Table 3.

Protection conferred by other dietary components

Study	Animal/Treatment	Diets	Meat	Carcinogen used	Findings	Conclusions
Allam et al.48	Fischer 344 rats Female 4 weeks old Study 1 (short term): 8 diets; n = 5/diet Study 2 (short term): 5 diets; n = 5/diet Study 3 (long term): 2 diets; n = 20/diet Fed 14 days (short term) Fed 3 months (long term)	Modified AIN76 diet Study 1: 60% meat (wet wt), freeze dried, and low calcium (20 mmol/kg) supplemented with calcium phosphate at 33, 55, 90, 150, 250 mmol/kg, or calcium carbonate or calcium gluconate at 250 mmol/kg; Study 2 calcium carbonate diets with hemoglobin(0.63 g/100 g diet); Study 3 calcium carbonate (33 and 100 mmol/kg) versus control	freeze dried beef meat (∼0.6 mmol heme/g meat)	Study 3 - 1,2-dimethylhydrazine (precursor of AOM)	In short term study, 150 and 250 mmol/kg calcium phosphate reduced fecal water cytotoxicity and TBARS. Calcium carbonate was more effective than calcium phosphate. In long term study, a diet containing 100 mmol/kg calcium carbonate did not promote ACF in the colon 101 d after a dimethylhydrazine injection	Calcium carbonate effectively mitigated impact of heme intake
Belobrajdic et al.49	Pigs Male 9 weeks old 2 diets; N = 5/diet Fed 4 weeks	Red meat control; red meat with arabinoxylan-rich fraction (AXRF) from wheat (370 g/kg diet)	Beef steak trimmed of fat (300 g/kg diet) Cooked until lightly browned, minced, frozen	None	AXRF supplemented pigs had increased SCFA in cecum compared to controls; AXRF lowered cecal pH and raised colon pH; AXRF lowered colonocyte DNA damage; cecal digesta p-cresol concentrations were lower with AXRF; AXRF group had lower phenol concentrations in the mid and distal colon compared to the control; AXRF reduced abundance of Prevotella clusters, Bacteroidetes phylum, and Clostridial clusters; control group elevated species of Proteobacteria phylum, Fusobacteria, Clostridial clusters I, II, XI, Bacteroidetes fragilis, and B. distasonis	There may be a beneficial effect due to consuming AXRF with meat in the diet.
de Vogel et al.50	Wistar Rats Male 8 weeks old Study 1: 4 diets; n = 8/diet Study 2: 4 diets; n = 8/diet Fed 14 days	Study 1: control, heme, spinach, heme + spinach Study 2: control, heme, heme + spinach, heme + chlorophyll	Heme (0.5 mmol/kg diet)		Heme fed rats had a 50% increase in rate of DNA replication compared to controls; spinach inhibited heme induced colonocyte proliferation; heme fed rats showed evidence of necrosis in surface cells; heme excretion increased with the addition of spinach; chlorophyll mimicked the effect of spinach in preventing heme induced epithelial proliferation; spinach and chlorophyll both inhibited the increased cytotoxicity of fecal water seen with heme treatment; chlorophyll blocked the formation of the “heme factor” almost completely (this effect was similar with spinach)	Spinach and chlorophyll inhibited heme induced stimulation of colonocyte proliferation; spinach, and the chlorophyll it contains may protect against changes induced by high levels of heme
de Vogel et al.50	SPF Wistar Rats Male 8 weeks old 5 diets; n = 8/diet Fed 2 weeks	Control, Heme, Heme + Na-chlorophyllin, Heme + Na, Cu-chlorophyllin, Heme + chlorophyll	Heme (0.5 mmol/kg diet)		Heme supplementation increased proliferation compared to controls; supplementation with natural chlorophyll but not chlorophyllins inhibited the heme effects on proliferation; Na-chlorophyllin decreased heme-induced cytotoxicity of fecal water and natural chlorophyll completely blocked heme induced cytotoxicity; heme excretion was low in the control, heme, and heme + chlorophyllin groups but chlorophyll groups had a >50% heme detection in the feces; TBARS of heme and chlorophyllin groups were increased 1.5-2 fold but chlorophyll addition reduced the formation of lipid radicals	Natural chlorophyll is able to induce a protective effect in a heme diet but chlorophyllins are not as effective
