Carrinogenesis vol.13 no.ll pp.2121-2125, 1992
Effect of dietary fiber on cytochrome P450IA1 induction in rat colonic mucosa
Sumio Kawata, Shinji Tamura, Yukihiko Matsuda, Nobuyuki Ito and Yuji Matsuzawa Second Department of Internal Medicine, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan
Introduction Epidemiologic studies on human cancer incidence under various environmental pollution conditions have suggested that populations consuming low fiber diets tend to have a greater incidence of colon cancer than those consuming higher fiber diets (1—3). The preventive effect of dietary fibers in colon carcinogenesis in animal models has been extensively studied (4-10). Strobel et al. (11,12) reported that the colon contains a mixed function oxidase system capable of activating carcinogens to their proximate reactive form. Their studies provided a good basis for the possibility that the cytochrome P450 system in the colon may play a role in tissue-specific carcinogenesis. Recently, a cytochrome P450IA1, an isoform of cytochrome P450, was found to be localized in the cytoplasm of the epithelium of colonic mucosa of rats treated with 3-methylcholanthrene (3MC*> (13) or j3-naphthoflavone (14). To clarify the effect of dietary fibers on colon chemical carcinogenesis, it is important to find whether dietary fibers can prevent the induction of the cytochrome P450-dependent mixed function oxidase system in the colonic mucosa by chemical inducers and carcinogens per se. In this study, we examined the effect of wheat bran in diet on cytochrome P450IA1 induction in the colonic mucosa of rats that received a single intragastric injection of 3MC by stomach tube. •Abbreviations: 3MC, 3-methylcholanthrene. © Oxford University Press
Immunohistochemical analysis Immunohistochemical analysis of cytochrome P450IA1 (PASQ^Q) in the colonic mucosa was performed as described previously (13). Wheat bran-fed 3MC-treated rats (n = 5), control diet-fed 3MC-treated rats (n = 5), wheat bran-fed untreated rats (n = 5), and control diet-fed untreated rats (n = 5) were made to fast 24 h before they were killed under diethylether anesthesia. Specimens were cut from proximal and distal portions of the colon and rinsed in ice-cold 10 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl (PBS), then embedded in Tissue-Tek (Miles Scientific) and frozen. Sections ~ 6 yia thick were sliced on Table I. Composition of rat diets administered Ingredient
% of total diet Control diet
Fiber diet
Casein" Safflower oil Mineral mixture1' Vitamin mixture1 Comstarch Wheat bran"1
27 10 6 2 55 0
27 10 6 2 50 5
Total ingredients
100
100
"Casein (HC1); edible extra grade hydrochloric acid casein. b Mineral mixture to supply the following minerals (g/100 g of mixture, Oriental Yeast Co.): sodium chloride, 4.66; potassium phosphate, 25.72; sodium phosphate, 9.35; anhydrous magnesium sulfate, 7.17; calcium lactate; 35.09; calcium phosphate, 14.56; ferrous citrate, 3.18; manganese sulfate, 0.12; potassium iodide, 0.01; zinc carbonate, 0.11; cupric sulfate, 0.03. Vitamin mixture (per 100 g of mixture, AIN-76, Oriental Yeast Co.): vitamin A, 40 000 IU; vitamin D 3 , 10 000 IU; vitamin E acetate, 500 mg; vitamin K3, 0.5 mg; choline, 20.0 g; vitamin B,, 60 mg; vitamin B2, 60 mg; vitamin R6, 70 mg; vitamin B| 2 , 0.1 mg; calcium D-pantothenate, 160 mg; folic acid, 20 mg; biotin, 2 mg; nicotinic acid, 300 mg. d Wheat bran was purchased from Oriental Yeast Co., Japan.
