Induction of tumors in the colon and liver of the immunodeficient (SCID) mouse by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ)—modulation by long-chain fatty acids

Elsayed I. Salim,3, Hideki Wanibuchi, Keiichirou Morimura, Takashi Murai1, Susumu Makino1, Taisei Nomura2 and Shoji Fukushima,4

First Department of Pathology, Osaka City University Medical School, 143 Asahi-machi, Abeno-Ku, Osaka 545-8585,
1 Aburahi Laboratory, Shionogi Co. Ltd, Shiga, and
2 Department of Radiation Biology, Osaka University Medical School, Osaka, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have recently shown that immunodeficient (SCID) mice, which lack functional T and B cells, are highly susceptible to low dose site specific induction of colon aberrant crypt foci (ACF), surrogates for colon tumors, by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ). To test whether long-term exposure to a high dose in the diet might prove carcinogenic to the SCID mouse colon, in contrast to other mice strains tested to date, the compound was administered at 300 p.p.m. in the diet to female 6–7-week-old SCID mice for 32 weeks. IQ induced high numbers of ACF, hyperplastic polyps, dysplasia, and colon adenomas, as well as hepatocellular altered foci and liver adenomas. Induction of colon tumors did not correlate with the main sites where ACF developed, the proximal colon, however, being seen mainly in the mid and distal colon. Induction of colon tumors correlated significantly with the incidence of dysplasia, crypt height, the mitotic index, cell proliferation and numbers of 8-hydroxydeoxyguanosine (8-OHdG)-positive cells in the colon crypt, particularly in mid and distal colon. Administration of 20% {omega}{omega}-6 polyunsaturated fatty acids (corn oil), {omega}{omega}-3 polyunsaturated fatty acids (perilla oil), or monounsaturated fatty acids (olive oil) simultaneously with IQ in the diet resulted in: (i) inhibition of colon and liver tumor induction by corn and perilla oil, whereas olive oil showed no effects; (ii) no reduction in total numbers of ACF by corn oil or perilla oil but significant suppression in the olive oil treated group; (iii) inhibition of tumor development particularly by {omega}{omega}-3 polyunsaturated fatty acids in perilla oil, correlating significantly with decreased cell proliferation in both colon and liver and a marked decrease in crypt heights and mitotic indices. Selective reduction in the numbers of 8-OHdG-positive nuclei, mainly in the middle and distal colon crypts, was also found to correlate with tumor inhibition. Thus, the results indicate carcinogenicity of IQ in the colon of the SCID mouse and preventive effects of polyunsaturated fatty acids.

Abbreviations: ACF, aberrant crypt foci; DNA–PK, DNA-dependent protein kinase; HAAs, heterocyclic aromatic amines; 13-S-HODE, 13-S-hydroxyoctadecadienoic acid; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; 15-LOX, 15-lipoxygenase; MeIQx, 2-amino-3,8-methylimidazo[4,5-f]quinoxaline; 8-OHdG, 8-hydroxydeoxyguanosine; 7-OHIQ, N-hydroxyl IQ; PBS, phosphate buffered solution; PC, phosphatidylethanolamine; PUFAs, polyunsaturated fatty acids; PhIP, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; SCID, severe combined immunodeficiency


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The risk of certain types of cancer is increasing, mainly due to dietary factors (1). Most epidemiological studies have found a positive correlation between dietary intake of foodstuff containing traces of heterocyclic aromatic amines (HAAs) or high fat diet and the risk of developing cancer (2). This provides a rationale for chemoprevention (3). 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), one HAA demonstrating high mutagenicity in vitro (4), was found to induce tumors in the liver, large intestine, small intestine, and zymbal gland of rats (5), and hepatocellular carcinomas in cynomolgus monkeys (6) with chronic feeding. Various experimental protocols with immunocompetent mice have shown that IQ can induce liver, lung or forestomach tumors (7,8). However, when administered at different dose levels and for different time intervals, no tumors were observed in the colon (9–11). In a previous study, we established that IQ when administered at low doses in the diet to immunodeficient (SCID) mice causes marked elevation in cellular proliferation in the colon epithelium, as well as a low dose-dependent increase in the incidence of putative preneoplastic aberrant crypt foci (ACF) (12). We therefore hypothesized that long-term or high dose exposure to IQ might elicit colon cancer development in these mice.

The mutagenicity and carcinogenicity of IQ is considered initially to involve oxidation of the exocyclic amino group to its corresponding N-hydroxyl-IQ by liver CYP1A1 and 1A2 (13). Subsequent O-acetylation or sulfonation of the exocyclic group (14), is believed to result in the formation of the ultimate genotoxic species, which is capable of binding to DNA, leading to formation of DNA–adducts, and in turn mutations and neoplastic transformation. Recently, it was shown that HAAs, including IQ, have the ability to induce DNA double-strand breaks and cellular transformation in the rat liver S9 and C3H/M2 fibroblast cell lines (15). Also DNA and protein adducts of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), another potent HAA, have been found in human blood and colonic tissues (16). Because daily exposure to IQ and other HAAs in food may present a potent carcinogenic risk to humans, it is important to understand the mechanisms underlying their induction of changes at the cellular level, and the impact of other dietary or biological factors such as fats and the immune system.