O’Callaghan et al.51	Sprague-Dawley rats Male Adult 12 diets; n = 8/diet Fed 4 weeks	15, 25, 35% red meat, 13, 22, 30% white meat, with or without HAMS	Beef steak trimmed of fat Cooked at 150℃ until lightly browned, dried at 45℃ for 48 h		Colonocyte telomere length decreased as the % of red meat increased and colonocyte telomere length was shortest with the highest red meat % and no HAMS treatment; addition of HAMS prevented telomere length shortening in the red meat group; white meat also showed a dose dependent decrease in telomere length but it was not significant; HAMS resulted in longer telomeres; telomere lengths were not different between the 15% red and 15% white meat groups; red and white meat increased DSB and SSB dose dependently and damage was greater with red meat; HAMS induced more SCFA production and lowered the concentrations of phenols and cresols; increased MDA concentrations was correlated with shorter telomeres; MDA and acetate concentrations impacted telomere length more than other variables studied	Increased red meat intake shortens colonocyte telomeres and RS is able to attenuate this reduction; levels of oxidative stress are related to the shortening of colonic telomeres; increased SCFA from RS is associated with a reduction of MDA levels and a decrease in telomere shortening and DNA damage
Paturi et al.52	Sprague-Dawley rats Male 4 weeks old 6 diets; n = 10/diet Fed 8 weeks	Cellulose, potato fiber, or potato-resistant starch for 2 wk without beef (Phase I diets), followed by feeding the same diets for 6 wk with 25% cooked beef (Phase II diets)	Cooked beef, 76.7% protein and 21.6% fat (DM basis)	None	Potato fiber resulted in lower Bacteroides-Prevotella-Porphyromonas. Colonic Bifidobacterium spp. and/or Lactobacillus spp. were higher with potato fiber and potato-resistant starch than with cellulose. Beneficial changes were observed in SCFA concentrations in response to potato fiber. Phenol and p-cresol concentrations were lower in the cecum and colon with potato fiber. An increase in goblet cells per crypt and longer crypts were found in the colon of rats fed potato fiber and potato-resistant starch diets. Fermentable carbohydrates had no effect on colonic DNA damage.	Potato fiber or potato-resistant starch has distinctive effects in the large bowel when fed in combination with red meat. Consuming nondigestible carbohydrates along with red meat is likely one of several dietary factors that contribute to maintenance of normal colonic health.
Pierre et al.22	Fisher 344 Rats Female 4 weeks old 8 diets; n = 10/diet Fed 100 days	Control (low Ca), Beef (Low Ca), control + Ca, Beef + Ca, control + olive oil, beef + olive oil, control + antioxidant, beef + antioxidant	Beef meat contained 0.6 umol/g heme; 60% of diet (wet wt, freeze-dried)	1,2-dimethylhydrazine	Calcium suppressed beef-meat induced ACF and MDF promotion but it did not reduce mean number of crypts per lesion; antioxidants and olive oil reduced MDF number but not to the extent of calcium and these factors did not affect ACF number; the high calcium control diet (no meat) had more MDF and ACF than control low calcium diets and more crypts per lesion; beef meat plus calcium showed little heme in the fecal water; calcium almost normalized lipid peroxidation, which was not seen with the addition of olive oil or antioxidants; neither calcium or olive oil reduced DHN-MA excretion in beef fed rats, but antioxidants did slightly decrease DHN-MA excretion	Perturbations associated with hemin consumption may be prevented by improved dietary calcium levels
Pierre et al.53	Fisher 344 Rats Female 5 weeks old Study 1: 7 diets; n = 5/diet Study 2: 3 diets; n = 16, 10, 10/diet Study 1: Fed 14 days Study 2: Fed 100 days	Study 1: experimental cured meat + protective agents (rutin, carnosol, alpha-tocopherol, calcium carbonate, inulin, trisodium pyrophosphate) or none (control) Study 2: control meat diet, control + calcium carbonate, control + alpha-tocopherol	Cured meat (dark cooked pork with nitrite and oxidized) added to AIN76 diet (40% protein, 15% fat, 0.27% calcium) Cured with 2 g salt/100 g, heated to 70℃, exposed to air for 5 days	Study 1: None Study 2: 1,2 dimethylhydrazine	Study 1: All tested additives decreased fecal water TBARS and urinary DHN-MA concentrations; addition of CaCO3, rutin, or alpha-tocopherol increased survival of Apc+/+ cells compared to Apc min/+ cells; only inulin