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Downloaded from http://carcin.oxfordjournals.org/ at McGill University Libraries on December 10, 2012
The effect of dietary fiber on the induction of cytochrome P450IA1 in rat colonic mucosa after a single intragastrk injection of 3-methykhoIanthrene (3MC, 20 mg/kg) was investigated by examining the drug-metabolizing enzyme activity, immunoblotting for cytochrome P450IA1 and immunohistochemistry. 7-Ethoxy coumarin-O-deethy lase activities were ~ 20-fold higher in mkrosomes from both proximal and distal portions of the colonic mucosa of control diet-fed 3MC-treated rats compared with those of control dietfed untreated rats. Strong immunofluorescence for cytochrome P450IA1 was localized in the cytoplasm of the colonk mucosa surface epithelium from the control diet-fed 3MC-treated rats. 7-Ethoxycoumarin-O-deethylase activity and cytochrome P450IA1 content determined by immunoblotting were significantly lower in wheat bran-fed 3MCtreated rats than in control diet-fed 3MC-treated rats. Immunohistochemical analysis showed much weaker immunofluoresence for cytochrome P450IA1 in the surface epithelium of the colonic mucosa of the wheat bran-fed 3MCtreated rats. These observations suggested that dietary fiber can affect the induction of cytochrome P450IA1 in colonic mucosa by dietary inducers or carcinogens.
Materials and methods Animals Adult male Wistar rats weighing —250 g were used. The rats were housed with free access to a nutritionally complete semisynthetic control diet or a high fiber diet containing 5% wheat bran (Table I). Water was supplied ad libitum at all times. Both diets were prepared by mixing in a blender. Each diet was packaged, marked with the appropriate color code, and stored as a dry powder until use. After 2 weeks exposure to the dietary regimens, the rats were randomly subdivided and received a single intragastric injection of 1.0 ml corn oil or 3MC, 20 mg/kg body weight, in 1.0 ml corn oil by stomach tube. After 48 h, the rats were killed under diethylethcr anesthesia. The animals were weighed immediately prior to death and the body weight was not significantly different among the four groups: control diet-fed untreated, control diet-fed 3MC-treated, wheat bran-fed untreated and wheat bran-fed 3MC-treated rats. Production of polyclonal antibody for cytochrome P450IA1 Liver mkrosomes were prepared from rats that received four daily i.p. injections of 3MC (25 mg/kg body weight) dissolved in olive oil as described previously (15). Cytochrome P4501A1 was purified as described elsewhere (16,17) with a minor modification of the original procedure (18). The specific content was 16.3 nmol/mg protein. Antiserum for the cytochrome P450IA1 was prepared in New Zealand white rabbits as described previously (13), and an immunoglobulin fraction was prepared from the immune serum by ammonium sulfate fractionation. Specificities of the ann-cytochrome P450IA1 antibody thus obtained were confirmed by Ouchterlony technique and Western blotting analysis as previously described (13). Western blotting analysis indicated that one major band (56 kDa) corresponding to the purified cytochrome P450IA1 was observed in colonic microsomes of 3MC-treated rats (13).
S.Kawata et at. a cryostat at —20°C and air-dried. The sections were incubated with anticytochrome P450IA1 antibody diluted to 1/50-1/500 (700-70 fig protein/ml) for 30 min at 37 °C in a moist chamber. After rinsing with washing buffer, the sections were incubated with goat FITC-conjugated anti-rabbit IgG antiserum for 30 min at 37°C. The sections were examined under a Nikon fluorescence microscope. For the control study, the anti-cytochrome P450IA1 antibody (500 fig protein/ml) was incubated with purified cytochrome P450IA1 (500 fig protein/ml) for 60 min at room temperature and the supernatant of the mixture was obtained by centrifugation at 10 000 g for 20 min. The absorbed antibody thus obtained or preimmune serum were used for control staining. Immunohistochemical analysis was performed on three sections for each rat. Photographs were taken under conditions of automatic exposure with the cable release system whjch allows 100% transmission of light to the camera. Exposure times for tissues stained with the antibody were recorded, and comparable exposure times were used when photographing tissue stained with the absorbed antibody or preimmune serum. The intensity of staining was interpreted blindly by visual inspection of photographs.
