This is an open access article published under an ACS AuthorChoice License, which permits
`copying and redistribution of the article or any adaptations for non-commercial purposes.
`
`Article
`
`pubs.acs.org/crt
`
`Role of Hepatic and Intestinal P450 Enzymes in the Metabolic
`Activation of the Colon Carcinogen Azoxymethane in Mice
`Vandana Megaraj, Xinxin Ding, Cheng Fang, Nataliia Kovalchuk, Yi Zhu, and Qing-Yu Zhang*
`Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany,
`Albany, New York 12201, United States
`
`ABSTRACT: P450-mediated bioactivation of azoxymethane (AOM), a colon
`carcinogen, leads to the formation of DNA adducts, of which O6-methylguanine
`(O6-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.), O6-mG and N7-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 N7-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.
`
`■ INTRODUCTION
`
`Azoxymethane (AOM) and its metabolic precursor, 1, 2-
`dimethylhydrazine (DMH), are commonly used carcinogens to
`study the molecular mechanisms of colon carcinogenesis in
`rodents.1−4 They are preferred model carcinogens because they
`induce tumors preferentially in the distal colon of rodents and
`because the tumors have pathological features known to be
`associated with human sporadic colorectal cancer.5 DMH and
`AOM are procarcinogens, which require metabolic activation
`by cytochrome P450 (P450) enzymes, primarily CYP2E1.6
`DMH undergoes N-oxidation to form AOM, which, upon
`hydroxylation, yields methylazoxymethanol (MAM). MAM is
`unstable, with a half-life of ∼12 h. It subsequently decomposes
`to yield formaldehyde and a highly reactive methyldiazonium
`ion, which alkylates the DNA bases, resulting in the formation
`including O6-methylguanine (O6-mG) and
`of DNA adducts,
`N7-methylguanine (N7-mG).4 Persistence of O6-mG can lead to
`mutation in oncogenes and initiation of tumorigenesis.7
`Previous in vitro studies have demonstrated that colon
`epithelial cells are capable of metabolizing DMH into
`carcinogenic metabolites, without the need for prior metabo-
`lism by other tissues or colonic bacteria.8−12 However, the
`prevailing hypothesis is that the liver plays a critical role in
`DMH/AOM bioactivation in vivo and that
`the reactive
`intermediates produced by the liver are transported to the
`colon via the blood or bile to induce carcinogenicity.13−15 This
`is a plausible hypothesis, given that
`the colon generally
`
`possesses much lower levels of P450 enzymes, relative to the
`liver. Nonetheless, the relative contributions of the liver and the
`intestine to DMH/AOM-induced DNA damage in the colon
`have not been directly determined, and the bioactivation in the
`target organ may also explain the organ-specific induction of
`tumors in the distal colon by AOM.
`In the present study, we determined the respective roles of
`hepatic and intestinal P450 enzymes in AOM metabolic
`activation in vitro and in vivo by studying the liver-specific
`Cpr-null (LCN) mouse and the intestinal epithelium-specific
`Cpr-null (IECN) mouse.16,17 The cytochrome P450 reductase
`(CPR or POR) is required for the monooxygenase activity of all
`microsomal P450 enzymes. The LCN and IECN mouse models
`have been found valuable for differentiating between hepatic
`and extrahepatic18−22 or between intestinal and extra-gut17,23,24
`P450 contributions to xenobiotic metabolism or toxicity. We
`chose to study AOM instead of its precursor DMH, owing to
`AOM’s higher potency and greater stability in dosing solutions.
`We compared tissue levels of O6-mG and N7-mG adducts in
`the liver, small
`intestine (SI), and colon among wild-type
`(WT), LCN, and IECN mice that were treated with AOM
`according to an established protocol for the induction of colon
`DNA damage.6,25,26 We further compared microsomal AOM
`metabolic activities in the various tissues among the three
`
`Received: December 26, 2013
`Published: February 19, 2014
`
`© 2014 American Chemical Society
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`strains of mice. Finally, we assessed AOM-induced colonic
`aberrant cryptic foci (ACF) formation, which represents the
`precursor lesions of colon cancers.4,27,28 Our findings indicate
`that knockout of liver Cpr or IE Cpr alone was not sufficient to
`block or reduce colonic DNA adduction and ACF formation by
`AOM and that P450 enzymes in both the liver and intestine
`likely contribute to AOM-induced O6-mG formation and the
`eventual colon carcinogenesis in WT mice.
