Role of hepatic and intestinal p450 enzymes in the metabolic activation of the colon carcinogen azoxymethane in mice

Abstract

P450-mediated bioactivation of azoxymethane (AOM), a colon carcinogen, leads to the formation of DNA adducts, of which O(6)-methylguanine (O(6)-mG) is the most mutagenic and contributes to colon tumorigenesis. To determine whether P450 enzymes of the liver and intestine both contribute to AOM bioactivation in vivo, we compared tissue levels of AOM-induced DNA adducts, microsomal AOM metabolic activities, and incidences of colonic aberrant crypt foci (ACF) among wild-type (WT), liver-specific P450 reductase (Cpr)-null (LCN), and intestinal epithelium-specific Cpr-null (IECN) mice. At 6 h following AOM treatment (at 14 mg/kg, s.c.), O(6)-mG and N(7)-mG levels were highest in the liver, followed by the colon, and then small intestine in WT mice. As expected, hepatic adduct levels were significantly lower (by >60%) in LCN mice but unchanged in IECN mice, whereas small-intestinal adduct levels were unchanged or increased in LCN mice but lower (by >50%) in IECN mice compared to that in WT mice. However, colonic adduct levels were unchanged in IECN mice compared to that in WT mice and increased in LCN mice (by 1.5-2.9-fold). The tissue-specific impact of the CPR loss in IECN and LCN mice on microsomal AOM metabolic activity was confirmed by rates of formation of formaldehyde and N(7)-mG in vitro. Furthermore, the incidence of ACF, a lesion preceding colon cancer, was similar in the three mouse strains. Thus, AOM-induced colonic DNA damage and ACF formation is not solely dependent on either hepatic or intestinal microsomal P450 enzymes. P450 enzymes in both the liver and intestine likely contribute to AOM-induced colon carcinogenesis.

Figure 1

Immunohistochemical analysis of CPR expression in colon. Paraffin sections of the proximal and distal colon from 2-month-old male IE-Cpr-null mice and WT littermates were processed for immunohistochemistry. The tissue sections were incubated with a polyclonal rabbit anti-rat CPR antiserum. Antigenic sites were visualized with a peroxidase-conjugated goat anti-rabbit secondary antibody, with Alexa Fluor 594-conjugated tyramide as the peroxidase substrate. Sections were mounted with Prolong mounting medium with DAPI counter stain. Fluorescent signals were detected with a tetramethylrhodamine isothiocyanate filter (for Alexa 594, red) and a DAPI filter (for DAPI, blue); scale bar, 100 μm. No signal was detected in negative control slides (data not shown), which were incubated with a normal goat serum in place of the anti-CPR antibody. Results shown are typical of three mice per strain analyzed.

Figure 2

AOM-induced colonic ACF formation. (A) Morphology of AOM-induced ACF in colon. ACF was detected in methylene-blue-stained colon, as described in Materials and Methods. Representative images of ACF (arrows) with 2 (left) or 8 (right) aberrant crypts are shown. (B) Numbers of ACF detected in colon. Male WT-A/J, IECN-A/J, and LCN-A/J mice were treated with saline or AOM at a dose of 7.5 mg/kg (s/c), once weekly for three weeks, and sacrificed 6 weeks later for the detection ACF in the proximal and distal colon. Saline-treated mice did not develop any ACF (not shown). Data (means ± S.D., n = 4 −8) for proximal and distal colons, as well as the combined data for the entire colon (total), are presented. There was no difference between the two mouse strains (p > 0.05, compared to WT; one-way ANOVA with Dunnett’s test).

Figure 3

Schematic summary of sources of AOM reactive metabolites in the colon.