The reaction mixture for 7-ethoxycoumarin O-deethylase activity contained rat colonic or small intestine microsomes (4 mg protein) and 0.5 mM 7-ethoxycoumarin as substrate in a final volume of 1 ml of 50 mM potassium phosphate (pH 7.4). After preincubation for 5 min at 37°C, the reaction was conducted by the addition of 0.5 fimol of NADPH, 125 fimol of glucose-6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase. The reaction was stopped after 5 min by the addition of 125 fil of 12% (v/v) TCA, and the fluorescence of 7-hydroxycoumarin was measured according to the method described by Greenke and Poland (21). The linearity of the assays was confirmed by both time and microsomal protein concentrations. Protein concentration was determined according to the method of Lowry et al. (22), using bovine serum albumin as standard. Western and dot immunoblotting Western blotting was carried out using anti-cytochrome P450IA1 antibody as described previously (13). Colonic microsomes (20 Mg protein/lane) were separated on a 10% SDS-polyacrylamide gel and blotted onto nitrocellulose membrane. The solubilized microsomal proteins on the membrane were incubated with anticytochrome P450IA1 antibody (primary antiserum) and with goat horseradish peroxidase-conjugated anti-rabbit IgG antiserum (secondary antiserum). Immunoreactive protein bands were detected using 0.05% (w/v) 4-chloro-l-naphthol and 0.0125% (v/v) HjCv All operations of dot-blotting were carried out at room temperature. After washing with 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl (TBS), a nitrocellulose membrane was pretreated with TBS containing 3% ovalbumin for 2 h to block non-specific binding sites. Dot-blotting was performed on the nitrocellulose membrane after solubilization of protein in TBS containing 0.1 % (v/v) Tween 20. After washing with TBS containing 0.1 % (v/v) Tween 20, the membrane was incubated with TBS containing 1/500 diluted (70 fig protein/ml) anti-cytochrome P450IA1 antibody for 2 h. After washing the membrane with TBS containing 0.1 % (v/v) Tween 20, the membrane was incubated with TBS containing ll5I-labcled donkey anti-rabbit IgG (3 x 105 c.p.m./ml) for 2 h. The membrane was washed, air-dried and exposed to X-ray film at —70°C. Statistical analysis The data were statistically analyzed using the Student's r-test. Chemicals Goat FITC-conjugated anti-rabbit IgG antiserum was purchased from Cooper Biomedical and goat horseradish peroxidase-conjugated anti-rabbit IgG antiserum from Miles Yeda Ltd. 123I-Labeled donkey anti-rabbit IgG was purchased from Amersham Corp. 3MC and 7-hydroxycoumarin were obtained from Wako Pure
2122
Results 7-Ethoxycoumarin O-deethylase activity of the colonic microsomes from control diet-fed untreated rats was significantly different between the proximal and distal portions (P < 0.01 compared to distal portion from wheat bran-fed 3MC-treated rats.
B
P450IAI—
1.0 p g protein 2.0 1.0 0.5 1
2
1
Fig. 1. Western Hot and dot-blotting analyses of microsomes from the distal portion of rat colon. (A) Western Wotting; lane 1, microsomes (20 fig protein/lane) from a control diet-fed 3MC treated rat; lane 2, microsomes (20 fig protein/lane) from a control diet-fed untreated rat. (B) Dot-blotting; lane 1, microsomes (0.5, 1, 2, 4 fig protein/dot) from a control diet-fed untreated rat; lane 2, microsomes (0.5, 1, 2, 4 fig protein/dot) from a control diet-fed 3MC-trcated rat; lane 3, microsomes (0.5, 1, 2, 4 fig protein/dot) from a wheat bran-fed 3MC-treated rat.
Downloaded from http://carcin.oxfordjournals.org/ at McGill University Libraries on December 10, 2012
Enzyme assay of small intestine and colon microsomes 7-Ethoxycoumarin O-deethylase activity was assayed in the small intestine and colon microsomes from wheat bran-fed 3MC-treated rats (n = 8), control dietfed 3MC-treated rats (« = 8), wheat bran-fed untreated rats (n = 8), and control diet-fed rats (n = 8). The colon was cut off from the end of the cecum to the end of the rectum and then divided into two portions: proximal and distal portions. Each portion of the colon was flushed with ice-cold 1.14% (w/v) KC1. Colonic microsomes were prepared from the proximal and distal portions as described previously (13) according to the method of Fang and Strobel (19,20), using phenylmethylsulfonyl-fluoride as a protease inhibitor. Colon mucosal cells were scraped from the everted colon with the edge of a glass slide into cold 10 mM Tris-HCl buffer (pH 7.4) containing 0.14 M KC1, 10 mM EDTA and 1 mM dithiothrehol. The cells were homogenized and centrifugal at 9000 g for 10 min. The 9000 g supernatant fraction was centrifuged at 105 000 g for 30 min to collect the microsomal fraction. A 25 cm portion of small intestine, distal to the pylorus, was removed and then small intestine microsomes were prepared by the same procedure. The washed microsomal pellet was resuspended in 0.25 M sucrose solution containing 10 mM EDTA and stored at - 8 0 ° C .