SCID mice congenitally lack functional T and B lymphocytes due to a mutation in the SCID gene that results in defects in the repair of DNA double-strand breaks, due to the lack of DNA-dependent protein kinase (DNA–PK) (17,18). However, recent experiments showed that SCID cells are able to activate p53 and p21 proteins, which in turn induce G1 and G2 arrest in the cell cycle or apoptosis in response to DNA damage (19), indicating some ability to repair double-strand break repairs, although at a reduced rate; the cells lack the ability to undergo repair of sublethal damage (20,21).

Epidemiological data have been obtained in the recent decades indicating a key role for the amount of dietary fat or red meat intake in the pathogenesis of different neoplasms in humans, such as cancers of the colon, breast and possibly the prostate (22). Studies on laboratory animals have also provided evidence incriminating the fatty acid composition of the diet as a major determinant of risk of tumor development (23). Recently, researchers have sought to identify the relative tumor-modulating capabilities of different types of dietary fats such as those containing high levels of {omega}-6 polyunsaturated fatty acids such as linoleic acid (c18:2, {omega}-6), which have been shown to enhance colon carcinogenesis in rodents (24), and oils rich in {omega}-3 polyunsaturated fatty acids which in contrast, display inhibitory effects (22). Moreover, a correlation between dietary fatty acids, particularly polyunsaturated fatty acids, and the activity of the immune system has emerged, indicating that consumption of particular forms can be used to reduce the severity of diseases resulting from a defective immune system, such as autoimmunity and certain types of cancers and to prolong survival of transplanted organs (25,26). However, the precise mechanisms underlying their anti-inflammatory, anti-tumor, and immunomodulating effects remain largely unknown (27).

Enhancement of cell proliferation is widely understood to be an important factor determining carcinogenic organ specificity by increasing the likelihood of genetic alteration (28). Genotoxic carcinogenic substances in general, and HAAs in particular, enhance cell proliferation in their target tissues. For example, 2-amino-3,8-methylimidazo[4,5-f]quinoxaline (MeIQx), another HAA that is a hepatocarcinogen, causes elevated cell division in the liver but not in the colon or kidney (29). In the colon, increased numbers of cycling cells or mitoses, leading to expansion of the cell proliferation zone and increased crypt height, as well as depressed apoptosis, are considered risk factors for tumor development (30).

There is also increasing evidence for an important role for free radicals in chemically induced carcinogenesis (31). Their generation with HAAs could act together with HAA-metabolites to cause DNA adducts (32). Generation of 8-hydroxydeoxyguanosine (8-OHdG) is thought accurately to reflect oxidative damage to DNA and is the most common among many endogenous oxidative DNA modifications formed under conditions of oxidative stress (33). Formation of 8-OHdG has been demonstrated in target organ DNA in a wide variety of in vivo experimental carcinogenesis models using either genotoxic or non-genotoxic carcinogens (34) and recently this parameter has attracted attention as an important biomarker of carcinogenic risk to humans (35). In the present experiment the possible involvement of 8-OHdG in oxidative stress caused by IQ or fatty acids was therefore also assayed.

The aim of the present study was to examine whether long-term administration of IQ would induce tumors in the colons of female SCID mice, and determine if supplementation with different fatty acids in the diet could exert modulatory effects when simultaneously administered with IQ, with special attention to mechanistic aspects.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and diet
IQ was purchased from the Nard Inst. Ltd (Amagasaki, Hyogo, Japan) and mixed in Oriental MF powdered diet (Oriental Yeast Co., Tokyo, Japan) at a concentration of 300 p.p.m., either alone or together with 20% {omega}-6 polyunsaturated fatty acids (corn oil), 20% {omega}-3 polyunsaturated fatty acids (perilla oil) or 20% monounsaturated fatty acids (olive oil). The oils obtained from Otayushi Co. Ltd (Okazaki, Aichi, Japan) were extra virgin extractions with extreme low acidity (0.14% for corn oil, 0.17% for both perilla and olive oils). The experimental diets were prepared once a week by adding the oils to the basal laboratory diets. The final w/w composition of the diets was 20% casein, 51% sucrose, 4% cellulose, 0.15% choline chloride, 4% mineral mixture, 1% vitamin mixture and 20% test oil. The diet had standard levels of nutrients. After processing, all diets were sterilized by {gamma}-irradiation and stored in vacuum-sealed bags at 4°C until use. The food in the animal cages was shaded from light and changed every two days. The concentrations of the main fatty acids in the oils are shown in Table IGo.


