Running title: Cured Meat Promotion of ColonCarcinogenesis
Key words: cured meat, colorectal cancer, prevention, calcium, tocopherol, preneoplastic lesions, fat oxidation, cytotoxicity, N-nitroso compounds.
Financial Support: This work was supported by ANR-French Agency (ANR-PNRA Heme Cancer project), by the COST B35 (European Cooperation in Science and Technology) and by NIH Grant R01-CA-1434600 from the United States National Cancer Institute. Abbreviation: ACF, aberrant crypt foci; DCNO, dark cooked meat with nitrite, oxidized; DHN-MA, 1,4-Dihydroxynonane mercapturic acid; DMEM, Dulbecco-modified essential medium; MDF, mucin depleted foci; TBARS, Thiobarbituric acid reactive substances. Names for PubMed indexing: Pierre, Martin, Santarelli, Taché, Naud, Guéraud, Audebert, Dupuy, Meunier, Attaix, Vendeuvre, Mirvish, Kuhnle, Cano, Corpet
Our second expectation, that the products of protein fermentation promote coloncarcinogenesis, however, was not supported by our studies. If the products of protein fermentation had been the promoters, we would have expected that the levels of fermentation products would have been reflected in the degree of promotion. This was not the case. Fermentation products reached a maximum for protein thermolyzed for one hour and were lower for longer thermolysis times (Figure 2), whereas promotion continued to increase with thermolysis time through two hours (Figure 3). Furthermore the thermolysis of soy and egg white proteins led to increased levels of protein fermentation products (Figures 4 and 5) but did not promote ACF growth (Figure 5). Thermolysis of the proteins thus appears to lead to reduced digestibility and increased colonic protein fermentation in each case, but only in one case to promotion of coloncarcinogenesis. The lack of association between the toxic protein fermentation products ammonia and the phenols with colon cancer promotion was unexpected. Previous studies suggested that ammo-nia given intrarectally can act as a colon tumor promoter (12). Perhaps the sudden exposure to intrarectal ammonia leads to more toxicity than exposure to ammonia that is continuously formed from the deamination of amino acids. The slow formation of phenols could also be less toxic than acute exposures to
This study also showed that the addition of a-tocopherol into cured meat inhibited the promotion of coloncarcinogenesis in rats. The MDF-number reduction by a-tocopherol was associ- ated with the normalization of urinary DHN-MA in rats fed cured meat (Table 2). We proposed that the promotion by heme iron would have been a result of fat oxidation end products (13, 14, 22); the Apc mutation renders cells resistant to 4-hydroxy-2- nonenal, which is an end product of heme-induced fat oxidation (25), which we measured by its urinary metabolite DHN-MA. Thus, the selection of Apc mutated cells by cytotoxic peroxides would explain the heme-induced promotion of coloncarcinogenesis (41). In the current trial, in the 14 d study, the reduction in fecal TBARS by a-tocopherol, calcium carbonate, and rutin (Table 1) was associated with reduced cytotoxicity against nonmutated Apc +/+ cells, which might have explained the reduced promotion. However, this reduction in fecal TBARS and cytotoxicity was not seen in the 100-d study (Table 2), which casted doubt that cyto- toxic peroxides and the selection of Apc mutated cells would explain promotion by cured meat. In contrast, the protection by a-tocopherol was associated with reduced fecal ATNC in rats (Table 2). This association supports the hypothesis that N-nitroso compounds are the major pro-cancer molecules from cured meat, which is a hypothesis supported by the studies of Mirvish et al (8, 12, 27, 42, 43) in rodents, of Bingham et al (7, 10, 28, 44) in volunteers, and a previous carcinogenesis study from this team (6). The addition of a-tocopherol to cured meat halved fecal ATNC in rats (Table 2) but did not reduce significantly fecal ATNC in volunteers. These results suggested that vitamin E would not be sufficient to reduce cured-meat toxicity in humans. Our previous carcinogenesis studies suggested that fat peroxides such as 4-hy- droxynonenal would explain the promotion by fresh red meat (13, 14, 22, 24), whereas this study
of purines. The lesions are repaired by O 6 -methylguanine-DNA
methyltransferase (Mgmt) and by enzymes of the base excision repair (BER) pathway, respectively. Whereas O 6 MeG is well es-
tablished as a pre-carcinogenic lesion, little is known about the carcinogenic potency of base N-alkylation products such as N3- methyladenine and N3-methylguanine. To determine their role in cancer formation and the role of BER in cancer protection, we checked the response of mice with a targeted gene disruption of Mgmt or N-alkylpurine-DNA glycosylase (Aag) or both Mgmt and Aag, to azoxymethane (AOM)-induced coloncarcinogenesis, using non-invasive mini-colonoscopy. We demonstrate that both Mgmt- and Aag-null mice show a higher colon cancer frequency than the wild-type. With a single low dose of AOM (3 mg/kg) Aag-null mice showed an even stronger tumor response than Mgmt-null mice. The data provide evidence that both BER initiated by Aag and O 6 MeG reversal by Mgmt are required for protection against
This study shows that a processed meat that contains heme and nitrite, and has been cooked at 70 C and exposed to air for five days at °
4 C, can increase the number of preneoplastic lesions in rats, which suggests coloncarcinogenesis promotion. This provides the first °
experimental evidence of promotion by cured meat, and it matches epidemiological results.