Chemicals Industries, Ltd, and 7-ethoxycoumarin from Aldrich Chem. Co. Ltd. All other chemicals were of the best grade available commercially.
Effect of dietary fiber on colonic P450 Induction
A strong positive reaction to immunostaining for cytochrome P450IA1 was seen in the distal portion of the colon of control diet-fed 3MC-treated rats (Figure 2). The staining was localized in the cytoplasm of the mucosal surface epithelium. However, in control diet-fed untreated, wheat bran-fed untreated and wheat bran-fed 3MC-treated rats, a faint immunofluorescence was observed in the surface epithelium. No reaction to immunostaining for cytochrome P450IA1 was obtained when the primary antiserum was replaced by the antibody absorbed with purified cytochrome P450IA1 or preimmune rabbit serum. Discussion Colonic mucosal cells have a cytochrome P45O-dependent mixed function oxidase system analogous to that of hepatic microsomes (11,12,19,20,23). Recently, Stralka and Strobel (24) reported the presence of a stable, active mixed function oxidase system in human colon mucosa which displays preferential activity toward nitrogenous compounds and provides a mechanism for the activation of carcinogens. Its distribution in the colon appears to parallel the reported incidence of human colonic carcinomas (24). In this study, 7-ethoxycoumarin O-deethylase activity in the distal portion of the colon was significantly higher than that
B
Fig. 2. Immunohistochemistry of the distal portion of rat colon for cytochrome P450IA1. (A) Immunohistochemistry of a section of colon of a control diet-fed 3MC-treated rat. A strong positive reaction to ami
Effect of dietary fiber on cytochrome P450IA1 induction in rat colonic mucosa
Sumio Kawata, Shinji Tamura, Yukihiko Matsuda, Nobuyuki Ito and Yuji Matsuzawa Second Department of Internal Medicine, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan
Introduction Epidemiologic studies on human cancer incidence under various environmental pollution conditions have suggested that populations consuming low fiber diets tend to have a greater incidence of colon cancer than those consuming higher fiber diets (1—3). The preventive effect of dietary fibers in colon carcinogenesis in animal models has been extensively studied (4-10). Strobel et al. (11,12) reported that the colon contains a mixed function oxidase system capable of activating carcinogens to their proximate reactive form. Their studies provided a good basis for the possibility that the cytochrome P450 system in the colon may play a role in tissue-specific carcinogenesis. Recently, a cytochrome P450IA1, an isoform of cytochrome P450, was found to be localized in the cytoplasm of the epithelium of colonic mucosa of rats treated with 3-methylcholanthrene (3MC*> (13) or j3-naphthoflavone (14). To clarify the effect of dietary fibers on colon chemical carcinogenesis, it is important to find whether dietary fibers can prevent the induction of the cytochrome P450-dependent mixed function oxidase system in the colonic mucosa by chemical inducers and carcinogens per se. In this study, we examined the effect of wheat bran in diet on cytochrome P450IA1 induction in the colonic mucosa of rats that received a single intragastric injection of 3MC by stomach tube. •Abbreviations: 3MC, 3-methylcholanthrene. © Oxford University Press
Immunohistochemical analysis Immunohistochemical analysis of cytochrome P450IA1 (PASQ^Q) in the colonic mucosa was performed as described previously (13). Wheat bran-fed 3MC-treated rats (n = 5), control diet-fed 3MC-treated rats (n = 5), wheat bran-fed untreated rats (n = 5), and control diet-fed untreated rats (n = 5) were made to fast 24 h before they were killed under diethylether anesthesia. Specimens were cut from proximal and distal portions of the colon and rinsed in ice-cold 10 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl (PBS), then embedded in Tissue-Tek (Miles Scientific) and frozen. Sections ~ 6 yia thick were sliced on Table I. Composition of rat diets administered Ingredient
% of total diet Control diet
Fiber diet
Casein" Safflower oil Mineral mixture1' Vitamin mixture1 Comstarch Wheat bran"1