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Table I. Chemical properties and fatty acids concentrations (%) of the oils used
 
Animals
Seventy-five female SCID (scid/Os Shi) mice, with a C.B-17-scid/scid background, were obtained at 3–4 weeks of age from Aburahi Laboratory (Shionogi Co. Ltd, Shiga, Japan). They were kept housed five per cage in sterilized plastic cages with wood chips for bedding in closed closets controlled at a temperature 22 ± 1°C and a relative humidity of 50.5 ± 5, with a 12 h light:12 h dark cycle. Cages were covered by Clean S filter caps specific for mice cages (Clea Japan Co. Ltd, Osaka, Japan), for further prevention of infection. Drinking water was autoclaved at 120°C for 20 min and changed every other day. Sterilized food and water were available ad libitum. Mouse body weights, food intake and water consumption were measured weekly.

Experimental procedures
After three weeks acclimation, 75 female SCID mice were randomized at 6–7 weeks of age into five groups of 15 mice each. Group 1 was administered the IQ alone in the basal diet. Group 2 received the IQ and 20% corn oil mixed together in the diet. Group 3 received IQ and 20% perilla oil and Group 4, IQ and 20% olive oil, while Group 5 received the basal diet and served as a non-treated control. The experimental period was 32 weeks under limited pathogen-free conditions, as described above. Female mice were chosen because they showed in our previous study (12) lower mortality than males and similar sensitivity regarding ACF induction. Data for the small number of mice that became moribund or died before the termination were not included in the present study.

The animals were killed under ether anesthesia at the termination and all the organs and tissues were investigated for the existence of tumors after being weighed. Sketches of all the tumors of the colon and liver and in other organs were drawn to facilitate histological quantitation. Colons were excised, flushed in saline, inflated by intraluminal injection of 10% phosphate buffered formalin, then slit opened along the longitudinal median axis from the caecum to anus and fixed flat between two pieces of filter paper in 10% phosphate buffered formalin solution for further investigation of tumors and ACF. All body organs were excised and fixed in the same fixative.

Histological evaluation
Main organs such as the liver, spleen, kidneys, lungs and the parts of the alimentary canal as well as all lesions were embedded in paraffin, sectioned at 3–4 µm in thickness, and prepared for routine histological examination with hematoxylin and eosin staining. The lesions and tumors found were classified according to the criteria of the International Agency for Research on Cancer (WHO) (36).

ACF count assay
After fixation for at least 24 h at 4°C, colons were stained with 0.2% methylene blue (in H2O) for 3–5 min, divided into proximal, intermediate, and distal segments, and examined for ACF by light microscopy at 40x and 100x magnification using the following criteria for identification: (i) increased size compared with normal crypts; (ii) enlarged pericryptal zone; (iii) slight elevation above the surrounding mucosa; (iv) more elongated shape of the luminal opening. The number of ACF in each segment, as well as the number of aberrant crypts in each focus, the ‘crypt multiplicity’, were counted. Immediately after scoring ACF, samples collected from the proximal, intermediate and distal colons were prepared for routine histological examination.

Immunohistochemical staining for PCNA
A mouse monoclonal antibody to PCNA (PC 10, Code no. M0744, Dako A/S, Denmark) was used with the avidin–biotin complex method. Briefly, sections were deparaffinized with xylene, hydrated through a graded ethanol series and incubated with 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. They were then incubated with 10% normal horse serum at room temperature for 30 min to block background staining, and overnight at 4°C with PCNA antibody diluted 1:1000 in TBS. After exposure for 30 min at room temperature to biotinylated horse anti-mouse IgG (Vector Lab. Inc., Burlingame, CA), sections were incubated with ABC at 1:25 dilution. Each step was followed by washing with TBS. Peroxidase activity was visualized by treatment with a 0.02% solution of diaminobenzidine tetrahydrochloride containing 0.05% hydrogen peroxide. Then nuclei were counterstained with hematoxylin.

Analysis of cell proliferation and mitosis
To score the cell proliferation parameters, cells stained for PCNA expression were counted randomly in the liver sections, or from the base up to the mouth of each side of complete mid-axially sectioned colon crypts in 4–5 sections from proximal, intermediate and distal colon segments of every mouse. The recorded parameters were: (i) total height of the crypt in number of cells from the base to the mouth (crypt height); (ii) PCNA indices to detect percentages of the total proliferating cells (PCNA index %), as well as to estimate the breadth of the proliferation zone; (iii) the frequency and spatial distribution of mitotic figures in prophase, metaphase, anaphase and telophase (37) within the colon crypts stained with hematoxylin and eosin. The numbers of mitotic cells were divided by the total numbers of crypt cells to generate the mitotic index (MI).

Immunohistochemistry for 8-OHdG
A mouse monoclonal antibody against 8-OHdG (IAb, IgG1, 10 µg/ml), code number (a) MOG-100 freezed type (Nikken Food Co. Ltd, Fukuri, Shizuka, Japan) (38), was used. After deparaffinization, the sections were sequentially treated with 0.3% H2O2 in methanol for 30 min for inactivation of endogenous peroxidase, 0.05 N NaOH in 40% ethanol for 12 min for denaturation of DNA, and 250 µg/ml RNase for 50 min at 37°C. After washing with phosphate buffered solution (PBS), the sections were sequentially exposed to avidin then biotin, 20 min for each step, to block nonspecific binding (Biotin/Avidin System Reagents, Vector Blocking Kit Cat. SP-2001, Vector Lab., Burlingame, USA). The sections were then incubated with 10% horse blocking serum for 40 min to block the background staining before incubation with diluted anti-8-OHdG antibody 1:300 overnight. The sites of peroxidase binding were demonstrated with diaminobenzidine. Sections were counterstained with Mayer’s hematoxylin for microscopic examination. Negative controls were immunostained as above, but with primary serum instead of the anti-8-OHdG antibody.