`
`■ MATERIALS AND METHODS
`
`Chemicals and Reagents. AOM, O6-mG, and formaldehyde were
`purchased from Sigma-Aldrich (St. Louis, MO). N7-mG was obtained
`from Santa Cruz Biotechnology (Dallas, TX). The sources of O6-
`methyl-deoxyguanosine (O6-m-dG) and O6-trideuteriomethyl-deoxy-
`guanosine (O6-CD3-dG) were the same as described previously.20 All
`solvents (acetonitrile, methanol, and water) were of high-performance
`liquid chromatography (HPLC) grade (ThermoFisher Scientific,
`Waltham, MA).
`Mouse Breeding. All studies with mice were approved by the
`Wadsworth Center Institutional Animal Care and Use Committee.
`WT B6, Vil-Cre(+/−)/Cprlox/lox (IE-Cpr-null or IECN) mice (on B6
`background), and Alb-Cre(+/−)/Cprlox/lox (liver-Cpr-null or LCN) mice
`(on B6 background)16 were obtained from breeding stocks maintained
`at the Wadsworth Center (Albany, NY) and used for CPR expression,
`AOM metabolism, and DNA adduct formation studies.
`WT, IECN, and LCN mice used for studying AOM-induced colonic
`ACF formation were on the susceptible A/J background. IECN-A/J
`and LCN-A/J mice were generated by backcrossing IECN-B6 and
`LCN-B6 to a congenic (A/J-N11) strain of Cprlox/lox mice for 3−5
`generations; the Cprlox/lox-A/J mice were produced by backcrossing the
`original Cprlox/lox-B629 to WT A/J mice (Jackson Laboratory, Bar
`Harbor, ME). WT-A/J mice used for experiments were produced from
`breeding pairs maintained at the Wadsworth Center.
`All animals were 2- to 3-month old at the beginning of each study
`described below. Mice were genotyped using tail DNA for the Cre
`transgene and the Cpr allele, as described previously.16,29
`Immunohistochemistry. The colon from male WT and IECN
`mice were obtained and cut into two equal halves, representing
`proximal and distal sections, and prepared as “Swiss rolls” for
`embedding and sectioning. Immunohistochemical analysis of CPR
`expression in paraffin sections of the colon was conducted as described
`previously for SI.17
`Preparation of Microsomes from Intestine and Liver. SI
`mucosa from two mice or colonic mucosa from four mice were pooled
`for each microsomal sample, prepared as reported previously.30 Liver
`microsomes were prepared from individual mice as described31 but
`with use of protease inhibitors.30 Microsomes were stored at −80 °C
`until use. Microsomal protein concentrations were determined using
`the bicinchoninic acid protein assay kit (Pierce Chemical, Rockford,
`IL) with bovine serum albumin as standard.
`Analysis of DNA Adducts in Tissue. For the determination of
`tissue levels of O6-mG and N7-mG, male WT, IECN, and LCN mice
`were treated with a single injection of AOM (at 14 mg/kg, s.c.)6 in
`saline. The liver, SI (duodenum,
`jejunum and ileum), and colon
`(proximal, distal) were obtained 6 h after AOM treatment. The SI and
`colon segments were slit open and rinsed with ice-cold saline before
`being stored at −80 °C until use. For DNA isolation, the middle lobe
`of
`the liver,
`the entire segments of duodenum,
`jejunum,
`ileum,
`proximal colon, and distal colon were homogenized in ∼4 mL of
`genomic DNA buffer (10 mM Tris-HCl, 100 mM NaCl, 2.5 mM
`EDTA, and 0.5% SDS). The homogenate corresponding to ∼100 mg
`of tissue was incubated with proteinase K (250 μg) at 55 °C for 2 h.
`DNA was extracted with phenol/chloroform/isoamyl alcohol
`(25:24:1) (Invitrogen), and precipitated with ethanol. The resus-
`pended DNA was incubated with RNase A (100 μg) and RNase T1
`(0.5 μL) for 1 h at 37 °C to remove RNA contamination. The final
`DNA preparations were stored at −20 °C until used for adduct
`analysis.