did not decrease fecal water cytotoxicty; fecal water of control + inulin was cytotoxic to the Apc +/+ but not the Apc min/+ cells Study 2: the addition of CaCO3 and alpha-tocopherol decreased MDF but not ACF per colon compared to control meat group; CaCO3 decreased all tested biomarkers but alpha-tocopherol only decreased heme concentration in fecal water and DHN-MA in the urine; fecal ATNC was decreased by both additives	The increase of early colon cancer lesions in rats by cured meat can be suppressed by dietary calcium or alpha-tocopherol; potential for cancer promotion due to cured meat could be negated by increased consumption of food rich in calcium
Toden et al.54	Sprague-Dawley Rats Male Adult 6 diets; n = 8/diet Fed 4 weeks	Either 15% casein, 25% casein, or 25% meat; +/−25% HAMS	Beef steak trimmed of fat or chicken breast trimmed of fat Cooked at 150℃ until lightly browned, dried at 45℃ for 48 hours		Cecal pH was lower with meat than casein in the absence of HAMS, but no pH difference when HAMS was included; HAMS increased fecal output relative to controls and significantly higher for the 25% meat diet group; pH of the feces was lowered by HAMS but not affected by protein type; 25% meat treatment had higher DNA damage compared to 15% casein; DNA damage was greater in non-HAMS groups; no difference in DNA damage among diets in the presence of HAMS; all SCFA were increased with the inclusion of HAMS	Substitution of red meat for casein elevates the extent of colon DNA damage; increased dietary protein (casein or beef) increases colonic DNA damage, especially in the absence of RS
Toden et al.55	Sprague-Dawley Rats Male Adult 12 diets; n = 8/diet Fed 4 weeks	Either 15, 25, or 35% cooked beef or 13, 22, or 30% cooked chicken, with or without 20% HAMS	Beef steak trimmed of fat or chicken breast trimmed of fat Cooked at 150℃ until lightly browned, dried at 45℃ for 48 hours		Dose dependent increase in colonic DNA SSB and DSB in control starch animals fed either red or white meat, and was higher for red than white meat; HAMS prevented the increase; red meat led to increased apoptosis compared to white control diets; cecal butyrate was higher in red meat HAMS (correlation between cecal butyrate and apoptosis was found)	Red meat leads to great increases in colonic DNA damage compared to white meat but HAMS protects against this damage
Toden et al.56	Sprague-Dawley Rats Male Adult 12 diets; n = 8/diet Fed 4 weeks	Either 15, 25, or 35% cooked beef or 13, 22, or 30% cooked chicken, with or without 20% HAMS	Beef steak trimmed of fat or chicken breast trimmed of fat Cooked at 150℃ until lightly browned, dried at 45℃ for 48 hours		Beef resulted in higher plasma levels of MDA relative to chicken, but levels were reduced by HAMS; leptin levels were higher with beef than chicken, and were reduced by HAMS; chicken, but not beef, increased IGF-1 and HAMS had no effect; chicken increased insulin, relative to beef diets containing HAMS; MMP-2 and TIMP-2 levels were higher with HAMS, but TIMP-2 was highest for chicken without HAMS; SSB and DSB DNA damage correlated with plasma MDA concentrations and DNA DSB correlated with colonic MDA concentrations; beef was positively associated with DSB for plasma and colonic MDA; hepatic portal plasma butyrate concentration was correlated with plasma MDA concentration	Changes in various factors that may affect cancer development were differentially affected by meat source, with some affected by beef and others affected by chicken. Including HAMS suppressed several impacts of either meat.
Van der Meer- van Kraaij et al.57	Wistar rats Male 9 weeks old 4 diets; n = 16/diet Fed 2 weeks	AIN-93 Control (20 mmol Ca/kg); heme (20 mmol Ca/kg and 0.5 mmol heme/kg); calcium (100 mmol Ca/kg); and heme + calcium (100 mmol Ca/kg and 0.5 mmol heme/kg)	None	None	Heme increased the cytotoxicity of fecal water and elevated colon epithelial proliferation. Calcium reduced cytotoxicity and inhibited heme-induced effects. Mptx was the strongest differentially expressed gene (down-regulated by dietary heme and up-regulated by calcium).	Calcium normalized many variables shown to respond to inclusion of heme in the diet.
