- Top of page
- Material and Methods
- Supporting Information
Diets high in red meat are established risk factors for colorectal cancer (CRC). Carcinogenic compounds generated during meat cooking have been implicated as causal agents. We conducted a family-based case-control study to investigate the association between polymorphisms in carcinogen metabolism genes (CYP1A2 -154A>C, CYP1B1 Leu432Val, CYP2E1 -1054C>T, GSTP1 Ile105Val, PTGS2 5UTR -765, EPHX1 Tyr113His, NAT2 Ile114Thr, NAT2 Arg197Gln and NAT2 Gly286Glu) and CRC risk. We tested for gene-environment interactions using case-only analyses (N = 577) and compared statistically significant results to those obtained using case-unaffected sibling comparisons (N = 307 sibships). Our results suggested that CYP1A2 -154A>C might modify the association between intake of red meat cooked using high temperature methods and well done on the inside and CRC risk (case-only interaction OR = 1.53; 95% CI = 1.19–1.97; p = 0.0008) and the association between intake of red meat heavily browned on the outside and rectal cancer risk (case-only interaction OR = 0.65; 95% CI = 0.48–0.86; p = 0.003). We also found that GSTP1 Ile105Val might modify the association between intake of poultry cooked with high temperature methods and CRC risk (p = 0.0035), a finding that was stronger among rectal cancer cases. Our results support a role for heterocyclic amines that form in red meat as a potential explanation for the observed association between diets high in red meat and CRC. Our findings also suggest a possible role for diets high in poultry cooked at high temperatures in CRC risk.
Colorectal (CRC) cancer is the third most common cancer and third leading cause of cancer death for both men and women in the United States.1 Red meat consumption has been reported as a “convincing” risk factor for CRC in a large review conducted by the World Cancer Research Fund.2 A meta-analysis of prospective studies published up to 2008 suggests that diets high in red meat or processed meat increase risk of CRC by about 20%.3 In contrast, no overall association was found between diets high in poultry and CRC risk.3 A few epidemiological studies, including our own, have taken into account cooking methods and doneness levels of red meat and poultry, and suggested positive associations between diets high in heavily browned red meat or red meat cooked using high temperature cooking methods and CRC.4–8
Carcinogens that form during the cooking or processing of meats have been postulated as potential culprits for the association between red meats and CRC risk. These include: heterocyclic amines (HCAs), polycyclic aromatic hydrocarbons (PAHs) and N-nitroso compounds (NOCs).9 High cooking temperature or prolonged duration of cooking favors the formation of HCA.9, 10 A few epidemiological studies have considered estimated levels of HCAs from diets high in well-done red meat and overall support a role for HCAs in CRC risk4, 6, 8, 11 PAHs are formed when meats are exposed to flames, such as when char-broiling and grilling,12 as well as during curing and processing of food with smoke.13 Exposure to NOCs can occur from exogenous sources, such as cured meats with nitrites, or from endogenous formation due to nitrosating agents that react with amines derived from red meat.9, 14, 15 The relative contribution of each of these 3 carcinogens to CRC is still uncertain.
Most absorbed dietary HCAs and PAHs are metabolized in the liver but are also transported back to the intestines via the bile acids and can be locally activated in the colon.16 N-nitrosamines can be directly activated in human colon.17 HCAs, PAHs and NOCs require metabolic activation before they can react with macromolecules. These carcinogens can also be detoxified and excreted, thus diminishing the amount of DNA damage induced by them. These metabolic reactions are carried out by specific combinations of Phase I and Phase II enzymes both in the liver and the colon. These enzymes vary in their metabolic activity in the human population; hence, it is biologically plausible to hypothesize that the inheritance of specific allelic variants of metabolizing genes may influence CRC risk. Whereas some epidemiological studies that focused on polymorphisms in Phase I and Phase II enzymes support this, overall results are inconclusive.18, 19 However, studies on the role of key polymorphisms in some of these enzymes jointly with meat intake, considering cooking practices and/or level of doneness, find overall support for the hypothesis that variation in metabolic enzymes might modify the effect of diets high in red meat.11, 20–27 However, few of these studies investigated potential interactions between these enzymes and diets high in poultry taking into account cooking methods.23, 24 Furthermore, most of these studies have focused on only a few of the most relevant metabolic enzymes.