27 10 6 2 55 0
27 10 6 2 50 5
Total ingredients
100
100
"Casein (HC1); edible extra grade hydrochloric acid casein. b Mineral mixture to supply the following minerals (g/100 g of mixture, Oriental Yeast Co.): sodium chloride, 4.66; potassium phosphate, 25.72; sodium phosphate, 9.35; anhydrous magnesium sulfate, 7.17; calcium lactate; 35.09; calcium phosphate, 14.56; ferrous citrate, 3.18; manganese sulfate, 0.12; potassium iodide, 0.01; zinc carbonate, 0.11; cupric sulfate, 0.03. Vitamin mixture (per 100 g of mixture, AIN-76, Oriental Yeast Co.): vitamin A, 40 000 IU; vitamin D 3 , 10 000 IU; vitamin E acetate, 500 mg; vitamin K3, 0.5 mg; choline, 20.0 g; vitamin B,, 60 mg; vitamin B2, 60 mg; vitamin R6, 70 mg; vitamin B| 2 , 0.1 mg; calcium D-pantothenate, 160 mg; folic acid, 20 mg; biotin, 2 mg; nicotinic acid, 300 mg. d Wheat bran was purchased from Oriental Yeast Co., Japan.
2121
Downloaded from http://carcin.oxfordjournals.org/ at McGill University Libraries on December 10, 2012
The effect of dietary fiber on the induction of cytochrome P450IA1 in rat colonic mucosa after a single intragastrk injection of 3-methykhoIanthrene (3MC, 20 mg/kg) was investigated by examining the drug-metabolizing enzyme activity, immunoblotting for cytochrome P450IA1 and immunohistochemistry. 7-Ethoxy coumarin-O-deethy lase activities were ~ 20-fold higher in mkrosomes from both proximal and distal portions of the colonic mucosa of control diet-fed 3MC-treated rats compared with those of control dietfed untreated rats. Strong immunofluorescence for cytochrome P450IA1 was localized in the cytoplasm of the colonk mucosa surface epithelium from the control diet-fed 3MC-treated rats. 7-Ethoxycoumarin-O-deethylase activity and cytochrome P450IA1 content determined by immunoblotting were significantly lower in wheat bran-fed 3MCtreated rats than in control diet-fed 3MC-treated rats. Immunohistochemical analysis showed much weaker immunofluoresence for cytochrome P450IA1 in the surface epithelium of the colonic mucosa of the wheat bran-fed 3MCtreated rats. These observations suggested that dietary fiber can affect the induction of cytochrome P450IA1 in colonic mucosa by dietary inducers or carcinogens.
Materials and methods Animals Adult male Wistar rats weighing —250 g were used. The rats were housed with free access to a nutritionally complete semisynthetic control diet or a high fiber diet containing 5% wheat bran (Table I). Water was supplied ad libitum at all times. Both diets were prepared by mixing in a blender. Each diet was packaged, marked with the appropriate color code, and stored as a dry powder until use. After 2 weeks exposure to the dietary regimens, the rats were randomly subdivided and received a single intragastric injection of 1.0 ml corn oil or 3MC, 20 mg/kg body weight, in 1.0 ml corn oil by stomach tube. After 48 h, the rats were killed under diethylethcr anesthesia. The animals were weighed immediately prior to death and the body weight was not significantly different among the four groups: control diet-fed untreated, control diet-fed 3MC-treated, wheat bran-fed untreated and wheat bran-fed 3MC-treated rats. Production of polyclonal antibody for cytochrome P450IA1 Liver mkrosomes were prepared from rats that received four daily i.p. injections of 3MC (25 mg/kg body weight) dissolved in olive oil as described previously (15). Cytochrome P4501A1 was purified as described elsewhere (16,17) with a minor modification of the original procedure (18). The specific content was 16.3 nmol/mg protein. Antiserum for the cytochrome P450IA1 was prepared in New Zealand white rabbits as described previously (13), and an immunoglobulin fraction was prepared from the immune serum by ammonium sulfate fractionation. Specificities of the ann-cytochrome P450IA1 antibody thus obtained were confirmed by Ouchterlony technique and Western blotting analysis as previously described (13). Western blotting analysis indicated that one major band (56 kDa) corresponding to the purified cytochrome P450IA1 was observed in colonic microsomes of 3MC-treated rats (13).
