Promotion of carcinogenesis and oxidative stress by dietary cholesterol in rat prostate

Yukio Homma1,4, Yasushi Kondo1, Masashi Kaneko1, Tadaichi Kitamura1, Wei Tak Nyou2, Makoto Yanagisawa2, Yorihiro Yamamoto2 and Tadao Kakizoe3

1 Department of Urology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, 2 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 and 3 National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan

4 To whom correspondence should be addressed Email: homma-uro{at}umin.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The association between prostate cancer risk and dietary fat consumption is well documented and explained partly by accelerated lipid peroxidation. We explored the possible effects of high dietary cholesterol on carcinogenesis and oxidative stress in the prostate of ACI/Seg rats. The rats develop prostate cancer spontaneously late in the life, providing an appropriate model to explore prolonged dietary conditions. Two groups of 20-week-old male rats, 28 each, were fed either a basal diet or a basal diet supplemented with 1% cholesterol (high cholesterol diet), and killed at 100 weeks of age. Rats on the high cholesterol diet developed adenocarcinoma in the ventral prostate more frequently (26 versus 4%, P = 0.023). In the repeat study, 26 rats each were treated similarly and killed at 80 weeks for histology and oxidative stress assay. Oxidative stress was assessed by measuring the plasma and intra-prostatic levels of vitamin E, vitamin C, uric acid and the oxidized and reduced forms of coenzyme Q9. The relative amount of oxidized form of coenzyme Q9 is a sensitive marker of oxidative stress. Rats on the high cholesterol diet demonstrated a higher incidence of atypical prostatic hyperplasia (24 versus 4%, P = 0.049). Also, the prostate showed a 2-fold increase (203% of the control) in the relative amounts of the oxidized form of coenzyme Q9 and reciprocal reduction of vitamin C (9.5% of the control) and uric acid (46% of the control) levels (P < 0.01), with a minimal change in vitamin E. The plasma levels of these compounds were not affected by dietary conditions. These results indicated that long-term feeding of a 1% cholesterol diet promoted carcinogenesis and tissue oxidative stress in rat prostate. The role of dietary fat and oxidative stress in prostate carcinogenesis needs further investigation.

Abbreviations: %CoQ-9, total coenzyme Q9


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prostate cancer is the most common malignancy in men in many western countries, but is far less common in Asia (1). To the contrary, the incidence of latent prostate cancer found at autopsy is similar between these areas (2,3). Furthermore, migrant studies have demonstrated that the incidence of clinical prostate cancer increases generation by generation after immigration in Japanese- or Chinese-Americans (4,5). These observations suggest strongly that the wide disparity in prostate cancer incidence worldwide may be attributable to environmental factors and high consumption of dietary fat has been regarded as the pertinent factor (6,7). Numerous cross-cultural and prospective comparison studies have indicated a positive association between prostate cancer mortality and dietary fat intake (810).

An assumed mechanism by which high dietary fat consumption contributes to prostate carcinogenesis is free radical-induced lipid peroxidation (11). Lipid peroxides cause damage to macromolecules, including enzymes and DNA (12), or increase synthesis of prostaglandins and leukotrienes that stimulate prostate cancer cell proliferation (13,14). These assumptions are supported by epidemiologic, interventional and laboratory studies that demonstrate protective effects of antioxidants such as vitamin E, lycopene or selenium in human prostate cancer (1517).

However, the linkage of high fat, oxidative stress and prostate carcinogenesis has not been confirmed in animal experiments. Feeding a high fat diet has not or has only marginally promoted prostate carcinogenesis in rats (7,1821). This would be partially because the experimental period was too short to detect the potential effects of the dietary condition. Lifelong exposure may be important in prostate carcinogenesis, as prostate cancer is heavily age-dependent in humans (1). In this regard, ACI/Seg rats, which develop prostate cancer spontaneously later than 80 weeks of age (22), may provide a suitable model (21). Another possible reason for the lack of association is that most animal studies used corn oil instead of cholesterol as the supplement fat. It is reasonable to choose not to use cholesterol in such experiments because most epidemiologic studies indicate no, or even negative, association between dietary or serum cholesterol and prostate cancer risk (23,24). Further, an animal study has shown no promotional effects of a high cholesterol diet on prostate carcinogenesis (25). Nonetheless, dietary cholesterol is possibly linked to prostate carcinogenesis. A high cholesterol diet promotes breast and colon cancer in rats (26,27); these are also more prevalent in western countries and may share common promotional mechanisms with prostate cancer. Additionally, high dietary cholesterol induces oxidative stress as demonstrated through increased plasma levels of 8-epi-prostaglandin F2{alpha}, a free radical oxidation product of arachidonate (28) or the accumulated content of oxidized low-density lipoproteins in intimal plaque (29). Oxidation of cholesterol initiates lipid peroxidation and by itself produces mutagenic cholesterol epoxides, which are detected at higher concentrations in prostate cancer (30).

