Metabolic proficiency and benzo[a]pyrene DNA adduct formation in APCMin mouse adenomas and uninvolved mucosa
Abid Sattar,
Alan Hewer1,
David H. Phillips1 and
Frederick C. Campbell2
University Department of Surgery, The Medical School, Framlington Place, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK and
1 Section of Molecular Carcinogenesis, Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK
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Abstract
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Tumour formation may involve interactions between genetic factors and environmental carcinogens. Adenoma formation in APCMin/+ mice is associated homozygous adenomatous polyposis coli (APC) gene mutation, but the effects on carcinogen susceptibility are unknown. This study tests the hypothesis that APCMin/+ adenoma formation is accompanied by changes in metabolic proficiency and carcinogen susceptibility. Cytochrome P450 (CYP)1A1/1A2, glutathione S-transferase (GST)
, µ and
classes and DNA adduct formation were assayed in adenomas and uninvolved mucosa from APCMin/+ mice, before and after benzo[a]pyrene (B[a]P) treatment. In untreated adenomas and mucosa, CYP1A1/1A2 and B[a]PDNA adducts were undetected but GST
, µ and
class enzymes were constitutively expressed. In adenomas, B[a]P only induced CYP1A1/1A2 to low level while GST
and
class enzymes were unaffected. A GSTµ band which was absent from mucosa, was induced in adenomas. In mucosa, B[a]P induced CYP1A1/1A2 and GST
and
, to high levels. B[a]PDNA adduct levels were 56 ± 15/108 nucleotides (median ± SE) in adenomas versus 89 ± 19/108 nucleotides in mucosa (P < 0.0001). APCMin adenomas show reduced bioactivation capacity and sustain less DNA damage from B[a]P exposure, than APCMin uninvolved mucosa. These properties could influence mutagenesis and subsequent neoplastic transformation of adenomas.
Abbreviations: AhR, aryl hydrocarbon receptor; APC, adenomatous polyposis coli; B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene 7,8-diol-9,10-epoxide; CYP, cytochrome P450; FAP, familial adenomatous polyposis; GST, glutathione S-transferase; HNPCC, hereditary nonpolyposis colorectal cancer; Min, multiple intestinal neoplasia; MIS, microsatellite instability.
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Introduction
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Complex interactions between genetic and environmental factors may influence multistage tumorigenesis. Sequential events involving cancer predisposing or suppressing genes may induce molecular changes, characteristic of individual stages of neoplastic transformation. Specific stages of tumorigenesis in different tissues may show distinctive metabolic profiles and susceptibility to chemical mutagens. For example, preneoplastic nodules arising from initiated hepatocytes display diminished expression of phase I carcinogen metabolizing enzymes and increased resistance to chemical carcinogens (1). Similarly, pulmonary hyperplastic foci and adenomas are associated with loss of inducibility of cytochrome P450 (CYP)1A1 which may reduce their functional capability for activation of potential carcinogens (2). However, data relating to metabolic properties of preneoplastic lesions of the intestine are lacking.
Mutations in the tumour suppressor adenomatous polyposis coli (APC) gene may represent the earliest step in development of human colorectal cancer (3). The APCMin/+ heterozygous mouse (Min = multiple intestinal neoplasia) carries a germline autosomal dominant mutation in murine APC, that predisposes to the development of adenomas, predominantly in the small intestine (4). The APCMin/+ heterozygous mouse makes an excellent model for the study of cellular and molecular makeup of preneoplastic stages of intestinal tumorigenesis. The APC gene exerts a `gatekeeper' function on intestinal crypt differentiation, and APC mutations are associated with a generalized increased rate of crypt fission (5). Adenoma formation is accompanied by inactivation of both APC alleles (6).
Effects of homozygous APC mutation and associated phenotype alterations on metabolism and susceptibility to environmental carcinogens are unclear. Elucidation of such relationships may yield valuable insight into mechanisms of stepwise intestinal tumorigenesis. Benzo[a]pyrene (B[a]P) is an environmental polycyclic aromatic hydrocarbon carcinogen, which may contaminate smoked or barbecued foods. B[a]P may induce a co-ordinated response regulated by the aryl hydrocarbon receptor (AhR), involving phase I and II xenobiotic metabolizing enzymes, namely CYP1A1, CYP1A2,
class glutathione S-transferases (GSTs), as well as others (7). This pathway of B[a]P metabolism generates the ultimate carcinogen, benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE) which binds to DNA, causing bulky adducts. B[a]P has high mutagenicity in murine small intestine and colon (8), causes diol-epoxideDNA adduct formation in human colon and may be implicated in human colorectal carcinogenesis (9).