S.Kawata et at. a cryostat at —20°C and air-dried. The sections were incubated with anticytochrome P450IA1 antibody diluted to 1/50-1/500 (700-70 fig protein/ml) for 30 min at 37 °C in a moist chamber. After rinsing with washing buffer, the sections were incubated with goat FITC-conjugated anti-rabbit IgG antiserum for 30 min at 37°C. The sections were examined under a Nikon fluorescence microscope. For the control study, the anti-cytochrome P450IA1 antibody (500 fig protein/ml) was incubated with purified cytochrome P450IA1 (500 fig protein/ml) for 60 min at room temperature and the supernatant of the mixture was obtained by centrifugation at 10 000 g for 20 min. The absorbed antibody thus obtained or preimmune serum were used for control staining. Immunohistochemical analysis was performed on three sections for each rat. Photographs were taken under conditions of automatic exposure with the cable release system whjch allows 100% transmission of light to the camera. Exposure times for tissues stained with the antibody were recorded, and comparable exposure times were used when photographing tissue stained with the absorbed antibody or preimmune serum. The intensity of staining was interpreted blindly by visual inspection of photographs.
The reaction mixture for 7-ethoxycoumarin O-deethylase activity contained rat colonic or small intestine microsomes (4 mg protein) and 0.5 mM 7-ethoxycoumarin as substrate in a final volume of 1 ml of 50 mM potassium phosphate (pH 7.4). After preincubation for 5 min at 37°C, the reaction was conducted by the addition of 0.5 fimol of NADPH, 125 fimol of glucose-6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase. The reaction was stopped after 5 min by the addition of 125 fil of 12% (v/v) TCA, and the fluorescence of 7-hydroxycoumarin was measured according to the method described by Greenke and Poland (21). The linearity of the assays was confirmed by both time and microsomal protein concentrations. Protein concentration was determined according to the method of Lowry et al. (22), using bovine serum albumin as standard. Western and dot immunoblotting Western blotting was carried out using anti-cytochrome P450IA1 antibody as described previously (13). Colonic microsomes (20 Mg protein/lane) were separated on a 10% SDS-polyacrylamide gel and blotted onto nitrocellulose membrane. The solubilized microsomal proteins on the membrane were incubated with anticytochrome P450IA1 antibody (primary antiserum) and with goat horseradish peroxidase-conjugated anti-rabbit IgG antiserum (secondary antiserum). Immunoreactive protein bands were detected using 0.05% (w/v) 4-chloro-l-naphthol and 0.0125% (v/v) HjCv All operations of dot-blotting were carried out at room temperature. After washing with 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl (TBS), a nitrocellulose membrane was pretreated with TBS containing 3% ovalbumin for 2 h to block non-specific binding sites. Dot-blotting was performed on the nitrocellulose membrane after solubilization of protein in TBS containing 0.1 % (v/v) Tween 20. After washing with TBS containing 0.1 % (v/v) Tween 20, the membrane was incubated with TBS containing 1/500 diluted (70 fig protein/ml) anti-cytochrome P450IA1 antibody for 2 h. After washing the membrane with TBS containing 0.1 % (v/v) Tween 20, the membrane was incubated with TBS containing ll5I-labcled donkey anti-rabbit IgG (3 x 105 c.p.m./ml) for 2 h. The membrane was washed, air-dried and exposed to X-ray film at —70°C. Statistical analysis The data were statistically analyzed using the Student's r-test. Chemicals Goat FITC-conjugated anti-rabbit IgG antiserum was purchased from Cooper Biomedical and goat horseradish peroxidase-conjugated anti-rabbit IgG antiserum from Miles Yeda Ltd. 123I-Labeled donkey anti-rabbit IgG was purchased from Amersham Corp. 3MC and 7-hydroxycoumarin were obtained from Wako Pure
2122
Results 7-Ethoxycoumarin O-deethylase activity of the colonic microsomes from control diet-fed untreated rats was significantly different between the proximal and distal portions (P < 0.01 compared to distal portion from wheat bran-fed 3MC-treated rats.
