In this study, we observed that consumption of more than three servings of red meat per week was associated with an increased risk of CRC. Similar findings were shown for red meat cooked by panfrying, oven broiling or grilling. When we modeled servings of red meat per week continuously, we observed an OR of 1.4 for an increase in 75 g/day of red meat consumption (~1.6 for every 100 g/day increase). The magnitude of this OR (1.6) is higher than the pooled summary statistic of 1.3 for every 100 g/day consumption of red meat in a pooled analysis of prospective studies (35
). We did not observe an association between consumption of heavily browned red meat (inside or outside) or between frequency of consumption of cooked poultry or heavily brown poultry and CRC. Further adjustment of our models by other dietary factors known to reduce CRC risk (e.g. fruits and vegetable intake) did not statistically significantly change our estimates.
In contrast to our findings, some studies (9
) reported a positive association between diets high in well-done red meat and CRC, whereas other studies reported an association only among carriers of specific metabolic phenotypes (12
). Studies done on colorectal adenomas, precursors of CRC, are also inconclusive, with one prospective study reporting an association between doneness of red meat and risk of adenomas (36
), whereas two other case–control studies failed to find an association (37
). Similarly, association studies that took into account different cooking methods and colorectal adenoma/cancer risk were inconclusive (9
). Overall, our findings suggest that components present in total red meat, and also in red meats cooked by broiling, grilling or barbecuing, may contribute to CRC risk. Our definition of total red meat included some processed meat items (e.g. sausages and bacon); therefore, our findings may suggest that NOCs present in cured meats or NOCs formed endogenously due to high red meat intake (6
), might be stronger candidates to explain the red meat and CRC association. In addition, PAHs and HCAs that formed in grilled and barbecued red meat may also explain this association. Nonetheless, we cannot discard other red meat components, such as heme-iron, that have also been suggested as cancer risk factors and that we did not evaluate in this study.
The main objective of our study was to identify potential effect modifiers among SNPs in the NER and MMR pathways. The results of our study based on case-only analyses suggest that the two SNPs we studied in the NER gene XPD
may modify the effects of level of doneness inside and outside of red meat (beef, pork, lamb and sausage), especially for rectal cancer. Our analyses using sibships generally supported these findings. In particular, our main finding was that subjects who frequently ate heavily browned red meat were at a higher risk of developing rectal cancer if they were carriers of two copies of the XPD
codon 312 Asp or XPD
751 Lys alleles but not if they carried at least one copy of the Asn321
alleles. Furthermore, our analyses considering poultry intake suggested that among carriers of the XPD
751 Lys allele, intake of poultry heavily browned on the outside might also increase risk of rectal cancer. To our knowledge, few previous studies have assessed interactions between NER genes and intake of meat or poultry in CRC risk or colorectal adenoma risk. In a Danish prospective study, Hansen et al.
) reported no interactions between intake of red meat, processed meat and white meat (fish and poultry) and genetic variants in the NER pathway on CRC risk. Berndt et al.
) did not find any statistically significant interaction between consumption of red meat and NER variants in the causation of CRC. However, both of these studies did not consider level of doneness or cooking methods, so we cannot fully compare with our results. Our finding of an XPD
by level of doneness interaction restricted to rectal cancer cases is in agreement with a study in Hawaii that reported an association between estimated levels of HCAs and rectal cancer but not with colon cancer among men (12
). It has been suggested previously that if the route of exposure to the colorectal mucosa is via the lumen, distal parts of the large intestine are more likely to encounter higher concentrations of the exposures due to the increase in concentration of lumen components as water absorption increases along the colon (34
). Therefore, our findings are consistent with this hypothesis.
It is biologically plausible that XPD
SNPs could modify the effect of well-done meats and CRC. Both carcinogens generated with charred meats (PAHs) and well-done meats (HCAs) can induce bulky adducts, which elicit NER. The XPD protein functions in NER as an adenosine triphosphate-dependent 5′–3′ DNA helicase. Its C-terminal domain (amino acids 478–759 that include codon 751) interacts with the p53 protein (24
). It has been reported that the XPD
polymorphism at codon 312, but not the one at codon 751, is associated with a 2.5-fold increase in ultraviolet-induced apoptosis among lymphoblastoid cell lines (24
). Interaction with p53 is known to reduce the helicase activity of the XPD gene, and it was hypothesized that inhibition of helicase activity may allow a stable formation of the complex of the damaged DNA and the NER machinery, resulting in a more efficient repair (42
). If either XPD
variant alleles we studied here (Asn312
), or another SNP in linkage disequilibrium with either of them, reduced p53 binding, this could explain why diets high in well-done or heavily brown red meat may have a detrimental effect among carriers of the more common Asp312
alleles, as these subjects would be less efficient in removing the damage induced by PAHs and HCAs. The literature for genotype–phenotype association studies for XPD
SNPs have been inconsistent, therefore no final interpretations can be made regarding our findings (43
). Further studies on the functional impact of genetic variants in the XPD
gene will help better understand our results.
The inherent advantage in this family-based study design is that it reduces the likelihood of confounding by population stratification. The population-based nature of this study is another advantage. Lastly, given that the USC Consortium is one of six members of the Colon-CFR, we will be able in the future to extend these studies to other centers in this large Consortium to validate and expand on our findings. Our study had three main limitations. First, 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, based on our findings, we cannot discard a potential role in CRC risk, or effect modifier role, of those NER genes for which we did not find associations. Furthermore, for the same reason, we are also unable to comment on which steps of the NER pathway might be most important for CRC risk. Second, we did not utilize summary measures of HCAs, PAHs and NOCs to determine which one might explain the association between consumption of cooked meat and CRC. Instead, we used data on frequency of intake of red meat or poultry cooked by panfrying, oven broiling and grilling, which serves as a surrogate measure for the formation of either HCAs or PAHs. The two variables that assessed level of doneness on the inside or outside of the meat may also serve as surrogates for the accumulation of carcinogens. Third, we were unable to investigate potential racial disparities in the role of meat intake, cooking practices and CRC risk, as most of the subjects in our study were white (75% probands). However, we did not find evidence that our main findings differed when restricting analyses to whites or non-whites (non-whites composition: 36% African-American, 34% Hispanics, 12% Asians, 17.5% other racial groups or unknown). Future larger studies using the entire Colon-CFR Consortium will allow us to better address any potential disparities.
In summary, our findings confirm a role for diets high in red meat as CRC risk factor and support the hypothesis that carcinogens that form in red meat heavily brown on the outside might play a role in this association, particularly for rectal cancer. Additional studies using a more comprehensive genetic approach in a larger study population as well as data from prospective studies are needed to confirm our results.