In situ detection of apoptosis
10% buffered formalin-fixed, paraffin-embedded colon tissue sections were used with an in situ apoptosis detection kit (peroxidase) from Oncor (Gaithersberg, MD) based on TdT dUTP–biotin nick end labeling of fragmented DNA (TUNEL assay), according to the manufacturer’s instructions. The number of apoptotic cells per crypt was recorded to estimate the apoptotic index (%) as for cell proliferation.

Statistical analysis
Significance of differences between group means values for lesions and immunohistochemistry findings were analyzed using SuperANOVA Duncan New Multiple Range analysis. Values for lesion incidence were analyzed using the X2 or exact probability tests (StatView v.4.5; Abacus Concepts Inc., USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Average body, liver and kidney weights (Table IIGo)
The final body weights of the IQ treated mice (Groups 1–4) were markedly decreased as compared with those for the non-treated mice of Group 5. Among the IQ treated groups, only mice fed olive oil showed significant decrease in body weights over Group 1. No differences in absolute or relative (ratio of organ weight:body weight %) spleen or kidneys weights were detected among the groups receiving IQ treatment, but all demonstrated significant increase in relative liver weights as compared with the non-treated mice (Group 5). Daily food intake showed no differences in IQ treated groups, but the animals of Group 5 showed higher food consumption than animals of Groups 1–4. Water consumption data did not vary among the groups.


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Table II. Final body and liver weights, food intake and water consumption
 
Incidence and multiplicity of epithelial lesions in the colon (Table IIIGo)
Continuous feeding of IQ to SCID mice induced a continuous histological spectrum of epithelial lesions in the colon (Figure 1a–cGo). The most pronounced were adenomas with proliferation into the submucosa, and adenomatous polyps. Adenomas were frequent in the distal colon and less frequent in the middle colon. Hyperplastic polyps had a similar distribution pattern, but were also sometimes seen in the proximal colon. Hyperplastic polyps were numerous, while only two cases of villous polyps were encountered. Dysplasia, and focal and diffuse hyperplasia were also noted. The incidence of adenomas in the colons of the IQ-only-treated mice (Group 1) was 50%, with a multiplicity (number per mouse) of 0.8 ± 1.1, no such lesions being observed in the non-treated control mice (Group 5). Treatment with polyunsaturated fatty acids in corn oil (Group 2) or perilla oil (Group 3) significantly inhibited the development of adenomas (incidences 9 and 0%, and multiplicity of 0.1 ± 0.3 and 0 respectively). The two oils also reduced the incidence and multiplicity of hyperplastic polyps and the dysplasia. Treatment with olive oil did not exert significant inhibitory effects on induction of epithelial lesions.


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Table III. Incidences and multiplicity of colon epithelial lesions
 


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Fig. 1. (a–c) Photomicrographs of IQ-induced lesions in colons of SCID mice. (a) Dysplastic crypts in a proximal colon of an IQ-treated mouse from group 1 (arrows). (x200, HE.) (b) A hyperplastic growth of colon mucosa towards the submucosa in the distal colon of a mouse from group 1 (arrow). (x100, HE.) (c) An adenoma in the distal colon of a mouse from group 1, exhibiting downward growth towards the submucosa (arrow). (x100, HE.) (d) Photomicrograph of 8-OHdG staining in colon of IQ-treated SCID mice. Note the high 8-OHdG positivity (arrow) in a hyperplastic polyp in the distal colon of a mouse of group 4. (x200, 8-OHdG.) (e,f) Photomicrographs of liver lesions of IQ-treated SCID mice. (e) A liver adenoma in a mouse of group 1. (x100, HE.) (f) High proliferative capacity of a liver adenoma indicated by increased numbers of PCNA-positive nuclei as compared with surrounding parenchyma (arrows), in a mouse of group 1. (x100, PCNA.).

 
ACF values (Tables IV and VGoGo)
The incidence of ACF was 100% in all IQ treated groups (Groups 1–4) with a distribution mainly in the proximal colon. Only one small aberrant crypt was seen in the colon of a non-treated mouse. Treatment with olive oil, but not corn oil or perilla oil, reduced the total numbers of ACF (i.e. 1–3 ACs or >=4 ACs) (Table IVGo). The inhibition was restricted only in the areas of the proximal colons but not in mid or distal colons (Table VGo). Figure 2Go shows the percentage ratio of adenomas to ACF incidence in all sites of the colon. Adenomas were significantly formed in the mid and distal colons when compared with proximal colons.