B
P450IAI—
1.0 p g protein 2.0 1.0 0.5 1
2
1
Fig. 1. Western Hot and dot-blotting analyses of microsomes from the distal portion of rat colon. (A) Western Wotting; lane 1, microsomes (20 fig protein/lane) from a control diet-fed 3MC treated rat; lane 2, microsomes (20 fig protein/lane) from a control diet-fed untreated rat. (B) Dot-blotting; lane 1, microsomes (0.5, 1, 2, 4 fig protein/dot) from a control diet-fed untreated rat; lane 2, microsomes (0.5, 1, 2, 4 fig protein/dot) from a control diet-fed 3MC-trcated rat; lane 3, microsomes (0.5, 1, 2, 4 fig protein/dot) from a wheat bran-fed 3MC-treated rat.
Downloaded from http://carcin.oxfordjournals.org/ at McGill University Libraries on December 10, 2012
Enzyme assay of small intestine and colon microsomes 7-Ethoxycoumarin O-deethylase activity was assayed in the small intestine and colon microsomes from wheat bran-fed 3MC-treated rats (n = 8), control dietfed 3MC-treated rats (« = 8), wheat bran-fed untreated rats (n = 8), and control diet-fed rats (n = 8). The colon was cut off from the end of the cecum to the end of the rectum and then divided into two portions: proximal and distal portions. Each portion of the colon was flushed with ice-cold 1.14% (w/v) KC1. Colonic microsomes were prepared from the proximal and distal portions as described previously (13) according to the method of Fang and Strobel (19,20), using phenylmethylsulfonyl-fluoride as a protease inhibitor. Colon mucosal cells were scraped from the everted colon with the edge of a glass slide into cold 10 mM Tris-HCl buffer (pH 7.4) containing 0.14 M KC1, 10 mM EDTA and 1 mM dithiothrehol. The cells were homogenized and centrifugal at 9000 g for 10 min. The 9000 g supernatant fraction was centrifuged at 105 000 g for 30 min to collect the microsomal fraction. A 25 cm portion of small intestine, distal to the pylorus, was removed and then small intestine microsomes were prepared by the same procedure. The washed microsomal pellet was resuspended in 0.25 M sucrose solution containing 10 mM EDTA and stored at - 8 0 ° C .
Chemicals Industries, Ltd, and 7-ethoxycoumarin from Aldrich Chem. Co. Ltd. All other chemicals were of the best grade available commercially.
Effect of dietary fiber on colonic P450 Induction
A strong positive reaction to immunostaining for cytochrome P450IA1 was seen in the distal portion of the colon of control diet-fed 3MC-treated rats (Figure 2). The staining was localized in the cytoplasm of the mucosal surface epithelium. However, in control diet-fed untreated, wheat bran-fed untreated and wheat bran-fed 3MC-treated rats, a faint immunofluorescence was observed in the surface epithelium. No reaction to immunostaining for cytochrome P450IA1 was obtained when the primary antiserum was replaced by the antibody absorbed with purified cytochrome P450IA1 or preimmune rabbit serum. Discussion Colonic mucosal cells have a cytochrome P45O-dependent mixed function oxidase system analogous to that of hepatic microsomes (11,12,19,20,23). Recently, Stralka and Strobel (24) reported the presence of a stable, active mixed function oxidase system in human colon mucosa which displays preferential activity toward nitrogenous compounds and provides a mechanism for the activation of carcinogens. Its distribution in the colon appears to parallel the reported incidence of human colonic carcinomas (24). In this study, 7-ethoxycoumarin O-deethylase activity in the distal portion of the colon was significantly higher than that
B
Fig. 2. Immunohistochemistry of the distal portion of rat colon for cytochrome P450IA1. (A) Immunohistochemistry of a section of colon of a control diet-fed 3MC-treated rat. A strong positive reaction to ami
