Promotion of colon carcinogenesis through increasing lipid peroxidation induced in rats by a high cholesterol diet

Cancer

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Letters

100 (1996)

CANCER LETTERS

8 l-87

Promotion of colon carcinogenesis through increasing lipid peroxidation induced in rats by a high cholesterol diet Tsui-Hwa Tseng a,*,Jeng-Dong Hsub, Chia-Yih Chua, Chau-Jong Wanga “Institute

of Biochemistry,

bDepartment

Chung

r!f Puthology,

Shun Medical and Dentul College, No. 113, Section Tuichung 402, Taiwan, ROC Chung Shun Medical und Den&l College, Taichung,

2, Ta-Ching Taiwan.

Street,

ROC

Received16November1995;accepted22 November1995

Abstract

To examine the influence of hypercholesteremia on 1,2-dimethylhydrazine (DMH)-induced rat colon cancer, SpragueDawley rats received dietary cholesterol (CH, O-2%) and cholic acid (CA, 0.25%) with or without DMH (20 mg/kg, S.C.injection) for 18 weeks. The rats receiving dietary cholesterol and cholic acid all significantly increasedtotal serum cholesterol and lipids but only a high cholesterol diet (2% CH plus 0.25% CA) decreasedthe activity of glutathione peroxidase (GSHPx) and increased the formation of peroxides in the colon (P < 0.01). The rats that received the combination of DMH and high cholesterol diet enhanced these effects. At the end of the experiment, the diet group administered DMH and high cholesterol (2% CH plus 0.25% CA) developed colon adenoma at 50% of incidence in pathological examination, but no colon adenoma formed in the rats treated with high cholesterol alone. It is supposed that a non-carcinogenic agent like cholesterol may potentiate the carcinogenicity of DMH in rats via an increaseof lipid peroxidation and decreasein the activity of peroxidase in the target organ. Keywords:

Colon cancer; Cholesterol; Lipid peroxidation; Dimethylhydrazine; Promotion

1. Introduction Dietary factors have been extensively studied for their correlation with the development of colon cancer. Experimental and epidemiologic studies in general have implicated dietary cholesterol as a factor in colon carcinogenesis [IA]. It is suggested that secondary cholesterol metabolites such as deoxycholic acid and lithocholic acid increase the proliferative status of rodent colonic epithelium but the mecha* Correspondingauthor

nisms are not fully known [5,6]. The correlation of serum cholesterol to some diseases has been assessed but the relationship between the level of serum cholesterol and colon cancer risk [7] is uncertain. Rats have been generally considered to be a useful animal for experiment but are resistant to the induction of hypercholesteremia by cholesterol feeding. However, the effect can be produced by feeding cholic acid along with a cholesterol rich diet [8]. Cholesterol may be oxidized in vivo chemically, enzymatically by specific enzymes, or by autooxidation and lipid peroxidation [9]. The products of lipid per-

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et al. /Cancer

oxidation may be decomposed to alkoxyl and peroxyl free radicals that can oxidize other cell components, resulting in the change of enzyme activity or the generation of mediators that can cause further cell damage [ 101. The association of lipid peroxidation with cell proliferation or differentiation is not clear 111,121. The murine model of colon carcinogenesis closely resembles human colon neoplasia in pathological features. A number of hydrazine derivatives are found in nature and in industry, agriculture and medicine [14]. Most of them have been shown to be carcinogenic and mutagenic. It has been reported that administration to rodent species of the potent procarcinogen 1,Zdimethylhydrazine (DMH, 20 mg/kg) results in the development of colon cancer around 24 weeks [ 151. In this experiment, we use dietary cholesterol (CH, O-2%) plus cholic acid (CA, 0.25%) to induce hypercholesteremia and colon peroxides. From the data, it is shown that all cholesterol diets increase total serum cholesterol and lipids but only the group fed with 2% CH plus 0.25% CA increases the formation of lipid peroxidation and decreases the activity of glutathione peroxidase (GSH-Px) in colon tissue. The rats receiving a combination of DMH and a high cholesterol (2% CH plus 0.25% CA) diet enhanced these effects and developed colon adenoma.