Promotion of carcinogenesis was evidenced on two putative precancerous endpoints: ACF and MDF ( 39 40 , ). Results for ACF and MDF were partially discordant. Actually, all tested cured meat diets increased the number of ACF per colon compared to control diet with no meat, whereas only the dark cooked meat with nitrite, oxidized diet increased the number of MDF per colon ( [ ] Table 4 ). Several cases of contradictory results between ACF and MDF results have already been published. Colonic MDF and tumors are suppressed by synbiotic, comprising the prebiotic Raftilose (a derivative of inulin) and two probiotic strains, a Lactobacillus and a Bifidobacterium , but ACF are not ( 40 ). Colonic MDF and tumors are promoted by cholic acid but ACF are not ( 42 ). MDF thus seem better predictors of coloncarcinogenesis than ACF are. However, we decided to show both MDF and ACF data, since ACF have already been used in more than one thousand published studies ( 43 ). The MDF-promoting dark cooked meat with nitrite, oxidized meat was a cooked shoulder of pork [ ]
Dietary guidelines advise to reduce the intake of red meat and/or of saturated fat, to the benefit of "white" lean meat (39). The need to develop strong supporting data in animal models before conducting intervention trials has recently been stressed by De Luca and Ross, in a commentary on the failure of the recent alpha-tocopherol, beta-carotene prevention studies (40). The present experimental study does not support the belief that in a high-fat diet context, red meat consumption promotes, or that white meat protects against, coloncarcinogenesis. This study has nevertheless two major limitations: 1) it was done in rodents, and we do not know if AOM-initiated rats are good models for human colon cancers, and 2) the endpoint was the development of putative precancerous lesions. The multiplicity of aberrant crypt foci correlates with the adenocarcinoma incidence in most rodents studies (41-43), but not all (44). In conclusion, this study has introduced a new and potentially important experimental finding concerning a possible beneficial effect of the water intake on colon cancer prevention.
Our results showed that fresh red meat intake signiﬁ- cantly increased the size of pre-cancerous lesion MDF in initiated rats (Table 1) and the number and size of intes- tinal tumors and tumor load in Apc Min mice (Fig. 1). More precisely, fresh red meat intake signiﬁcantly increased the number of medium and large tumors (Supplementary Table S7). In the two carcinogenesis animal models, meat-induced promotion was associated with a signiﬁcant increase in fecal heme and lipoperoxidation biomarkers (TBARS, HNE in fecal water, and urinary DHN-MA in rats [Table 1]; TBARS in Apc Min mice [Fig. 2]). In mucosa, promotion in initiated rats was positively associated with the level of ferritin and annexin (Supplementary Table S6), two proteins for which expression was positively associated with the degree of dysplasia (50, 51). In our study, the level of ferritin protein in the colon mucosa correlated with the size of MDF (r ¼ 0.497, P ¼ 0.002), with the TBARS level in fecal water (r ¼ 0.511, P ¼ 0.001) and with the DHN- NA content in urine (r ¼ 0.409, P ¼ 0.013). The level of annexin protein in mucosa positively correlated with the size of MDF (r ¼ 0.452, P ¼ 0.006), with the heme content in fecal water (r ¼ 0.394, P ¼ 0.017) and with the TBARS level in fecal water (r ¼ 0.402, P ¼ 0.015). These results on fecal and urinary biomarker modulations are consistent with previous studies showing a promoting effect of hemoglobin or lyophilized red meat on pre- cancerous lesions or on tumors in association with increased fecal lipoperoxidation (16, 18–20). In human volunteers, the present crossover study showed that eating fresh red meat for 4 days was sufﬁcient to increase lipoperoxidation biomarker concentrations in fecal water, compared with the control period without meat (Fig. 4). We did not observe a signiﬁcant increase in urinary DHN-MA, which is consistent with our previous results with red meats (22). In humans, unlike in rodents, meat intake correlates with endogenous forma- tion of fecal ATNC (52, 53). In this study, we did not observe a signiﬁcant increase of fecal ATNC (Supplemen- tary Fig. S6) and the level of ATNC was low overall, but this result is consistent with other human studies in which an increase in fecal ATNC was found only in subjects consuming at least 240 g of red meat per day and for a longer consumption period (52, 54, 55). Thus, the intake of fresh red meat can modulate lipoperoxida- tion biomarkers associated with the promotion of coloncarcinogenesis in initiated rats and Apc Min mice, which gives experimental support to the epidemiology-based conclusion that red meat may be a cause of colorectal cancer (1, 2, 4).