This study has assessed metabolic changes and susceptibility to B[a]P induced DNA adduct formation, associated with adenoma formation in APCMin/+ mice. Expression of CYP1A1/1A2, GST
, µ and
class xenobiotic metabolizing enzymes was assessed before and after B[a]P exposure, in macroscopic adenomas and uninvolved mucosa from APCMin/+ mice. BPDEDNA adduct formation was also assessed by 32P-post-labelling (10).
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Materials and methods
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Chemicals
All chemicals were of analytical grade and readily available commercially.
Antibodies
Antibodies had been raised, and cross reactivity characterized, by methods described previously for cytochrome P450 (11) and GSTs (12). Polyclonal antibodies raised against CYP1A1, CYP1A2, GST
, µ and
class enzymes were used. Although some antibody cross reactivity between CYP1A1 and CYP1A2 occurred, induction of each of these two enzymes by B[a]P was assessed separately.
Animals and treatment
APCmin heterozygous C57BL/6J mice (gift from Prof. J.C.Mathers, University of Newcastle, Newcastle upon Tyne) were used in the study. Animal numbers were kept to the minimum compatible with statistical evaluation of the data, in accordance with the UK Coordinating Committee on Cancer Research guidelines (13). Tail DNA genotyping was carried out by allele-specific PCR (14) to select nine female heterozygotes at age 3034 weeks, to provide sufficient adenomas and mucosal specimens for analysis of enzyme expression, induction and DNA adduct formation. Animals were kept at 22°C and allowed rat and mouse no. 1 diet (SDS) and water ad libitum. Animals received either a single i.p. dose of B[a]P (Sigma, Poole, UK) 100 mg/kg in 250 µl corn oil (Mazola, Surrey, UK) (n = 4 animals) or corn oil (250 µl) only (n = 1) or no treatment (control; n = 4) animals.
Tissue retrieval
Small intestines were excised from the duodenum to ileocaecal valve and the smooth muscle layer was carefully excised by a combination of sharp and blunt dissection. Intestines were then opened longitudinally and the mucosal layers were spread out over tissue paper. Adenomas were identified by naked eye as tumour-like excrescences which stood proud from the surrounding mucosa. These were counted by one observer (A.S.), dissected free from the surrounding mucosa using a scalpel and then snap frozen in liquid nitrogen, until use. After all visible adenomas had been excised, all remaining mucosa was minced then snap frozen, as above. Adenomas were pooled for analyses pertaining to enzyme expression and induction while measurements of DNA adducts were assayed in relation to individual adenomas and mucosa specimens.
Preparation of microsomes
Tissue samples of pooled adenomas or macroscopically uninvolved mucosa were thawed, diced, resuspended in isolation medium (85.6 g sucrose, 0.48 g HEPES, 0.38 g EDTA) and centrifuged at 400 g for 3 min at 4°C. The pellet was resuspended in 1 ml isolation medium and homogenized on ice using an ultra-turrex T8 (IKA Laboratories, Stauffen, Germany). The homogenate was then centrifuged at 20 000 r.p.m. for 20 min at 4°C (Rotor: Kontron TFT 50.3Ti). The supernatant was filtered over muslin and ultracentrifuged at 50 000 r.p.m. for 1 h at 4°C. The final pellet was resuspended in 200 µl of isolation medium and stored at 20°C. Protein content was estimated using a modification of the Bradford protein assay (15) with bovine serum albumin as a standard.
Western blot
Western blots were performed using a modified SDSPAGE and transblotting (16) method, utilizing the mini-Protean II system (Bio-Rad, Herts, UK). Electrophoresis of microsomal preparations was carried out against molecular weight markers (Novex, San Diego, CA). Following SDSPAGE and transblotting, filters were blocked with skimmed milk protein overnight and incubated with 1:2000 primary antibodies (polyclonal rabbit anti-mouse IgG for P450 and GST) for 2 h. Secondary antibodies [1:2000] were goat anti-rabbit IgG conjugated with horseradish peroxidase for cytochrome P450 and GST. Secondary antibodies were applied for 2 h. Microsomes prepared from untreated or corn-oil-treated intestinal APCmin mucosa or adenomas were used as controls. Proteins were visualized by enhanced chemoluminescence (Amersham, Bucks, UK) with exposure times of 5 s to 20 min. Intensity was assessed semi-quantitatively by densitometry. Blots were scanned and analysed with an Aries (Relysis) scanner and Picture Publisher LE software (Micrografx). Band intensity was quantified using TINA 2.09d quantification software (Raytest). All values were calculated as the averages of three separate experiments. Enzyme induction was assessed by comparison of densitometry values for B[a]P-treated and control tissues, in each blot.