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Table IV. Effect of dietary administration of IQ and/or different fatty acids on the induction of ACF in the colons of SCID mice after 32 weeks
 

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Table V. Distribution of ACF in different colonic regions of SCID mice treated with IQ and/or different oils
 


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Fig. 2. Percentage ratio of adenomas:ACF in all sites of the colon. Percentages of adenomas:ACF were significantly higher in mid and distal colons when compared with areas of proximal colons. *P < 0.05, **P < 0.05 columns from left to right are; IQ only; , IQ + corn oil; , IQ + perilla oil; , IQ + olive oil; , non-treatment.

 
Average colon crypt heights (Figure 3Go)
Figure 3Go shows the combined data for crypt heights in the entire colon. In Group 1, the average number of cells per crypt was dramatically increased compared with the non-treated control value (Group 5). Treatment with perilla oil significantly blocked this increase and average crypt heights in the corn oil treated group also showed a tendency to decrease. Comparing crypt heights in different colonic regions (data not shown), the corn oil exerted significant effects in the middle and distal regions of the colon, while values remained almost unchanged in the proximal region. On the other hand, the average colon crypt heights in the olive oil-treated mice were essentially the same as for Group 1.



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Fig. 3. Average mucosal crypt heights for the entire colon. *P < 0.05 versus the non-treatment group; **P < 0.05 versus the IQ alone group. n = 148, 134, 138, 178 and 174, and SE = 1.75, 1.22, 0.82, 1.9 and 0.76 in columns from left to right, respectively.

 
Effects of IQ and lipids on colon crypt cell proliferation (Figure 4Go)
PCNA positive cells were markedly more common in the colon crypts of IQ-treated Groups 1–4 compared with the non-treated control Group 5. The three oils investigated here were found significantly to decrease the PCNA index in all cases. The inhibition of cell proliferation was found only in the middle and distal colon, the proximal colon demonstrating no differences in PCNA indices among the groups (data not shown).



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Fig. 4. PCNA indices for the entire colon. *P < 0.05, versus the non-treatment group; **P < 0.05 versus the IQ alone and non-treatment groups. Number of crypts counted (n) are 178, 133, 125, 172 and 170, and SE = 0.89, 0.87, 0.91, 0.94 and 0.83 in columns from left to right, respectively.

 
Effects of IQ and lipids on colon crypt cell mitosis (Figure 5Go)
Crypt cell mitosis, expressed by percentage (%) or as mean number per colon crypt (Figure 5Go), was significantly increased in mice given IQ either alone or in combination with lipids compared with the control mice in Group 5. However, the corn and perilla oils decreased significantly the crypt cell mitosis with the latter having the most pronounced effect. Olive oil tended to decrease the mitotic level, but, with no significant differences compared with Group 1.



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Fig. 5. Mitotic indices for the entire colon. (A) Mitotic indices (%) for colonic crypts, *P < 0.05 versus the non-treatment group; **P < 0.05 versus the IQ alone and non-treatment groups. (B) Mitotic cells/colonic crypt, *P < 0.05 versus the non-treatment group; **P < 0.05 versus the IQ alone and non-treatment groups. n = 281, 286, 304, 341 and 324, and SE = 0.07, 0.04, 0.06 and 0.03 in columns from left to right, respectively.

 
Effect of IQ and lipids on 8-OHdG staining in colon crypts (Figure 6Go)
Colon crypts of mice of Group 1 demonstrated marked increase in the numbers of 8-OHdG positive cells as compared with the non-treatment group. The middle and distal colon had the highest levels of expression (P < 0.001 proximal colon versus mid and distal colons in Group 1) (Table VIGo). Positive cells were present in the middle and basal parts of the crypts as well as the upper region. Atypical or dysplastic colon crypts, in addition to those found in the hyperplastic polyps, adenomas and adenomatous polyps, all showed extensive staining (Figure 1dGo). Treatment with corn oil and perilla oil significantly decreased the numbers of 8-OHdG-positive cells. Perilla oil supplementation had the greatest effect (P < 0.05 versus corn oil), reducing the numbers almost to the control level. The inhibitory effects of perilla and corn oils were most pronounced in the mid and distal colons (P < 0.001), although perilla oil also depressed the expression levels in the proximal colon (P < 0.05). Olive oil-fed mice had high numbers of positive cells, with expression levels slightly higher than those in the IQ control Group 1 (Figure 6Go).



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Fig. 6. 8-OHdG-positive cell counts (%) for colonic crypts. *P < 0.05 versus the non-treatment group; **P < 0.05 versus the IQ alone group. Number of crypts counted (n) are 171, 184, 211, 189 and 175, and SE are 0.52, 0.38, 0.08, 0.65 and 0.05 in columns from left to right, respectively.