`
`Article
`
`Assay for DNA Adduct Formation in Microsomal Reactions
`in Vitro. The assay for AOM-induced in vitro DNA adduct formation
`was based on a published method.32 Briefly, microsomes (0.5−2.0 mg/
`mL) were incubated with calf thymus DNA (1 mg/mL) and AOM
`(200 μM) in a total volume of 1.0 mL. The assay buffer consisted of
`0.1 M Tris−HCl (pH 7.4), 1 mM EDTA, 20 mM MgCl2, 0.3 M KCl,
`and 1.5 mM NADPH. Incubations were carried out at 37 °C for 60
`min in a shaking water bath. An additional 30 nmol of NADPH was
`added after the first 30 min. The reaction was stopped by the addition
`of 0.5 mL of ice-cold 7.5 M ammonium acetate. DNA was then
`extracted as described above for
`tissue homogenates. Control
`incubations were performed without NADPH.
`Detection of DNA Adducts by LC-MS/MS. Levels of O6-mG
`and N7-mG were determined, for both in vivo and in vitro DNA
`samples, essentially as described20 with minor modifications. Briefly,
`DNA samples (100−200 μg) were fortified with the internal standard
`O6-CD3-dG (6 pmol) and hydrolyzed in 0.1 N HCl at 80 °C for 90
`min. The samples were allowed to cool, neutralized with NH4OH, and
`analyzed using LC-MS. Control genomic DNA from corresponding
`tissues were used for the preparation of calibration curves for the
`quantification of O6-mG and N7-mG, with O6-mG and N7-mG
`standards added in 40 to 1000 nM. Blank controls for the solvent and
`matrix were included in each set of calibration samples. The LC-MS
`method for the detection of O6-mG was according to ref 24. N7-mG
`was detected using the same method; the parent/product ion pairs
`were monitored at m/z 166/149 and m/z 166/124, using the MRM
`scan mode. The retention time was 7.2 min for N7-mG and 7.6 min for
`O6-mG and O6-CD3-dG. The detection limits for N7-mG and O6-mG
`were 0.02 pmol and 0.04 pmol (on column), respectively, with an
`injection of 6 μg of hydrolyzed DNA. The DNA adduct levels were
`normalized to guanine levels for all samples, determined using HPLC,
`as described previously.20
`(MAM) Formation in
`Assay for Methylazoxymethanol
`Microsomal Reactions in Vitro. MAM formed from AOM in
`microsomal
`incubations was detected as formaldehyde using the
`chromotropic acid method.9 The microsomal
`incubations were
`performed at 37 °C, using 500 μM AOM and 0.2 mg/mL microsomal
`protein for 10 min. Control assays were performed in the absence of
`either AOM or microsomes to correct
`for any nonenzymatic
`conversion of AOM to formaldehyde.
`AOM Induced Colonic ACF Formation. Male, 8−10 week old,
`WT-A/J, IECN-A/J, and LCN-A/J mice (8 per group) were treated
`with either saline or AOM (7.5 mg/kg BW, s.c.),33 once weekly for 3
`weeks. Mice were sacrificed 6 weeks post-treatment for ACF detection,
`as described previously.33 The entire colon (from the cecum to anus)
`was excised (within 4 min from the time of euthanasia). A longitudinal
`incision was made along the entire length of the colon, which was
`further cut into two equal-length segments, representing proximal and
`distal portions of the colon. The segments were dipped in PBS to
`remove fecal pellets and then kept flat between filter papers in 10%
`buffered formalin for at least 24 h. Subsequently, the colons were
`immersed in freshly prepared 0.1% methylene blue for 10 min and
`rinsed briefly in deionized H2O to remove excess dye. The colon was
`mounted carefully on a microscope slide with the mucosal surface side
`up and viewed under a light microscope (Nikon TE2000) with 40×
`magnification. The ACF in the entire mucosal surface of the colon
`were counted blindly and independently by two investigators and
`recorded.
`Other Methods. Statistical significance of differences among the
`three mouse strains in various parameters was examined using one-way
`analysis of variance (ANOVA), followed by Dunnett’s post-hoc test for
`pair wise comparisons, with the use of Graph Pad Prism 5 (Graph Pad,
`San Diego, CA). In all cases, p < 0.05 was considered statistically
`significant.