DNA isolation
Samples of mucosa or adenomas were snap frozen in liquid N2, powdered, thawed in 10 mM EDTA, homogenized and suspended in 10% SDS (0.1 vol). DNA was isolated by phenolchloroform extraction (10). RNA contaminants were removed by adding 3 µl of 50 U/µl RNase T1 (Sigma) and 3 µl of 10 U/µl RNase A (Sigma) for 30 min at 37°C.
32P-post-labelling
Post-labelling was carried out as previously described by Randerath et al. (17). DNA was digested by spleen phosphodiesterase (Boehringer Mannheim, East Sussex, UK) and micrococcal nuclease (Sigma) in Na succinate and CaCl2 (pH 6.0) at 37°C overnight, to release deoxyribonucleoside 3' monophosphates. Samples were digested for 1 h with nuclease P1, then labelled by incubation with carrier-free [
-32P]ATP (50 µCi) and T4 polynucleotide kinase (6 U) (17).
Thin layer chromatography (TLC)
Resolution of 32P-labelled adducts was performed on PEI-cellulose TLC sheets using a multidirectional anion-exchange solvent system. Adduct spots on chromatograms were visualized by autoradiography for 18 h at 85°C, using screen intensifiers. A microchannel array detector which images the 32P activity in real time, was used in conjunction with an Autograph 2.1 two-dimensional radioisotope imaging system (Oxford Positron Systems, Oxford, UK) to quantify adduct levels. Appropriate blank areas of the chromatogram were counted to obtain background levels, which were then subtracted.
Statistics
Statistical analysis of adduct formation was performed using the MannWhitney two sample test.
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Results
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Tissues
Assays of cytochrome P450 and GST
, µ and
class enzyme expression were carried out by western blot in microsomes from pooled uninvolved APCMin/+ mouse small intestinal mucosa samples and adenomas (n = 73), from control animals. Similar assays were also carried out in pooled small intestinal mucosa samples and adenomas (n = 78), from B[a]P-treated (test) APCMin/+ mice. DNA adduct formation was assayed by 32P-post-labelling in 11 adenomas and eight mucosa specimens after corn oil treatment only (control) and 38 adenomas and 32 mucosa specimens, from B[a]P-treated (test) animals.
Xenobiotic metabolizing enzyme expression in mucosa and adenomas
Untreated animals.
CYP1A1 and CYP1A2 were undetected in untreated APCMin/+ mouse intestinal mucosa or adenomas (Figure 1
). GST
,
and µ class enzymes were constitutively expressed in mucosa and adenomas (Figures 24

). In adenomas, an additional GSTµ band probably representing a GSTµ isoenzyme was identified. This band was undetected in mucosa (Figure 4
).

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Fig. 1. CYP1A1/1A2 expression in untreated APCMin/+ tissues by western blot. Lanes 1 and 2, affinity purified protein standards for CYP1A1 and CYP1A2, respectively; lanes 35, represent untreated mucosa; lanes 68, untreated adenomas. No expression detected.
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Fig. 2. GST class enzyme expression in untreated treated APCMin/+ tissues by western blot. Lanes 14, untreated mucosa; lanes 58, untreated adenomas. Figures denote densitometry values.
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Fig. 3. GST class enzyme expression in untreated treated APCMin/+ tissues by western blot. Lanes 14, untreated mucosa; lanes 58, untreated adenomas. Figures denote densitometry values.
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Fig. 4. GST µ class enzyme expression in untreated treated APCMin/+ tissues by western blot. Lanes 14, untreated mucosa; lanes 58, untreated adenomas. Note the appearance of an additional band in adenomas, which was undetected in uninvolved mucosa. In lanes 48, densitometry values are shown in two rows, corresponding to main and additional bands, respectively.
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B[a]P-treated animals.