Oxidative stress is defined as a disturbance in the prooxidant–antioxidant balance in favor of the former. Oxidative stress can be sensitively detected through the redox status of coenzyme Q, as oxidation of coenzyme Q is one of the early events preceding lipid peroxidation during spontaneous oxidation (31). We have developed a sensitive and reliable method to measure reduced and oxidized forms of coenzyme Q (32) and demonstrated increased oxidative stress in newborn babies (33), and in patients with chronic hepatitis, hepatocellular carcinoma or acute myocardial infarction (34,35). This method is applicable to the measurement of tissue oxidative stress.

In this study we examined the linkage between a high fat diet, oxidative stress and prostate carcinogenesis in long-term in vivo animal experiments by feeding ACI/Seg rats on high dietary cholesterol, using the redox status of coenzyme Q as the marker of oxidative stress.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and diets
Male ACI/Seg rats, were purchased from Harlan Industries (Cumberland, IN) at 4–6 weeks of age and housed in plastic cages with woodchip bedding, with 5–6 rats in each cage, in an animal room maintained at a temperature of 22 ± 2°C and a humidity of 55 ± 15% with a 12-h light 12-h dark cycle. Animals were cared for in accordance with the institution's guidelines. The rats were fed either a natural basal diet, MF-2 (Nippon Bio-supply Center, Tokyo) or a high cholesterol diet, with 1% cholesterol added to the basal diet. The basal diet contained 54.0% carbohydrates (w/w), 23.8% protein, 5.1% fat, 3.2% fiber and 13.9% other components. The cholesterol content was 0.075%. These diets were supplied every 4 weeks and stored at 4°C. Food and tap water were given ad libitum.

Experimental protocol
At 20 weeks of age, the rats were arbitrarily divided into two groups of 28 rats each and fed either the basal diet or the high cholesterol diet. At 100 weeks of age the rats were killed and both lobes of the ventral prostate were fixed in 10% phosphate-buffered formalin for histological examination (experiment 1). An additional two groups of 26 rats each were fed either the basal diet or the high cholesterol diet from 20 weeks of age and killed at 80 weeks. The left ventral lobe of the prostate was fixed in formalin for histopathology. The right ventral lobe and the plasma drawn from the vena cava were frozen at –80°C until a chromatographic assay of oxidation-related compounds could be performed. Plasma testosterone levels were measured using radioimmunoassay at a Special Reference Laboratory (Hachioji, Tokyo) (experiment 2).

Histopathology
For histologic evaluation, three 5 µm thick sagittal slices were made at 1 mm intervals of the formalin-fixed specimen and stained with hematoxylin and eosin. The presence of atypical hyperplasia and adenocarcinoma was determined according to the criteria proposed by Bosland (36). Atypical hyperplasia was defined as a focal hyperplastic lesion at least 3–5 cells thick, involving one or more alveoli. The lining cells showed no or minimal pleomorphism and tended to be arranged in a cribriform pattern, but the normal alveolar architecture was not disturbed by capsular compression. Adenocarcinoma was defined as an epithelial proliferation occupying three or more alveoli, with a cribriform or solid growth pattern. The cells were clearly pleomorphic and often associated with inflammatory infiltrates. They compressed or sometimes invaded the adjacent alveoli and the normal architecture was distorted. The assigned group of each rat was not blinded during autopsy, although histologic evaluations were performed independently in a blinded fashion by two researchers (Y.H. and Y.K.). When the two investigators were found to have made different diagnoses from their examination of a sample, they would discuss the diagnosis and come to agreement before breaking the key.

