Chemoprevention of colon cancer: systematic review of preclinical studies in rats & mice.
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Azoxymethane & Dimethylhydrazine
Why? Rats and mice have almost no spontaneous colon cancer. To test diets and agents which could prevent cancer, one needs animals with tumors. This is why rodents are given a carcinogen. Commonly-used carcinogens are dimethylhydrazine (DMH) derivatives: Most published rats' studies were done in animals initiated with DMH or its metabolite AOM (237 studies out of 283 cited by Corpet & Taché, 2002).
DMH, 1,2-dimethylhydrazine CH3-NH-NH-CH3 (also called symmetrical N,N'-DMH) is metabolized in the liver to
AOM, azoxymethane . . . . . . CH3-[O]N=N-CH3 (see AOM structure aom.pdf, and aom use) Then to
CH3-[O]NH-NH-CH2OH , which leads to
CH3+ methylcarbonium ion, believed to be the ultimate carcinogen which binds stem cells DNA in the colon.
AOM supply stops: shift to DMH?
- No more AOM: in 2002, the Sigma Chemical company (St. Louis, MO), and Ash Stevens Inc. (Detroit, MI), stopped to provide azoxymethane to customers in Europe. Most European scientists had to turn back to DMH to induce ACF or cancer in rodents. How to chose the dose of DMH? Use our database: a column shows the carcinogen doses, another shows ACF or tumor yield. Click on the heading "carcinogen": table will be sorted by carcinogen & doses.
- DMH dose & ACF number: the standard initiation protocol (2 AOM injections, 15 mg/kg BW, at a week interval) yields 145 ± 41 ACF, 8-14 weeks later (35 studies in Fisher 344 male rats). A similar number of ACF is obtained with 2 x 25 mg/kg DMH injections (114 ACF, 2 studies). Twice more ACF are obtained with 4 x 20 mg/kg DMH (278 ACF, 4 studies).
- DMH dose & tumor incidence: Fisher 344 male rats initiated with 2 x 15 mg/kg of AOM have a 78% tumor incidence 42 weeks later (49 studies, range 46-100% incid., 31-54 wks). Three 15 mg/kg AOM doses gives 67% tumor incidence, 33 weeks after initiation (19 studies, range 36-100%, 28-40 wks). The tumors database reports 12 DMH studies in F344 rats. Cumulated doses of injected DMH ranges from 160 to 400 (median 200), given in one to 20 separated injections. Thirty-four weeks after first DMH injection, the tumor incidence is 74% (range 40-94%, 20-48 wks).
- DMH one shot: a single dose of DMH seems OK (200 mg/kg of DMH-HCl) according to Glauert, Beaty and Karkare. No side effect was observed with this dose (H. Glauert, personal communication).
- Regression analyses: In an attempt to clarify dose-effect and time-effect relationships, we performed multiple regression analysis on subsets of data.
- DMH in S.D. rats : Tumor incidence = (0.19 * total dose) + (1.54 * no. of weeks) (19 studies)
- DMH in F344 rats: Tumor incidence = (0.13 * total dose) + (1.19 * no. of weeks) (12 studies), and after excluding an outlier:
- DMH in F344 rats: Tumor incidence = (0.16 * total dose) + (1.00 * no. of weeks) (11 studies)
- AOM in F344 rats: Tumor incidence = (0.72 * total dose) + (1.26 * no. of weeks) (96 studies)
Correlation coefficient was higher than 0.93 in each case, p value for each coefficient was lower than 0.002, and F value for the multiple regression analysis was higher than 60 (p<0.000). However, the residuals ranged from -35 to +35%. Thus prediction of tumor incidences was not accurate: predicted 100% incidence might give 65% incidence. Residuals looked smaller in F344 than in SD rats (e.g., + or - 20%). Usual strategies to detect the growth of tumors in vivo should thus be used before final sacrifice (e.g., fecal occult blood testing, sacrifice of sentinel rats, colonoscopy)
- How to use DMH? most studies use DMH dihydrochloride. The database reports the doses as given by authors. Doses should be adjusted accordingly (MW: DMH=60, DMH-HCl=133). DMH is a powder that has to be weighed and dissolved in saline. Care must be taken so that the powder does not fly away from the scale or be inhaled. Also, DMH stability depends on the solvent, and phosphate buffers should be avoided (R. Bird, personal communication). DMH-HCl unadjusted solution is too acidic and painful to the animals (H. Glauert, personal communication), pH must be adjusted to 6.7 with NaOH, but it is not so easy to do.
CONCLUSION on AOM/DMH shift
- According to ACF data, one should use twice more DMH than AOM
- According to tumor data, one should use six times more DMH than AOM (2x100 dmh replaces the usual 2x15 aom).
- After two sad studies with not enough tumors, we decided to use 2 x 150 mg/kg of DMH-HCl to induce tumors, like Femia & Caderni, 2005.
PhIP & other colon carcinogens: other carcinogens are used less frequently that AOM or DMH. They include specific nitrosamines (MNU, MNNG), and heterocyclic amines like 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), IQ and MeIQx. Compared to AOM, nitrosamines are less convenient to use (e.g., MNU must be infused intra-rectally), less specific (e.g., i.p. MNU induces tumors at many sites). Compared with AOM, heterocyclic amines are more expensive (see PhIP quotation at TRC site), and less potent (e.g., in PhIP studies, 3 to 6 ACF are typically scored per colon).
Mutations in chemically-induced tumors: AOM-induced tumors share many histopathologic characteristics with human tumors, but they have a low tendency to metastasize. They, like human tumors, are often mutated on K-ras and beta-catenin genes and show microsatellite instability. However, unlike human tumors, they are seldom mutated at the adenomatous polyposis coli (Apc) gene (15%), are never mutated at the p53 gene. However, like Apc mutated human tumours, rat tumours accumulate beta-catenin in the nucleus. This is due to Ctnnb1 mutation, which produces a B-catenin resistant to degradation (Femia 05). Alternatively, a mutation in the GSK3 phosphorylation motif of the B-catenin gene can reduce B-catenin degradation (Takahashi 04). PhIP induces the Apc and B-catenin mutations frequently (15 and 50%, respectively) and microsatellite instability, but no K-ras or p53 mutations. Thus, Wnt/B-catenin/Tcf pathway plays a major role in carcinogen-induced rat tumours like in human tumors. Like in humans too, COX-2 and iNOS are over-expressed in these tumours.
Heterocyclic amines (PhIP, IQ, MeIQ, MeIQx) are produced when a muscle (meat or fish) is heated at a high temperature. Cooking meat on a grill, a pan, or in oil thus generates heterocyclic amines, and a huge literature is devoted to these carcinogens. PhIP is thus present in our daily diet, though AOM is not. Heterocyclic amines were discovered by Takashi Sugimura. The way of cooking is sensible, as explained by S. Robbana, but the total dose we ingest seems very small compared to the carcinogenic dose in rats as discussed by Stavric, and as demonstrated with the HERP index (Human Exposure/Rodent Potency) of Lois Gold and Bruce Ames.
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Corpet DE & Taché S, 2002, Nutrition & Cancer - & - DE Corpet & F Pierre, 2003, Cancer Epidemiol. Biomarkers Prevention - http://www.inra.fr/reseau-nacre/sci-memb/corpet/indexan.html & Mirror site