CYP1A1 and CYP1A2 were induced in mucosa and adenomas of APCMin/+ mice, by B[a]P treatment. Induction of CYP1A1 in mucosa (Figure 5
; lanes 35) by B[a]P was 3-fold higher, while that of CYP1A2 was 2-fold higher, than induction in adenomas (Figure 5
; lanes 68). GST
and
class enzymes were induced by B[a]P exposure, by ~1.52-fold in mucosa (Figures 6 and 7
; lanes 35), whereas GSTµ class enzyme expression appeared unaffected (Figure 8
; lanes 35). In adenomas, GST
and
enzymes were unaffected by B[a]P exposure (Figures 6 and 7
; lanes 68). Expression of the additional GSTµ isoform which was detected in untreated adenomas, increased by 56-fold after B[a]P exposure (Figures 8
; lanes 68).

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Fig. 5. CYP1A1/1A2 expression in B[a]P treated APCMin/+ tissues versus untreated controls by western blot. Two bands are shown corresponding to CYP1A2 (top band) and CYP1A1 (bottom band). Lane 1, corn-oil-treated mucosa; lane 2, corn-oil-treated adenomas; lanes 35, B[a]P-treated mucosa; lanes 68, B[a]P-treated adenomas. Figures denote densitometry values.
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Fig. 7. GST class enzyme expression in B[a]P-treated versus untreated APCMin/+ tissues, by western blot. Lane 1, corn-oil-treated mucosa; lane 2, corn-oil-treated adenomas; lanes 35, B[a]P-treated mucosa; lanes 68, B[a]P-treated adenomas. Densitometry values are shown.
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Fig. 8. GST µ class enzyme expression in B[a]P-treated versus untreated APCMin/+ tissues, by western blot. Lane 1, corn-oil-treated mucosa; lane 2, corn-oil-treated adenomas; lanes 35, B[a]P-treated mucosa; lanes 68, B[a]P-treated adenomas. A consistent additional band was observed in untreated and B[a]P-treated adenomas. Other faint or inconsistent bands could represent protein degradation products or other weakly expressed GST µ class isoforms. Densitometry values relate to main and additional bands only.
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DNA adduct formation.
No adducts were detected in APCMin/+ mouse intestinal mucosa or adenomas, after corn oil treatment. B[a]P treatment produced DNA adducts in both mucosa and adenomas. Adduct levels were significantly higher in mucosa than in adenomas [89 ± 19/108 nucleotides (median ± SE) in uninvolved mucosa versus 56 ± 15/108 nucleotides in adenomas; P < 0.0001] (Figure 9
).

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Fig. 9. DNA adduct levels in B[a]P-treated APCMin/+ mucosa versus adenomas. P < 0.0001 by MannWhitney test.
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Discussion
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Development of intestinal cancer involves the accumulation of mutations in tumour suppressor genes and oncogenes, which associate with distinct morphological stages of preneoplastic progression (18,19). Homozygous mutation of the APC gene accompanies the earliest stages of adenoma formation in familial adenomatous polyposis (FAP) (20) and are frequent in sporadic adenomas (21). Patients with FAP develop benign colonic tumours numbering up to many thousands, but each progresses slowly towards malignancy (22). Similarly, spontaneous progression of adenomas to carcinomas is rare in APCMin/+ mice (23). Administration of activation-dependent carcinogens to this mouse model tends to increase adenoma numbers, rather than induce malignant change in existing adenomas (24).
In any tissue, the rate of accumulation of mutations varies with both genotoxic injury and capacity for DNA repair. Microsatellite instability (MIS) may be considered indicative of large increases in the rate of accumulation of all classes of mutation (25). However, the incidence of MIS is low in sporadic colorectal adenomas (26). In contrast, hereditary non polyposis colorectal cancer (HNPCC) is associated with an inherited deficiency in DNA mismatch repair, has a high incidence of MIS (27) and rapidly sustains fixed mutations (22). Adenomas associated with HNPCC progress rapidly to malignancy, while sporadic and FAP adenomas show a slow rate of neoplastic progression. Hence, high mutation frequency and resultant accumulation of genetic aberrations may facilitate rapid progression of malignant transformation.