Van Hecke et al.58	In vitro digestions, used in Caco-2, HT-29 and HCT-116 cultures 80 digests; n = 3 replicates per digest	Low fat or high fat meat diets combined with 0, 5, 10, 15, or 20 mg α-tocopherol; quercetin; silibinin; ascorbic acid; gallic acid, ferulic acid, chlorogenic acid, or caffeic acid	Beef (low fat); Beef with added pork fat (high fat)	None	Lipid peroxidation products in the digesta was higher with high fat meat, and induced greater cytotoxicity in the cell lines; lipophilic compounds were all antioxidants, but hydrophilic compounds were either antioxidants or pro-oxidants depending on the doses and fat content	Numerous dietary factors influence lipid peroxidation product formation during cooking and digestion; some mitigate cytotoxicity and genotoxicity associated with digests from high or low-fat beef/pork.
Winter et al.59	C57BL/J Mice Male 8 weeks old 6 diets; n = 12/diet Fed 4 weeks	Low casein; high casein; low casein + RS; Low meat; High meat; High meat + RS	Lean, minced steak Cooked at medium temp with mixing to prevent burning; oven dried, ground into a powder		No affect of protein level on fecal pH; red meat increased p-cresol, propionate and total SCFA concentrations compared to casein; RS lowered pH, ammonia, and phenol concentration and increased SCFAs; no effect of amount or type of protein on cell proliferation or apoptosis; RS increased proliferation and reduced apoptosis; DNA adducts were higher in mice consuming red meat compared to casein but protein amount had no effect; RS reduced DNA adduct formation; there was a positive relationship between DNA adduct formation and the levels of p-cresol and fecal pH; propionate, butyrate and total SCFA correlated inversely with distal colon apoptosis; and apoptosis positively correlated with fecal pH	High protein diets increase DNA adducts in the colon and the type of protein has a greater effect than the amount of protein; Supplementation with fermentable CHO reduced formation of DNA adducts
Winter et al.60	C57BL/J Mice Male 8 weeks old 4 diets; n = 16/diet Fed 4 weeks (short term); 18 months (long term)	Modified AIN-76 diets (15% casein) Control, Control + 10% HAMS, Heme, Heme + 10% HAMS	Heme (0.2 µmol/g)- approximates heme content of a high red meat diet Diets placed in sealed containers and stored at 4℃	None	Long term heme mice weighed less; heme increased proliferation in short term but not long term study; in long term study there was no change in apoptosis, or DNA adducts with heme, but HAMS increased proliferation; heme lowered apoptosis in older mice, cell proliferation increased with age; heme led to increased DNA adducts	Heme increased DNA adducts and cell proliferation, and reduced apoptosis; HAMS promoted good bacteria and reduced formation of toxic products from protein over short periods but changes are not sustained over time; heme was not sufficient to induce colon cancer

ACF:: aberrant crypt foci; AIN: American Institute of Nutrition; AOM: azoxymethane; Apc min: adenomatous polyposis coli gene minus; AXRF: arabinoxylan-rich fraction; ATNC: apparent total nitrso compound; BrdU: Bromodeoxyuridine; CLA-FFA: conjugated linoleic acid free fatty acid; CLA-TG: conjugated linoleic acid triglyceride; CMT: cell media type; CRC: colorectal cancer; DHN-MA: 1,4-Dihydroxynonane Mercapturic Acid; DSB: double strand breaks; HAMS: High amylose maize starch; IFG-1: insulin-like growth factor 1; IL-6:interlukein 6; LAMS: low amylose maize starch; MDA: Malondialdehyde; SCFAs: short chain fatty acids; Mptx: Mucosal pentraxin; MMP-2: matrix metalloproteinase; SSB: single strand breaks; TBARS: thiobarbituric acid-reactive substances; TIMP-2: tissue inhibitor of matrix metalloproteinase; TNFα: tumor necrosis factor; TXB2: Thromboxane B2.