`
`■ RESULTS
`
`Effects of Tissue-Specific Cpr Deletion on AOM-
`Induced DNA Adduct Formation in the Liver and
`Intestine. The respective roles of the liver and intestinal
`
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`Chemical Research in Toxicology
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`microsomal P450 enzymes in the bioactivation of AOM in vivo
`was determined by comparing levels of AOM-induced DNA
`adducts in WT, IECN, and LCN mice (Table 1). Levels of O6-
`
`Table 1. AOM-Induced DNA Adduct Formation in the Liver
`and Intestinea
`
`tissue
`
`liver
`
`duodenum
`
`jejunum
`
`ileum
`
`pmol/μmol guanine
`O6-mG
`N7-mG
`strain
`413 ± 53
`3062 ± 954
`WT
`448 ± 68
`2926 ± 564
`IECN
`1046 ± 311b
`159 ± 24b
`LCN
`43 ± 10
`168 ± 18
`WT
`63 ± 10b
`19 ± 4b
`IECN
`71 ± 12b
`189 ± 32
`LCN
`13 ± 2
`41 ± 6
`WT
`17 ± 3b
`6 ± 1.2b
`IECN
`19 ± 4
`59 ± 16
`LCN
`5 ± 1.4
`24 ± 7
`WT
`9 ± 2b
`2 ± 0.4b
`IECN
`7 ± 1.3
`28 ± 5
`LCN
`343 ± 102
`72 ± 15
`WT
`61 ± 12
`375 ± 148
`IECN
`984 ± 294b
`108 ± 15b
`LCN
`84 ± 17
`480 ± 147
`WT
`67 ± 11
`384 ± 140
`IECN
`901 ± 232b
`128 ± 16b
`LCN
`aMale WT, IECN, and LCN mice were injected with AOM at a dose
`of 14 mg/kg (s.c.), and levels of O6-mG and N7-mG, as well as total
`guanine, were determined in the liver, SI (duodenum, jejunum, and
`ileum), and proximal and distal colon at 6 h after the injection. Data
`represent the means ± SD of eight determinations, each of a separate
`mouse. bP < 0.05, compared with the corresponding WT tissue; one
`way ANOVA with Dunnett’s post-hoc test.
`
`Article
`
`The loss of intestinal P450 activity, in IECN mice, did not
`change adduct levels in the liver, but it led to significant
`decreases in O6-mG and N7-mG levels in the duodenum,
`jejunum, and ileum to <50% of the WT level. Interestingly, in
`the proximal and distal colons,
`there was no significant
`difference in the abundance of DNA adducts formed between
`the IECN and WT mice (Table 1).
`Previous studies have shown through immunoblot analysis
`that CPR protein expression was abolished in colonic
`microsomes from IECN mice.17 To be sure, we further
`examined CPR expression in the colon of WT and IECN mice
`by immunohistochemical analysis. As shown in Figure 1, the
`CPR protein was abundantly detected in the colon epithelium
`in both proximal and distal colons from WT mice, but it was
`absent in colons from IE-Cpr-null mice.
`
`proximal colon
`
`distal colon
`
`mG and N7-mG, which are known to be produced by AOM
`treatment,6,34 were determined in the liver, duodenum,
`jejunum, ileum, proximal colon, and distal colon at 6 h after
`AOM treatment, at a dose (14 mg/kg) that was shown
`previously to be effective in inducing adduct formation in
`mice.6 The levels of N7-mG were (3−9 times) greater than O6-
`mG levels in the various tissues examined, which is consistent
`with previous reports.6,26,34 Regardless of
`the strain,
`the
`amounts of O6-mG and N7-mG produced by AOM were
`highest in the liver, followed by proximal and distal colons,
`which had similar levels, and then by duodenum, jejunum and
`ileum (Table 1). For O6-mG, the WT levels in the liver DNA
`was ∼5 times higher than that in colon DNA, ∼10 times higher
`than that in duodenal DNA, and ∼32 and 83 times higher than
`that in jejunum and ileal DNA.
`in the LCN mice,
`The loss of P450 activity in the liver,
`caused a significant decrease in hepatic O6-mG and N7-mG
`levels to <40% of
`the WT level, confirming the role of
`microsomal P450 enzymes in AOM bioactivation in the mouse
`liver in vivo. In contrast, there was a significant increase in O6-
`mG levels (by ∼1.7-fold) in the duodenum and significant
`increases in both O6-mG (by ∼1.5-fold) and N7-mG (by ∼2.1−
`2.9-fold) levels in the proximal and distal colons of LCN mice
`compared to those in WT mice (Table 1). These data indicated
`that the AOM-induced DNA adduct formation in the SI and
`colon does not depend on bioactivation by hepatic P450
`enzymes.