 

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Table VI. 8-OHdG expression levels (%) in different colonic regions of SCID mice treated with IQ and/or different oils
 
Effects on colon crypt apoptosis
The number of apoptotic cells per crypt was evaluated either by means ± SD or as percentages in the colon crypts. The position of apoptotic cells varied from the upper to lower compartments with no obvious differences among the crypt regions. Mean values for Groups 1–5 were 0.49 ± 0.09, 0.412 ± 0.09, 0.35 ± 0.08, 0.483 ± 0.08 and 0.321 ± 0.07 respectively, while the percentages per colon crypt were, 0.70, 0.71, 0.72, 0.70 and 0.64% respectively. No significant effects on apoptosis were detected.

Effects on liver carcinogenesis (Table VIIGo)
IQ treatment in Group 1 animals induced a 58% incidence of liver tumors (mainly hepatocellular adenomas and one hepatocellular carcinoma), with a multiplicity of 1.6 (Figure 1eGo), as well as a 100% incidence of foci of cellular alteration (preneoplastic lesions). Non-treated control mice (Group 5) had no such lesions in their livers, except one mouse with a small eosinophilic altered focus that caused an incidence of 8% in this group. Treatment with corn oil and perilla oil again reduced the incidence of liver adenomas to 0 and 8%, and the multiplicity to 0 and 0.3 per effective mouse, respectively. Olive oil treatment showed a tendency to inhibit the incidence and multiplicity of the liver adenomas, but without statistical significance.


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Table VII. Effect of dietary administration of IQ and/or different fatty acids on induction of liver lesions and tumors in SCID mice after 32 weeks
 
Cell proliferation in the liver
All mice treated with IQ had markedly higher PCNA indices (Figure 1fGo) for liver cells than the non-treated control value (Figure 7Go). Similar to the colon case, the three investigated oils reduced the values as compared with Group 1, with the perilla oil-treated group showing the lowest levels.



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Fig. 7. PCNA indices for liver. *P < 0.05 versus non-treatment group; **P < 0.05 versus the IQ alone and non-treatment groups. Number of sections counted (n) are 66, 62, 57, 60 and 51, and SE are 1.3, 0.88, 0.71, 0.82 and 0.46 in columns from left to right, respectively.

 
Incidence of tumors in the other organs (Table VIIIGo)
A number of mice had thymomas or splenic lymphomas that metastasized in a few cases to lymph nodes, liver or lung, with no difference among the groups. Spontaneous tumors such as a lung adenoma and an adenocarcinoma, a thyroid follicular adenoma, and a skin squamous cell papilloma also developed with no difference among the groups. No mammary tumors were observed.


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Table VIII. Incidences of other tumors induced in SCID mice after dietary treatment with IQ and/or different fatty acids for 32 weeks
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, the administration of IQ to female SCID mice continuously for 32 weeks clearly induced a spectrum of colon epithelial lesions, including adenomas and hyperplastic polyps. This was associated with an increase in cell proliferation, elevated mitotic indices and increased crypt heights along with elevated levels of 8-OHdG in the colon mucosa. The neoplastic development was noticed mainly in the middle and distal colon, in contrast to the ACF which were predominantly located in the proximal colon. Also in the liver, IQ induced increased levels of cell proliferation along with a continuous histopathological spectrum of hepatic cell adenomas and altered foci.

Colon adenomas showing growth towards the submucosa and various types of colon polyps have different importance as precursors of carcinoma development in rodents (39). Adenomas and adenomatous polyps undergo malignant conversion and demonstrate a quantitative relation to carcinomas (40). Hyperplastic polyps, although less frequent, appear readily to undergo malignant transformation and the appearance of dysplastic crypts in hyperplastic mucosa is correlated with subsequent development of carcinomas (40). Deschner et al. (41) described extension of the normal proliferative compartment in DMH-treated mice colon, from the lower two thirds to the cryptal surface, to be characteristic as a stage I abnormality. A shift in the major zone of the DNA synthesis to the upper region of the crypts (stage II), and elevated labeling indices in the whole crypt areas (stage III), leading to elongation of crypts, also facilitate expression of a neoplastic phenotype. Therefore, IQ treatment for a longer time might be expected to induce colon carcinomas in these SCID mice. However, administration of IQ by other investigators (42,43) at 300 p.p.m. to immunocompetent mice such as C57BL6Bya wild-type and Eµ-pim-1 transgenic mice for 24 or 40 weeks did not exert any carcinogenic effect.

It has been reported previously that the dietary fatty acid composition can influence the initiation, promotion and progression stages of experimental carcinogenesis in mice (44). In the present study, the corn and perilla oils caused clear inhibitory effects on both colon and liver carcinogenesis, while olive oil showed a non-significant tendency to inhibit in the liver, and a possible slight stimulatory effect on colon tumor development despite decreasing ACF numbers. Corn oil is rich in linoleic acid (60%) which is a {omega}-6 polyunsaturated fatty acid (cis-C18:2 {omega}-6). Perilla oil has ~63% of {alpha}-linolenic acid which is a {omega}-3 polyunsaturated fatty acid (cis-C18:3 {omega}-3), while olive oil mainly contains (65%) the monounsaturated fatty acid, oleic acid (cis-C18:1 {omega}-9).