In this study, we investigated the role of polymorphisms in genes encoding 7 enzymes that play key roles in the metabolism of the 3 main meat-induced carcinogens: CYP1A2 (HCA activation16), CYP1B1 (HCA28 and PAH29 activation), CYP2E1 (NOC activation30), GSTP1 (HCA, PAH and NOC detoxification31), EPHX1 (PAH activation29), PTGS2 (also known as COX-2, HCA1 and PAH32 activation) and NAT2 (HCA activation33). We considered their overall association with CRC risk and their potential modifier role on the effect of diets high in red meat or poultry, taking into account cooking practices and doneness levels. All these SNPs were chosen based on their known impact on protein function and previous reports on their role on CRC risk.
- Top of page
- Material and Methods
- Supporting Information
We investigated the role of polymorphisms in 7 metabolic enzymes that are relevant for the activation or detoxification of carcinogens formed in meats. These polymorphisms were selected due to their known impact on protein function, and due to the key roles these enzymes play in the metabolism of the main carcinogens formed in cooked red meats and poultry. Nevertheless, we cannot ignore that in our study we have conducted many different comparisons and some of these findings might be false positives due to chance. When taking into account Bonferroni corrections for multiple testing, and the comparison of IORs from proband-only analyses to those obtained from proband-sibling analyses, we found our strongest and most consistent findings were the modifier role of CYP1A2 -154A>C on the effect of red meat level of doneness on the inside on CRC risk and on the outside of red meat on rectal cancer risk, and the modifier role of GSTP1 Ile105Val on the effect of diets high in poultry cooked at high temperature on CRC risk. Overall, results were generally stronger for rectal than colon cancer.
The observed allelic frequencies of the SNPs we investigated were comparable to those previously reported.40 We did not find strong evidence for an association between any of the 6 SNPs and the NAT2 predicted phenotype and CRC risk. However, our results suggest that the CYP1A2 (-154A>C) SNP might modify the association between inside or outside level of doneness of red meat and CRC risk, with results suggesting an overall stronger effect for rectal cancer. Among individuals carrying the C allele, we found an ∼ 30% increased risk associated with diets high in red meat well-done on the inside with no such association among individuals carrying A allele. Furthermore, our results suggested that among carriers of the C allele, diets high in red meat heavily browned on the outside might increase rectal cancer risk, but not colon cancer risk. CYP1A2 is an inducible phase I metabolizing enzyme and it plays a key role in the metabolism of HCAs.16 The CYP1A2 (-154A>C) polymorphism is common among Caucasians41 and it may explain the reported variation in CYP1A2 inducibility.42 The A allele is associated with higher enzymatic activity compared with the protein coded by the C allele.42 Therefore, an effect modification of this SNP on the effect of HCAs on CRC risk is plausible. Our results suggest that the carcinogenic effects of diets high in red meat well done on the inside or outside would be greater in individuals carrying one or 2 copies of the C (slower) allele than individuals carrying 2 copies of the A (faster) allele. HCAs formation in red meat is a function of temperature and cooking time, and it is known to accumulate in meats cooked at high temperatures for longer periods of time, such as those heavily brown on the outside. Once absorbed in the colon, HCAs are rapidly transported to the liver where they can serve as substrates for N-oxidation by CYP1A2, or N-glucuronidation by UGT enzymes, or they can be converted to sulfamyl-HCAs. Sulfamyl-HCAs and HCA-N-glucuronides can be excreted back into the intestines via the bile acids, where they can be converted back into parent HCAs, which can undergo further activation into reactive species directly in the colon and rectum.43 Therefore, it is possible that slower activation of HCAs in the liver by CYP1A2 might contribute to more or longer availability of HCAs in the colorectum, by the above mentioned mechanisms. This could explain our finding of stronger effects of red meat heavily browned among individuals who carry a slower CYP1A2 allele. The finding of a stronger, or exclusive effect modification on the rectum might be explained by the fact that distal parts of the large intestine are more likely to encounter higher concentrations of the carcinogenic exposures due to increased water absorption along the colon.44 Recently, we have reported a similar finding for red meat level of doneness among carriers of polymorphisms in the XPD gene.7
In our interpretation of the CYP1A2 findings we cannot ignore that this enzyme is also able to locally metabolize NOCs in colon, even though CYP2E1 and CYP3A4 are considered the primary enzymes responsible for NOCs hydroxylation.30 Hydroxylated forms of NOCs can react and induce DNA damage.45 However, the existing evidence suggests that exposure to NOCs would occur via high intake of red meat, regardless of cooking method.14, 15 In our results, we do not observe evidence of effect of modification of CYP1A2 on total red meat intake. The effect modification seems to be most relevant for red meat heavily browned on the outside. Therefore, our findings seem to implicate HCAs more strongly than NOCs. Red meat heavily browned on the outside could also accumulate PAHs, if the meat is grilled or barbecued with flames. The fact that CYP1A2 plays a more central role in HCA than in PAH metabolism offers less support for a role of PAHs in the association between red meat heavily browned and rectal cancer risk.