2. Materials and methods 2.1. Chemicals DMH, cholesterol, cholic acid, glutathione (GSH), glutathione reductase and malondialdehyde were purchased from Sigma Chemical Co., St. Louis, MO. Total serum lipid and cholesterol were measured with enzymatic kits from Beckman. 2.2. Animals and treatment Male Sprague-Dawley rats (90-100 g body weight) purchased from Taichung Veterans General Hospital Animal Center were used for the experiment. The animals were housed four per cage in an environmentally controlled animal room. The rats were divided into two groups (with or without DMH injection) and then in four groups (8 rats/group) for each family. The first group with or without DMH

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injection was fed a commercial diet, while the second group was fed a commercial diet supplemented with CA (0.25% w/w), the third group with CH (1%) plus CA (0.25%), and the fourth group with CH (2%) plus CA (0.25%) for 18 weeks. Food and water were provided ab libitum. DMH was dissolved just prior to subcutaneous (s.c.) injection (20 mg/kg weekly) in 1 mmol/l EDTA (pH 6.5) [ 161. 2.3. Serum lipids assay The animals were sacrificed by decapitation. The blood was collected and plasma was separated by centrifugation. The levels of serum total lipid and cholesterol were measured by biochemical autoanalyzer using enzymatic kits (Beckman) [17,18]. 2.4. Determination of peroxides Rats were killed by decapitation and colon tissue was promptly removed and placed immediately on ice. Tissue homogenates were made in a 50 mmol/l phosphate buffer containing 0.1 mmoY1 EDTA and centrifuged at 10 000 X g at 4°C for 20 min. Aliquots of the supernatant were prepared for the following assays [18]. Colon hydrogen peroxide was measured using a scopoletin fluorescence assay in which H,O, oxidizes scopoletin to a non-fluorescent state in a reaction catalyzed by horseradish peroxidase [19]. Colon lipid peroxide was estimated with malondialdehyde (MDA) as a standard according to an improved thiobarbituric acid (TBA) fluorescence method (E,/E,,, = 515 nm/554 nm) [20]. 2.5. Enzyme activity assay of GSH-Px GSH-Px activity was determined in the colon homogenated using the spectrophotometric assay of Paglia et al. [21]. Protein concentration was determined using a standard commercial kit (Bio-Rad Laboratories, Watford, UK) with bovine serum albumin as a standard [22]. 2.6. Pathological histology of the colon Immediately after rat death, a complete necropsy was performed and the colon was examined for gross abnormalities. Colon tissues were fixed in 10% buff-

T.-H. Table

Tseng et al. /Cancer

Letters

100 (1996)

83

RI-87

1

Influence

of dietary

cholesterol

Serum

Treatmenta

Normal 0% CH 1% CH 2% CH

with

diet + 0.25% + 0.25% + 0.25%

CA CA CA

and without cholesterol

the administration

of DMH

on serum

lipids

of

Serum

(mg/dl)

rats total lipid (mg/dl)

-DMH

+DMHb

-DMH

+DMH

3O*F 68 * 10** 129i39 131*31

32 + 5 85 + 21W 154*31 186?51#

294 k 47 374+51* 545 * 90** 556* 131

330 f 21 437 k 50# 597 + 52## 640? 108’

aMale Sprague-Dawley rats were fed with CH and CA as described in the text. bDMH was given by weekly S.C. injection of 20 mg/kg for 18 weeks. CValues are means + SD for eight rats per group. *P < 0.01, **P < 0 001, compared with normal diet without DMH injection by Student’s t-test. with normal diet with DMH injection by Student’s t-test. #P < 0.01, HP < 0.001, compared

ered formalin, processed for histological examination according to conventional methods and stained with hematoxylin and eosin (H&E).

little dependence on DMH injection in normal diet rats but an obvious increase of total serum lipids and cholesterol in rats on a cholesterol diet with DMH administration (Table 1).