In earlier reports, high levels of dietary corn oil rich in {omega}-6 polyunsaturated fatty acids exerted strong tumor enhancing effects on mammary and colon cancer incidence in rats and mice (45). Therefore, the significant reduction in IQ-induced colon and liver tumors observed here might be considered not in line with expectation. However, a search of the literature revealed that a diet containing 5 or 20% corn oil in combination with 10% pectin (46) inhibited significantly the development of colon adenocarcinomas in the rat descending colon. In explanation of the mechanism of action of linoleic acid, Shureiqi et al. (47), showed that human colon cancers are associated with a down regulation in 13-S-hydroxyoctadecadienoic acid (13-S-HODE), the product of 15-lipoxygenase (15-LOX) metabolism of linoleic acid, and that 13-S-HODE can suppress cell proliferation and induce apoptosis in transformed colonic epithelial cells.

Sex differences could also play a role in this respect. It was shown previously that heterocyclic aromatic amines, for example PhIP, can induce cell proliferation in the colons of male rats but not in females although both sexes had the same levels of DNA adducts (48). Also, the response of female rodents to corn oil was found to be different from the males with regard to organ specificity (49). The modifying effects of corn oil are also known to depend on whether it is given during the initiation or promotion phases of carcinogenesis. Reddy (50) showed that when rats were fed 23.5% corn oil during the initiation stage, there was no increase in the incidence of colon tumors as compared with controls, but, when 23.5% corn oil was fed during the post-initiation stage (after carcinogen treatment), there was a significant increase in colon tumor incidence. Administration of corn oil here was simultaneous with long term IQ treatment, so that we might expect a different response in the present model. The inhibitory effects were not due to a decrease in the consumed IQ dose because: (i) food intake did not show significant differences among the IQ treated groups; (ii) IQ induced as high numbers of ACF in the colons of mice treated with corn oil as in Group 1, these lesions being known to be induced dose-dependently by chemical carcinogens (51); (iii) induction of liver preneoplastic foci was 100% with a dramatic increase in cell proliferation in the corn oil group similar to the other IQ-administered groups.

The inhibitory effects of perilla oil containing {omega}-3 polyunsaturated fatty acids observed here are in line with previously reported data for rats and mice (37,48). In the present study, perilla oil inhibited adenoma and liver tumor induction, along with a selective and a significant reduction in the 8-OHdG levels, particularly in the middle and distal colon. Modulation of epithelial cell turnover in the colonic crypts may be an important mechanism in the protection afforded against IQ-induced carcinogenesis by {omega}-3 polyunsaturated fatty acids (37). Recently consumption of oils rich in {omega}-3 polyunsaturated fatty acids was shown to attenuate the activity of a number of biochemical pathways implicated in the aberrant regulation of proliferation or cell death during tumorigenesis (52). In the present study, the non-significant promoting effects of olive oil on colon carcinogenesis were accompanied by increase in the mitotic index, crypt heights and high levels of 8-OHdG-positive cells, particularly in mid and distal colons. Although epidemiological studies have indicated a low mortality from colon cancer in the areas where people consume large amounts of olive oil (53), this is not always the case in the animal experiments. Onogi et al. (54) earlier discussed that olive oil containing ~81% of oleic acid had a promoting effect on ACF, while perilla oil containing 56% {omega}-linolenic acid reduced numbers in male rats. Moreover, another study revealed no effect on colon tumor incidence or growth in rats fed diets supplemented with olive oil rich in oleic acid (40%), despite evidence of mammary tumor-promoting properties (55).

w-6 polyunsaturated fatty acids (PUFAs) are highly susceptible to lipid peroxidation resulting in highly reactive substances including superoxide and hydroxyl radicals and a complex range of peroxidized and/or oxygenated lipids that may be broken down to yield various further metabolites (56). In the present study, although linoleic acid is associated more with lipid peroxidation which gives rise to oxidative stress, it should be assumed that the administration of corn oil should increase adenoma formation. But contrary to the predictions, it was found protective. In living cells, {omega}-6 PUFA are not directly responsible for oxidative stress and exhibit lowered antioxidant defense within the cells. Neoplastic cells, as compared with normal cells, are resistant to lipid peroxidation due to impaired or absence of 6-desaturation of essential fatty acids in cancer cells (57). Addition of excess linoleic acid to mice may have lowered antioxidant defense within the cells resulting in a protective effect in this group {omega}-9 fatty acids in olive oil, although not associated with oxidative stress (56), were not protective to adenomas in the present study. It is well known that many neoplastic and cancer cells contain increased levels of antioxidants, including oleic acid and vitamin E. Additional levels of oleic acid in the diet seemed to increase free radical generation in dividing cells, leading to a non-protective and maybe slight promoting effect in this group. From another point of view, administration of IQ poses an acute oxidative stress to the colonic mucosa which is not modulated by oleic acid (58). Oleic acid does not partake in lipid peroxidation therefore it does not affect the 8-OHdG levels in the colonocytes. Linoleic acid does, therefore it scavenges free radicals and reduces 8-OHdG levels.