1 Introduction: inflammatory bowel disease and colon cancer
With respect to a functional interplay between chemistry and biology, the intestinal tract is one of the most complex organs in the body. In the intestine, local epithelial cells closely interact with the body’s immune system and meet with a complex bacterial microflora and a plethora of endogenous metabolites and xenobiotic substances. All of these factors need to be tightly balanced to ensure intestinal homeostasis and to guarantee efficient uptake of nutrients and excretion of metabolites. If this balance is disturbed, pathological states can appear, such as inflammatory bowel disease (IBD). As a major risk factor for colon cancer, IBD entails chronic, relapsing inflammation of the gastrointestinal tract that affects millions of people worldwide, with >1 million new patients each year in the US alone . The paucity of safe and effective therapies for IBD amplifies its public health impact and motivates the hunt for the molecular etiology of the disease and the mechanisms linking colitis with coloncarcinogenesis. This review addresses recent developments in chemical and biological mechanisms underlying inflammation-induced colon cancer, starting with a general overview of the reactive chemical species generated during colonic inflammation followed by a discussion of the mechanistic interplay between chemical and biological mediators of inflammation and the role of microbial pathogenesis and genetic toxicology in the etiology of inflammation-induced colon cancer. The reader is referred to several recent reviews of molecular aspects of IBD and cancer development not covered here, in particular immunological and genetic studies [1-7].
The pluronics, or poloxamers, are a family of nonionic surface active agents. They are block copolymers of hydrophobe propylene oxide sandwiched between two hydrophile ethylene oxide blocks (Schmolka, 1994). 5 pluronics, PEG, and 3 other PEG-like agents with a hydrophobe moiety, were tested here (Table 1). Among the 9 agents we have tested, only the most water-soluble, PEG and PLU, were very potent against ACF (Table 1). The mechanism by which PLU decreases coloncarcinogenesis is not known.
Denis E. Corpet *, Fabrice Pierre
UMR1089 Xenobiotiques, INRA & ENVT, Ecole Nationale Veterinaire Toulouse, BP-87614, 23 Capelles, 31076 Toulouse, France
Tumours in rodent and human colon share many histological and genetic features. To know if rodent models of coloncarcinogenesis are good predictors of chemopreventive efficacy in humans, we made a meta-analysis of aspirin, beta- carotene, calcium, and wheat bran studies. Controlled intervention studies of adenoma recurrence in human volunteers were compared with chemoprevention studies of carcinogen-induced tumours in rats, and of polyps in Min (Apc(+/-)) mice: 6714 volunteers, 3911 rats and 458 mice were included in the meta-analyses. Difference between models was small since most global relative risks were between 0.76 and 1.00. A closer look showed that carcinogen-induced rat studies matched human trials for aspirin, calcium, carotene, and were compatible for wheat bran. Min mice results were compatible with human results for aspirin, but discordant for calcium and wheat bran (no carotene study). These few results suggest that rodent models roughly predict effect in humans, but the prediction is not accurate for all agents. Based on three cases only, the carcinogen-induced rat model seems better than the Min mouse model. However, rodent studies are useful to screen potential chemopreventive agents, and to study mechanisms of carcinogenesis and chemoprevention.
Our results confirm the occurrence of a lymphangiogenic switch occurring during the course of carcinogenesis. This is in line with the previous study showing a proliferation of lymphatic endothelial cells during the early stages of neoplastic progression 42 . Large and dilated lymphatic vessels appeared associated with the epithelia of dysplasic lesions. The exact mechanisms by which tumor cells recruit and invade lymphatic vessels are still unknown. Since vascular remodeling associated with lymphangiogenesis and angiogenesis seems to involve a similar process 43 , we hypothesized that PAI-1 could influence the lymphangiogenic reaction occurring in this multistage carcinogenesis model. However, the lack of PAI-1 did not affect either the lymphatic vessel density or lymphatic vessel architecture. This observation is in line with our previous study showing that PAI-1 deficiency or a pharmacological inhibitor of serine proteases did not affect the endothelial cell sprouting from thoracic duct explants in the in vitro lymphatic ring assay 44 .