`
`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.
`
`Effects of Tissue-Specific Cpr Deletion on AOM-
`Induced ACF Formation in the Colon. AOM is known to
`induce ACF in the colon as a downstream event to AOM-
`induced DNA adduct formation and a precursor to the eventual
`tumorigenesis.4,27 Thus, we further compared the extent of
`AOM-induced ACF formation in WT, IECN, and LCN mice
`after they were backcrossed 3−5 generations to the susceptible
`A/J background.
`Irrespective of the mouse strain, no ACF was detected in the
`colons of saline-treated mice; in contrast, colonic ACF was
`detected in all three strains of AOM-treated mice (Figure 2). As
`shown in Figure 2A, the aberrant crypts in ACF, which are
`preneoplastic formations, are distinguished from normal crypts
`by their larger size, increased pericryptal area, irregular lumen
`
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`Article
`
`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).
`
`Table 2. Microsomal Activity toward AOM in Vitroa
`
`rates of microsomal metabolism
`
`nmol formaldehyde/min/mg protein
`
`pmol N7-mG/μmol G/h/mg protein
`colon
`SI
`liver
`colon
`SI
`liver
`strain
`6.4 ± 4.1
`12.6 ± 7
`34 ± 13
`6.4 ± 1.8
`10.3 ± 3
`153 ± 57
`WT
`27 ± 11
`1.3 ± 2.6b
`121 ± 15
`<LOQc
`<LOQc
`<LOQc
`IECN
`7.7 ± 4
`18.6 ± 7.3
`4.8 ± 2.6
`10.2 ± 1.1
`6.4 ± 6.3b
`<LOQc
`LCN
`aThe rates of formation of formaldehyde or DNA adducts were measured for hepatic, SI, and colon microsomes of WT, IECN, and LCN mice on a
`B6 background. Each intestinal microsome preparation was obtained from pooled tissues from 2 to 4 adult male mice; three microsomal preparations
`were analyzed for each group. Hepatic microsomes were obtained from individual animals (n = 4). For DNA adduct formation, reaction mixtures
`contained 0.5−2.0 mg of microsomal protein, 200 μM AOM, 1.0 mg of calf thymus DNA, and other components as described in Materials and
`Methods in a total volume of 1.0 mL. Reactions were carried out at 37 °C for 60 min in the presence or absence of 1.5 mM NADPH. O6-mG
`formation was not detected under the conditions used (limit of detection was ∼2 pmol/μmol G/h/mg protein). For formaldehyde formation,
`reaction mixtures contained 0.2 mg of microsomal protein, 500 μM AOM, and other components as described in Materials and Methods in a total
`volume of 1.0 mL, and reactions were carried out for 10 min. Values represent the means ± SD (n = 3−4). bp < 0.01, compared with corresponding
`WT microsomes; one way ANOVA with Dunnett’s post-hoc test. cBelow the limit of quantification (LOQ, ∼1).
`
`with slit shaped appearance, greater staining intensity, and/or
`elevation above adjacent normal crypts.
`No significant difference in the total ACF counts was found
`among the strains for either the proximal or the distal region of
`the colon (Figure 2B). These results are largely consistent with
`the DNA adduct data, showing that colonic ACF formation is
`not critically dependent on AOM bioactivation by hepatic or
`intestinal microsomal P450 enzymes.
`Effects of Tissue-Specific Cpr Deletion on Rates of
`Microsomal AOM Metabolism. To further confirm that the
`liver and intestinal Cpr deletion did reduce the capacity of the
`respective tissues to activate AOM to DNA-reactive electro-
`philes, we analyzed the alkylation of calf thymus DNA by
`reactive metabolites of AOM formed in incubations with the
`
`liver, SI, and colon microsomes from WT, IECN, and LCN
`mice (Table 2). Notably, although we performed analysis to
`detect both O6-mG and N7-mG, the levels of O6-mG were
`below the limit of detection. Therefore, only N7-mG levels are
`presented.
`On an equal protein basis, hepatic microsomes were much
`more active than SI and colon microsomes in NADPH-
`dependent AOM bioactivation and N7-mG adduct formation.