2.7. Statistical analysis 3.2. Influence of diet on colon peroxides Data were analyzed using Student’s t-test. Statistical significance was assigned when P c 0.05.

3. Results 3. I. Influence of diet on total serum lipids Total serum lipids and cholesterol in the rats were significantly (P < 0.01 and P < 0.001) increased in all dietary cholesterol (O-2%) and cholic acid (0.25%) groups with or without DMH injection. There was

Although the source of H202 generation is not clear, it is suggested that DMH could generate Hz02 in the presence of copper ion [lo]. MDA is a low molecular weight product which is formed during the peroxidative degradation of polyunsaturated fatty acids with three or more double bonds. The DMH administration increased colon hydrogen peroxide (P < 0.01) but not formation of MDA. High cholesterol diet (2% CH plus 0.25% CA) significantly (P < 0.01) increased colon H,Oz and MDA and po-

Table 2 Influence

of dietary

Treatmenta

cholesterol

on rat colon MDA

(nmollg

-DMH Normal 0% CH 1% CH 2% CH

diet + 0.25% + 0.25% + 0.25%

CA CA CA

115+. 120 i 121 f 136 +

11 15 10 15*

with and without

the administration

colon)

of DMH

on malondialdelyde Hz02

+DMH

-DMH

126 + 21 131*10 137 f 17 151* 15#

18.9 22.6 26.3 31.4

“All treatments and DMH administration were as described in Table 1. bValues arc means + SD for eight rats per group. *P < 0.01, compared with normal diet without DMH injection by Student’s t-test #P < 0.01, compared with normal diet with DMH injection by Student’s t-test.

(nmol/g

and hydrogen

peroxide

colon) +DMH

* i & +

3.5 5.0 l.O* 5.5*

30.2 29.6 30.9 38.0

+ + * lr

4.3* 3.6 4.0 5.6#

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tentiated H202 and MDA increase in the group with DMH combination (Table 2). 3.3. Influence of diet on colonic GSH-Px activity As a consequence of HzOz and MDA increase, the balance between oxygen radical generation, normally associated with cellular metabolism and its dissipation by cellular defense systems is disturbed, allowing reactive oxygen species to attack biomolecules. GSH-Px, one defense enzyme, prevents lipid peroxidation by scavenging hydrogen peroxide and reducing hydroperoxides of fatty acid. From the data, we found that a high cholesterol diet (2% CH plus 0.25% CA) could decrease the activity of GSH-Px (P < 0.01). DMH administration had no influence on the activity of GSH-Px in normal diet rats but a cholesterol diet (l-2% CH plus 0.25% CA) with DMH rats decreased colonic GSH-Px activity significantly (P < 0 05, P < 0.01; Table 3). 3.4. Colon cancer incidence The DMH injection rats fed a commercial diet for 18 weeks were normal but those with a high cholesterol diet (2% CH plus 0.25% CA) developed colonic adenoma (50% incidence, Table 4). All colon sections from rats in the experiment were compared. The tumors were adenomatous polyps measuring up to 3 mm and showing a distinct tubular structure (upper Table 3 Influence of dietary cholesterol with and without DMH administration on the activity of GSH-Px in rat colon Treatmen?

GSH-Px (nmol/min per mg protein) -DMH

Normal 0% CH 1% CH 2% CH

diet + 0.25% CA + 0.25% CA + 0.25% CA

+DMH

18.59 f 3.09

18.21 + 3.10

17.92 f 2.01 17.62 f 2.99

15.96 f 3.86 14.87 f 2.21# 14.57 f 2.05”

15.29 f 1.70*

“All treatments and DMH administration methods are described in Table 1. bValues are means + SD for eight rats per group. *P < 0.01, compared with normal groups without DMH injection by Student’s f-test. #P < 0.05, ‘#P < 0.01, compared with normal group with DMH injection by Student’s r-test.