The relationship between adduct formation, mutations (2830) and cancers (31) has been established in various tissues. Genotoxic injury due to activation-dependent carcinogens like B[a]P, varies between tissues (8) and may be influenced by tissue metabolic capacity (32). In metabolically competent tissues, B[a]P induces increased expression of CYP1A1, CYP1A2,
class GSTs and other enzymes (7), partly through activation of the AhR. This response to chemical toxins invoked by AhR, involves a bioactivation/detoxification equilibrium of xenobiotic response genes. CYP1A1/1A2 bioactivates B[a]P to the ultimate carcinogen, BPDE. This class of reactive intermediates are substrates for GST isoenzmes and may be detoxified by conjugation, particularly by GST
and µ class enzymes (33,34).
In addition to its role in regulation of expression of xenobiotic metabolizing enzymes (35), AhR performs key functions in murine tissue differentiation and development (36). Expression of AhR may be differentiation dependent (37). The normal intestine is metabolically competent to interface with an adverse environment. Xenobiotic metabolizing enzymes are highly expressed in intestinal epithelium, along a differentiation gradient. Cytochrome P450 and GST enzymes are abundantly expressed in terminally differentiated functional cells lining the villus, but are scant in undifferentiated crypt epithelium (38). Intestinal crypt differentiation and fission is regulated partly by the APC gene (5), which mediates signal transduction through ß-catenin (39). In the human colon and in the APCMin/+ mouse intestine, adenoma formation is frequently accompanied by homozygous APC mutation (20) and differentiation changes (40,41). Effects of differentiation changes associated with adenoma formation in the APCMin/+ mouse intestine, upon proficiency for xenobiotic metabolism were unknown.
This study has shown that adenoma formation in the APCMin/+ mouse intestine is accompanied by minor changes in constitutive expression of xenobiotic metabolizing enzymes but greater alterations of metabolic responses to B[a]P. In untreated control animals, CYP1A1/1A2 enzymes were undetected while expression of GST
and
enzymes were similar in adenomas and mucosa. An additional GSTµ band was detected in untreated adenomas but not in uninvolved mucosa. At least four murine GSTµ isoenzymes have been cloned, similar to those in rat, which may be induced by AhR dependent and independent pathways (42).
Treatment of animals with B[a]P, only induced CYP1A1/1A2 to low levels and had no discernible effect on GST
or
, in adenomas. However, the additional GSTµ band observed in untreated adenomas was induced by B[a]P, in adenomas. Conversely, B[a]P induced CYP1A1/1A2, GST
and
to high levels in uninvolved mucosa. The additional GSTµ band found in adenomas, was undetected in mucosa. While the mechanistic basis of these differences in metabolic responsiveness is unclear, differentiation-dependent factors including the AhR could be implicated.
Any alteration of the capacity for B[a]P bioactivation or detoxification within tissues may influence the ultimate yield of BPDE available for adduct formation, genotoxic injury and promotion of tumorigenesis (43). 32P-post-labelling analysis in this study, revealed the adduct pattern typical of BPDE:DNA reaction (43) in both adenomas and uninvolved mucosa. However, adduct levels were significantly higher in the mucosa. These findings could be causally linked to relative metabolic proficiency, namely robust bioactivation responses in mucosa versus weak responses in adenomas. These properties could impart some resistance of adenomas to further accumulation of carcinogen induced mutations and consequent progression to malignancy.
Since many carcinogens are activation dependent, chemically induced tumorigenesis may be inhibited by blockade of metabolic activation. Cytochrome P450 inhibitors may impede mutagenesis and tumour formation (44). The present study has shown greater CYP1A1/1A2 expression, responsiveness and greater genotoxic injury after B[a]P, in uninvolved mucosa than in adenomas. Future work may ascertain whether cytochrome P450 inhibitors have greater antimutagenetic effects in mucosa than in adenomas and whether they may have a role in prevention of adenoma formation and recurrence after removal.
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Acknowledgments
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We are grateful to Prof. J.C.Mathers (Department of Biological and Nutritional Sciences, University of Newcastle) for supply of APCMin/+ mice, and to Prof. J.D.Hayes and Prof. C.R.Wolf (Biomedical Research Centre, Dundee) for kind supply of antibodies to GST
, µ and
class enzymes and CYP1A1/1A2, respectively. These studies were funded by the Scottish Home and Health Department and the Cancer Research Campaign.
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Notes
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2 To whom correspondence should be addressed Email: f.c.campbell{at}newcastle.ac.uk 
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Received October 26, 1998;
revised February 16, 1999;
accepted February 26, 1999.