`Interestingly, colon microsomes were competent
`in this
`reaction, showing ∼60% of the activity seen in SI microsomes
`in WT mice (Table 2). Microsomes of the liver (but not those
`of the SI or colon) of LCN mice showed significantly lower
`activity in N7-mG formation compared to that of WT mice.
`Similarly, microsomes of the SI and colon from IECN mice
`
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`showed minimal or no activity in the formation of N7-mG,
`whereas the liver microsomes from IECN mice had activity
`similar to that of WT in N7-mG formation (Table 2).
`We also compared the liver, SI, and colon microsomes from
`WT, IECN, and LCN mice for their ability to metabolize AOM
`to formaldehyde via the formation of MAM (Table 2). In
`accordance with the in vitro N7-mG formation data, hepatic
`microsomes showed the highest activity in the hydroxylation of
`AOM, followed by SI and colon microsomes. With the loss of
`hepatic CPR, liver microsomes of LCN mice had no activity in
`formaldehyde formation, whereas the activities of the intestinal
`microsomes of LCN mice were comparable to those of WT
`mice. In IECN mice, neither SI nor colon microsomes had
`detectable activity in formaldehyde formation from AOM, while
`the liver activity was similar to that of WT (Table 2).
`
`■ DISCUSSION
`
`Considering the high incidence of cancers in the colon and the
`fact that the gastrointestinal tract is a major portal of entry for
`myriad chemical carcinogens and toxicants, it is important to
`determine the metabolic mechanisms of chemical carcino-
`genesis
`in the colon,
`including the source of
`reactive
`intermediates, and the role of target tissue metabolic activation.
`Although the liver is the most abundant in P450 enzymes, some
`extrahepatic tissues, such as the lung and small intestine, have
`been shown to be highly efficient in target-tissue bioactivation
`of carcinogens and other toxicants, leading to tumorigenesis or
`tissue toxicity.20,23,24,35,36 The colon appears to possess a
`somewhat unique profile of P450 expression, both in terms of
`expression levels and isoforms present,12,37−39 and the role of
`colonic P450s in xenobiotic metabolism and toxicity is not well
`understood.
`We studied AOM as a model colon carcinogen. Although
`human exposure to AOM, derived from a rare plant,
`is
`uncommon, our exposure to other,
`structurally related
`hydrazine derivatives that are found in mushrooms, tobacco,
`herbicides, rocket fuels, and drugs are frequent.40 Hence, the
`metabolic mechanisms of AOM may have broad implications.
`In that regard, while several studies had reported efficient in
`vitro metabolism of AOM by colon microsomes from rodents,
`hamsters, and humans,8,9,11,12 others failed to detect such
`activity in the colon.41 When non-P450-mediated bioactivation
`of MAM in colon was investigated in vitro, alcohol dehydrogen-
`ase (ADH) was implicated;42 however, a role for ADH was not
`confirmed in vivo.13,43
`We determined the influence of tissue-specific suppression of
`P450 activity, via conditional Cpr deficiency, in the liver and
`intestine on AOM metabolism in vitro and AOM-induced DNA
`adduct
`formation in vivo. For
`in vivo studies, we chose
`subcutaneous AOM administration since it was reportedly the
`most effective route for producing colon tumorigenesis.44 We
`collected tissues for analysis at 6 h after AOM administration, a
`time point
`that was previously found to yield maximal
`alkylation of target tissue DNA.25,26 All bioactivation studies
`were conducted with mice on the B6 genetic background. In
`that connection, it should be noted that although mouse strain-
`related differential sensitivity was reported for AOM-induced
`carcinogenesis, with A/J being the most sensitive strain,28,33,45 a
`strain difference between B6 and A/J mice was not observed in
`AOM-induced DNA adduct formation (data not shown).
