done red meat as well as significant associations between recurrence of multiple adenomas and DiMeIQx and MeIQx. These data support meat processing methods, such as charring and curing, as important modifiable dietary exposures that if avoided may reduce the recurrence of colorectal adenomas, particularly large and multiple lesions. Future modification of CHARRED to capture exposures to recently characterized meat HCAs (40) and/or inclusion of determinants of NOC exposures may help to further clarify food preparation methods and meat types, which act as primary sources of diet-derived carcinogens.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Public Health Service grants (CA-41108, CA-23074) and the Specialized Program of Research Excellence in Gastrointestinal Cancer from the National Cancer Institute (CA95060); KO7 Career Development Award from the National Cancer Institute (CA106269) to E.T.J.


   Acknowledgments
 
We wish to thank E.Graver, J.Guillén-Rodríguez, V.Hartz and R.Whitacre for their expert assistance. We are indebted to the staff at the Phoenix and Tucson clinic study sites as well as the Analytical Core staff for their valuable contribution.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received May 31, 2007; revised July 23, 2007; accepted July 27, 2007.


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S. Rohrmann, S. Hermann, and J. Linseisen
Heterocyclic aromatic amine intake increases colorectal adenoma risk: findings from a prospective European cohort study
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