This comprehensive integrative analysis of 224 colorectal tumour and normal pairs provides a number of insights into the biology of CRC and identifies potential therapeutic targets. To identify possible bio- logical differences in colon and rectum tumours, we found, in the non-hypermutated tumours irrespective of their anatomical origin, the same type of copy number, expression profile, DNA methylation and miRNA changes. Over 94% had a mutation in one or more members of the WNT signalling pathway, predominantly in APC. However, there were some differences between tumours from the right colon and all other sites. Hypermethylation was more common in the right colon, and three-quarters of hypermutated samples came from the same site, although not all of them had MSI (Fig. 2). Why most of the hypermutated samples came from the right colon and why there are two classes of tumours at this site is not known. The origins of the colon from embryonic midgut and hindgut may provide an explanation. As the survival rate of patients with high MSI-related cancers is better and these cancers are hypermutated, mutation rate may be a better prognostic indicator.
have previously performed similar orthotopic-implantation of parental MC38 cells or MC38-fLuc in the colon of B6 mice however the two profiles of CRC development seen here have not been previously described. 40 – 42 These studies have reported a rather low tumor incidence (25% to 40%) between 4 and 6 weeks post-implantation, which was explained by a poor tumor intake. However, in the absence of longitudinal monitoring of tumor growth by bioluminescence detection, these authors were unable to observe that tumor-cell implanta- tion was similar in all mice and most probably missed the rejection phase that we detected, in 70% of mice, during the second-week post-implantation (see Figure 1 ). Thus, we conclude that longitudinal monitoring of tumor progression in MC38-orthotopic model is crucial for the characterization of a spontaneous CRC rejection. In addition, we initially opti- mized CRC development by injecting 1 × 10 6 MC38 cells, which represents a lower tumor-cell dose than used in the above-cited studies (i.e. 2 × 10 6 MC38 cells). 40 – 42 Injecting three times more cells (3x10 6 ), to optimize the chances of tumor implantation, did not dramatically increase the percen- tage of mice developing a progressive CRC profile, thus imply- ing that the number of injected cancer cells has a negligible impact on the two CRC developmental profiles. Although rarely observed in B6 mice, 43 increased immunogenicity linked to the expression of luciferase in tumor cells was previously described in other tumor mouse models. 44 However, since the outcome of orthotopic parental MC38 tumors was the same as that of MC38-fLuc IC-tumors, we concluded that luciferase immunogenicity could not explain the CRC rejection profile.
of each step in the sequence of carcinogenesis. 11 Whether the metaplasia in these models is de novo, originating from the esophageal glands, is derived by entrapment of duodenal mucosa to the esophageal wall during the anastomosis, or occurs by creeping substitution (proximal migra- tion of duodenal cells through the anastomosis) remains unclear. 8,12,13 Doubt has been cast on whether these models reflect the development of BE and EA. 8 Moreover, many investigators have also reported adenosquamous or pure squamous cell carcinoma, questioning the model’s ability to elucidate mechanisms of tumorigenesis. 9,13 Finally, the respective roles of duodenal and gastroduo- denal reflux in the model remain unsolved. 5,7 These questions raise the validity of the animal model as a reliable tool in understanding the human carcinogenic sequence. 8,12,13
acid (TNBS)-induced rodent model of colitis ( 39 ). Although topiramate is known to enhance GABAergic neurotransmission, it also has a broad spectrum of action such as blocking AMPA and kainate receptors, and sodium and calcium channels as well as inhibiting carbonic anhydrase ( 40 , 41 ). Thus, it is hard to rule out the possibility that other factors contribute to the therapeutic effects of topiramate to TNBS-induced colitis. Additional studies are needed to determine the precise mechanism of topiramate in colitis. Work by Aggarwal et al. ( 42 ) recently reported that GABA level decreased in serum and colon biopsy of UC patients, although GABA A R π subunit increased significantly. The authors speculated that reduced GABA level contributed to pathogenesis of UC. One explanation for the discrepancy may be due to differ- ent models or different clinical course of the disease. In addition, as the authors had not studied the effect of blocking GABAergic signal in UC, extreme caution should be exercised when draw- ing the conclusion of GABA reduction as a contributory factor for UC. Thus, further scientific efforts are needed to clarify the specific contribution of GABAergic system in UC.