`Our in vitro studies clearly demonstrated the predominant
`role of P450 enzymes in microsomal metabolism of AOM, in
`the liver, SI, and colon. It is interesting that SI and colon
`
`Article
`
`microsomes showed somewhat similar ability to bioactivate
`AOM, as indicated by their rates of
`in vitro formation of
`formaldehyde and N7-mG, even though the total P450
`concentration in SI is reportedly several
`folds higher than
`that in the colon.46 This finding may be related to the reported
`presence of CYP2E1 in the colon,37,39,46 and the known activity
`of CYP2E1 toward AOM.6,47 The apparent absence of O6-mG
`adduct formation in vitro by microsomes from any of the mouse
`tissues analyzed was consistent with the fact that N7-mG was
`much more abundant than O6-mG in vivo,
`in AOM-treated
`mice (Table 1), which has been noted previously with adducts
`formed by another compound.48
`The results of AOM-induced DNA adduct formation in vivo
`(Table 1) are more complex to explain. In AOM-treated LCN
`mice, compared to similarly treated WT mice, hepatic O6-mG
`and N7-mG levels were substantially decreased, consistent with
`a major role of hepatic P450 enzymes in AOM bioactivation in
`the liver. Note, however, that the contributions of hepatic
`microsomal P450s might have been underestimated by the data
`from the LCN mice, where the loss of hepatic microsomal
`P450-mediated AOM clearance would lead to increases in
`AOM concentrations in the liver and extrahepatic tissues, and
`consequently increased DNA adduct formation via alternative
`metabolic pathways and/or in extrahepatic tissues.
`The DNA adduct levels were significantly increased, rather
`than decreased, in most parts of the intestine, including the
`colon, by the loss of hepatic microsomal P450 activity (Table
`1). This result clearly indicated that hepatic microsomal P450-
`mediated metabolism is not required for AOM to induce DNA
`adduct formation in the colon. However, the data could not tell
`us whether liver-produced reactive AOM metabolites con-
`tributed any part to DNA adduct formation in the colon, given
`the likely increase in AOM bioavailability in the target organ
`(and thus a possible overestimation of local contribution to
`DNA adduct formation) in the LCN mice.
`Given that AOM was administered subcutaneously and based
`on previous studies on pharmacokinetics of other compounds
`in the IECN mouse,17 we believe that the bioavailability of
`AOM was unchanged in IECN compared to that of WT mice.
`As expected,
`the lack of microsomal P450 activity in the
`intestine resulted in much reduced levels of AOM-induced
`DNA adducts in the SI but not in the liver. Surprisingly, DNA
`adduct
`levels in the colon were also unchanged, which
`contrasted with results of in vitro microsomal assays showing
`substantial decreases in AOM bioactivating activity in the IECN
`mice relative to that in WT mice (Table 2). The reasons for this
`apparent discrepancy between in vitro and in vivo results remain
`to be determined. However, it is worth noting that although the
`colon had the lowest in vitro activity among the three tissues
`analyzed (Table 2), the level of DNA adducts in the colon was
`the highest among all intestinal segments (Table 1). The latter
`fact was at
`least partly related to a possible colonic
`accumulation of
`the reactive metabolite derived from the
`proximal SI or to colonic bacterial β-glucuronidase activity,
`which can act on MAM-glucuronide (derived either from the
`liver or the SI) to release free MAM in the colon, as depicted in
`Figure 3. The regenerated MAM, once absorbed, can either
`undergo further bioactivation or spontaneously decompose, to
`reactive methyldiazonium ion in the colon, leading to adduct
`formation.49 The bacterial source of reactive AOM metabolites
`might have overshadowed the amounts of MAM generated
`directly from AOM by intestinal P450, thus obliterating any
`decreases in colonic DNA adducts resulting from the loss of
`
`660
`
`dx.doi.org/10.1021/tx4004769 | Chem. Res. Toxicol. 2014, 27, 656−662
`
`

`

`Chemical Research in Toxicology
`
`■ ACKNOWLEDGMENTS
`
`Article
`
`We thank Dr. Robert Turesky for helpful discussions and Ms.
`Weizhu Yang for help with mouse breeding. We gratefully
`acknowledge the use of the Biochemistry, Histopathology, and
`Advanced Light Microscopy and Image Analysis Core Facilities
`of the Wadsworth Center.
`
`■ ABBREVIATIONS
`P450, cytochrome P450; CPR, cytochrome P450 reductase;
`NADPH, reduced β-nicotinamide adenine dinucleotide phos-
`phate; HPLC, high performance liquid chromatography; LC-
`liquid chromatography−mass spectrometry; WT, wild-
`MS,
`type; B6, C57BL/6; IECN, intestinal epithelial Cpr-null; LCN,
`liver specific Cpr-null; O6-mG, O6-methylguanine; N7-mG, N7-
`methylguanine; AOM, azoxymethane; MAM, methylazoxyme-
`thanol; DMH, 1,2-dimethyl hydrazine; ACF, aberrant cryptic
`foci
`
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