Letters

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Table 4 Effect of dietary cholesterol with and without DMH administration on the incidence of colon adenoma in rats Treatmen?

Normal 0% CH 1% CH 2% CH

diet + 0.25% CA + 0.25% CA + 0.25% CA

% Incidence (no. of rats with colon adenomakotal no. of rats) -DMH

+DMH

0 (018)

0 0 0 50

0 (O/8) 0 (O/8)

0 (O/8)

(O/8) (O/8) (O/8) (4/8)

‘All treatments and DMH administration methods are described in Table 1.

parts of fields; Fig 1) in contrast to normal glands (H&E stain X40 and x 100). 4. Discussion While epidemiological and experimental data indicate a direct correlation between dietary fat intake and the development of colon cancer, the effect of dietary cholesterol on this disease is still not clear Recent studies suggest an inverse relationship between serum cholesterol and the risk for colon cancer [23]. Many studies have shown that metabolites of cholesterol in colonic lumen may act to promote colon carcinogenesis [13,24]. In this experiment, we found that dietary cholesterol and cholic acid are involved in DMH carcinogenesis perhaps via an increase of peroxides and a decrease of protecting enzyme activity of the target. Although cholic acid, the primary metabolite of cholesterol, has been shown to be a promoter of colon carcinogenesis [25], the group of rats fed only cholic acid with DMH for 18 weeks did not develop colon cancers at increased rates. DMH can be metabolized to a methyl free radical and generate hydroxyl radical or hydrogen peroxide in the presence of metal ions that may be contribute to the initiation of lipid peroxide [ 141. MDA, the termination product of lipid peroxidation, has been shown to be biologically active with cytotoxicity and genotoxicity [26,27]. Hydrogen peroxide, generated in several human biosynthetic pathways, can transiently or permanently damage nucleic acids lipids or proteins [28,29]. This study showed that high cholesterol diet (2% CH plus 0.25% CA) significantly increased

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the formation of lipid peroxide and hydrogen peroxide of colon tissue. It suggests that a high cholesterol diet potentiates the reactive oxygen species (ROS) propagation of carcinogen in the colon of rats (Table 2). ROS may cause oxidative DNA lesions which

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lead to cytotoxicity followed by regenerative proliferation [ 291. The body has a range of extracellular and intracellular protection systems to combat excessive production of ROS. GSH-Px is a selenoprotein enzyme that

Fig. 1. Rat colon adenoma resulting from a high cholesterol diet and received H&E, x40). (B) Tubular adenoma of colon (upper part of field, H&E, X 100).

DMH.

(A) Tubular

adenoma

of colon

(upper

part of field,

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catalyses the reduction of a large number of hydrogen peroxides ranging from H202 to the spectrum of organic hydrogen peroxide (ROOH). From the data, we found that high cholesterol (2% CH plus 0.25% CA) could decrease the activity of GSH-Px (Table 3). It is more likely that tissues constantly exposed to carcinogens, which did not develop harmful effects were protected by their own host systems. However, these systems could be affected by non-carcinogenic agents. Chen et al. demonstrated that an 80% incidence of rat colon cancer induced by DMH was detected by colonoscopy around 24 weeks [ 151. In this experiment, it seems that the time of tumor production (Table 4) was advanced for high cholesterol (2% CH plus 0.25% CA) diet along with DMH. In conclusion, a high cholesterol diet may potentiate the carcinogenicity of DMH in rats via an increase in lipid peroxidation and decrease of the activity of defense enzymes in the colon. Acknowledgements We thank Chung-Shan Medical and Dental College Research Fund (CSMC 82-B017) for partial support and a National Science Council Grant (NSC 84-233 1-B-040-003), Republic of China.

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