However, IQ is reported to compromise several antioxidant mechanisms in the intestine: (i) lowering manganese superoxide dismutase activity; (ii) reducing constitutive levels of glutathione peroxidase; and (iii) decreasing the ability to induce catalase (59). Incorporation of highly unsaturated {alpha}-linolenic acid may further compromise the antioxidant status of the cell via increased lipid peroxidation, leading to cell antioxidant viability. Begin et al. (60) showed that {alpha}-linolenic acid and linoleic acid exert similar potency and selectivity in killing cancer cells in vitro due to lipid peroxidation. Therefore, {alpha}-linolenic acid showed inhibitory effects due to its capability in scavenging free radicals and reducing 8-OHdG levels in the colon cells. In line with our results, it was shown recently (61) that dietary {omega}-3 polyunsaturated fatty acids in fish oil modulated methylation-induced DNA adduct levels in the colon during the initial stages of malignant transformation in the rat AOM carcinogenesis model by influencing the expression of O6-methylguanine-DNA-methyltransferase.

On the other hand, data from the present study may indicate that lipid peroxidation is protective against the formation of adenomas but not ACF. On the oleic acid-rich diet, lipid peroxidation cannot occur and therefore ACF formation is significantly reduced. Because of this, many of the ACF formed may become adenomas due to higher 8-OHdG levels in the colonic crypt cells. Previously, it was supposed that olive oil might positively influence metabolic activation in the liver (62), but this did not appear to be the case here since there was no marked effect on liver carcinogenesis. In our previous study using SCID mice (12), we showed that the induction of ACF mainly in the proximal colon was correlated with a dose-dependent elevation in cell proliferation in the three colon segments, but particularly in the proximal colon. In the present and a previous study (63), no correlation was detected between the site of most frequent ACF and site of tumor induction, leading us to suggest that these lesions are a direct and early biological response to cellular proliferation in the colon, while malignant transformation is also associated with 8-OHdG formation in the colon crypts, and possibly with decreased apoptosis levels in the early stages of initiation.

From another point of view, although the initial activation step of IQ to its mutagenic 7-keto derivative, the N-hydroxyl IQ (7-OHIQ), occurs in the liver (13), unchanged IQ is known to be present in the gut after oral exposure (64). Complete conversion of IQ to 7-OHIQ takes place by gut microflora (65). Proximal colons exhibit higher numbers of ACF. Activation of IQ to a direct-acting carcinogen by colonic microflora (65), or direct absorption through the lumen (66) may take place in the first site of exposure to unchanged IQ, the proximal colons, giving rise to more ACF. Further study to address the rates of conversion of IQ by gut microflora in different sites of the colon of SCID mouse appears warranted.

Enhanced peroxidation of polyunsaturated fatty acids in cell membranes by intracellularly produced free radicals, with an altered cellular redox potential and activation of protein kinases, are often linked with persistent oxidative stress and consequent changes in transcription factors, with enhanced development of malignant disease (67). To our knowledge, the membranes of the SCID mouse are not reported to differ in structure or function from the membranes of other mice strains. Robblee et al. (68) concluded that high-fat diets did not affect the phospholipid content of the colonic mucosa or muscles of mice. The quantity of fat in the diet appeared significantly to increase some phospholipid profiles of the colonic mucosa such as the levels of phosphatidylethanolamine (PC). The small polar head group of PC allows very close packing and reduces the permeability and fluidity of the membrane (68). However, it is not possible to conclude from this study if the plasma membrane composition of colonic mucosal cells was altered or if there were any changes in phospholipid content that might reflect changes found in the colonocytes. Further studies may be interesting if conducted to compare the phospholipid profiles of SCID mice normal and neoplastic colonic membranes when treated with different long-chain fatty acids. The present study is the first to provide evidence of a selective modulation by polyunsaturated fatty acids of 8-OHdG formation due to IQ in the mouse colon mucosa.

In conclusion, the susceptibility to IQ-carcinogenesis in the colon demonstrated here suggests an involvement of the immune system in the development of colon tumors. Various data in human (69) and in animal models (70) point to the existence of immune responses to cancers and the present data support the findings of Pierre et al. (71), who showed that Min mice depleted of CD4+ and CD8+ lymphocytes developed twice as many tumors as immunocompetent mice.


    Notes
 
3 Present address: Department of Zoology, Faculty of Science, Tanta University, Laboratory of Experimental and Molecular Carcinogenesis, Tanta 31527, Egypt Back

4 To whom correspondence should be addressed Email: fukuchan{at}med.osaka-cu.ac.jp Back


    Acknowledgments
 
This work was performed partly in the First Department of Pathology, Osaka City University Medical School, Osaka, Japan, and partly in the laboratories of the Zoology Department, Faculty of Science, Tanta University, Tanta, Egypt. This investigation was supported in part by funding from Core Research for Evolutional Science and Technology, Japan, and by a grant-in Aid from the Ministry of Education, Science, Sports and Culture, Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 6, 2001; revised April 2, 2002; accepted April 9, 2002.