In support of our findings, one previous study by Le Marchand et al. reported that the combination of the CYP1A2 and NAT2 predicted phenotypes, assessed using a caffeine-based test, exert interactions with well-done red meat, only among ever smokers.20 To best compare our findings to those of Le Marchand et al., we also investigated a potential effect modification by smoking of the observed interaction between CYP1A2 and well-done meat on the inside, analyses we consider exploratory given the sample size of our study. Similarly to Le Marchand et al.,20 we observed that the CYP1A2 x well-done meat interaction was restricted to ever smokers (interaction OR = 2.1; 95% CI = 1.42–3.06; p = <0.001) and absent among never smokers (interaction OR = 1.1; 95% CI = 0.76–1.66; p = 0.557)(case-only CYP1A2 x smoking status interaction p = 0.027). In contrast, 2 recently published studies investigating the CYP1A2 -154A>C SNP did not find evidence that this SNP modified the relationship between red meat or doneness level of red meat and CRC.22, 46 Possible explanations for the discrepancy between our study and these previous ones might include differences in meat variable definitions, and lack of stratification by tumor subsite in these previous studies. In our study findings were stronger for rectal cancer.
We also found a statistically significant interaction between the GSTP1 Ile105Val and diets high in pan-fried, oven-broiled or grilled poultry. Altogether, our results suggest that diets high in poultry cooked using high temperature methods associated with increased CRC risk only among carriers of the Ile allele. This effect modification seemed stronger for rectal cancer cases. HCAs are known to accumulate in poultry cooked at high temperature.47 GSTs are a supergene family of Phase II metabolism genes, that catalyze the binding of a large number of electrophiles to the sulfhydryl group of glutathione.48, 49 Carcinogens formed in meats cooked at high temperatures, such as HCAs and PAHs, become electrophilic after activation; therefore, GSTs become crucial in their detoxification process. Experimental studies suggest that proteins coded by the Ile allele have reduced enzyme activity compared to those coded by the Val allele,50 which has been reported to have approximately up to 3-fold activity towards PAH bay-region diol epoxides.51 Therefore, our findings are plausible as they indicate that among subjects who carry the less efficient GSTP1 enzyme, diets high in poultry cooked at high temperatures might have a more detrimental effect on CRC risk due to deficient excretion of activated carcinogens. To our knowledge, this is the first study to report a modifier role of GSTP1 in the relationship between cooked poultry intake and CRC risk.
We did not find any evidence of effect modification related to red meat or poultry intake for the polymorphisms we investigated in EPHX1, CYP1B1, CYP2E1 and PTGS2 enzymes. In contrast, one study suggested that CYP1B1 variants significantly interacts with red meat doneness intake22 and another study reported a modifier role for a CYP2E1 insertion variant on the association between processed meats and rectal cancer.21
The use of proband-only analysis allowed us to maximize statistical power by using data from all available probands regardless of the availability of siblings. Analyses of proband-sibling pairs allowed us to internally validate our results using a family-based design that eliminates the need for gene-exposure independence in addition to confounding by population admixture. However, our study has a few limitations. First, our sample size was not large enough for detecting gene-environment interactions of small effects, which we may have missed. In particular, sample size was smaller for analyses by tumor subsite. Second, we did not consider direct measures of carcinogens but instead we considered information from the questionnaire with respect to the frequency of meat intake and the meat-cooking methods, which indirectly captures the formation of the carcinogens. Last, we only considered SNPs presumed to impact protein function based on prior knowledge, rather than a comprehensive tag SNP-based approach that would capture most of the genetic variation in each gene. Therefore, we cannot discard a role for the genes for which we report no associations with overall CRC risk.
In conclusion, our findings suggest that diets high in red meat well-done on the inside or outside may increase CRC risk, particularly rectal cancer, presumably through the formation of HCAs. Furthermore, our results indicate that diets high in poultry cooked at high temperature might also be detrimental for CRC risk, perhaps through the formation of PAHs or HCAs.