Assay of oxidative stress
Plasma and tissue levels of ascorbic acid and uric acid were determined as described previously (37). In brief, plasma or tissue homogenate (50 µl) was mixed vigorously with 200 µl of cold methanol in a 1.5 ml polypropylene tube. After centrifugation at 10 000 g for 3 min at 4°C, 50 µl of the methanol layer (corresponding to 10 µl of plasma) was injected immediately onto a high performance liquid chromatograph (HPLC), equipped with an aminopropylsilyl column (Type Supelcosil LC-NH2, 5 mm, 250 x 4.6 mm i.d., Supelco Japan, Tokyo) and a UV detector (260 and 460 nm). The mobile phase consisted of methanol/40 mM sodium monobasic phosphate (=9/1, v/v) delivered at a flow rate of 1.0 ml/min. The levels of coenzyme Q, vitamin E (mixture of {alpha}- and {gamma}-tocopherols), free cholesterol and cholesterol esters were determined using a published method (32) with modifications. In brief, the extracts with 2-propanol were analyzed with HPLC, equipped with an analytical column (Type Supelcosil LC-8, 5 µm, 250 x 4.6 mm i.d., Supelco), a reduction column (Type RC-10-1, Irica, Kyoto), a UV detector (210 nm) and an amperometric electrochemical detector (Model {sigma}985, Irica). The oxidation potential for amperometric electrochemical detector was +600 mV (versus Ag/AgCl) on a glassy carbon electrode. The mobile phase consisted of 50 mM sodium perchlorate in methanol/tert-butyl alcohol (8:2, v/v) delivered at a flow rate of 0.8 ml/min. All samples were assayed and decoded by a blinded investigator.

Statistical analysis
Student's t test was used for the comparison of body weight and plasma and intra-tissue levels of substances. The proportions of rats having specific pathologic lesions were determined using Fisher's exact test. P values of <0.05 with two-tailed analysis were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In both experiments, one rat each on the high cholesterol diet was excluded because of early death from unknown reasons. Body weight was significantly greater in rats on the high cholesterol diet throughout. The average body weights (±SE) at autopsy of 100 weeks were 374.4 (±7.2) and 427.2 (±8.3) g in rats on the basal diet and the high cholesterol diet, respectively (P < 0.001). The average weights of the prostatic ventral lobes (±SE) were 284.6 (±4.2) and 324.2 (±5.3) mg in rats on the basal diet and the high cholesterol diet, respectively (P< 0.001). The relative weights of the ventral lobes corrected by body weight (±SE) were 0.77 (±0.04) and 0.76 (±0.03) mg/g, respectively (N.S.). At autopsy, fatty liver, intraperitoneal deposition of adipose tissue and abundant fatty tissue around the prostate were commonly seen in rats on the high cholesterol diet.

Histologically, rats fed the high cholesterol diet demonstrated significantly higher incidences of atypical hyperplasia at 80 weeks (24 versus 4%; P = 0.049) and prostatic adenocarcinoma at 100 weeks (26 versus 4%; P = 0.023) (Table I). These neoplastic lesions were focally distributed and no macroscopic cancer was found. Plasma and intra-prostatic concentrations of cholesterols and antioxidants are summarized in Tables II and III, respectively. Rats fed the high cholesterol diet showed an ~4-fold increase in plasma levels of total cholesterol with only minimal changes in levels of uric acid, vitamin C and percentage content of oxidized coenzyme Q9 in total coenzyme Q9 (%CoQ-9). In contrast, the assay of intra-prostatic levels of these compounds demonstrated only a marginal increase in cholesterol level, marked reductions in uric acid (46%) and vitamin C (9.5%), and an ~2-fold increase (203%) in %CoQ-9, indicating exaggerated oxidative stress in the prostate. It should be noted that there was almost no difference in plasma and intra-prostatic concentrations of vitamin E between the two groups of rats. Plasma testosterone at 80 weeks of age was higher in rats on the high cholesterol diet (mean ± SE; 0.72 ± 0.09 ng/ml) than the control rats (0.51 ± 0.06 ng/ml), but the difference was not statistically significant (P = 0.057).


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Table I. Histologic findings of ventral lobes of the prostate

 

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Table II. Plasma concentrations of cholesterol and antioxidants and redox status of coenzyme Q9

 

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Table III. Intra-prostatic concentrations of cholesterol and antioxidants and redox status of coenzyme Q9

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The association between prostate carcinogenesis and high dietary fat consumption has been shown in numerous studies (110), and tissue oxidative stress is presumed to be one of the mechanisms involved (1114,16,17). The association has not been well reproduced in animal studies (7,1821) however, and the role of oxidative stress in prostate carcinogenesis has yet to be elucidated. In this study we investigated the correlation between high dietary cholesterol, oxidative stress and prostate carcinogenesis in ACI/Seg rats.

In experiment 1 we administered the 1% cholesterol diet to the rats from 20 to 100 weeks of age, resulting in a significantly higher incidence of prostate adenocarcinoma (26 versus 4%; P = 0.023). In the repeat study (experiment 2), which was terminated earlier at 80 weeks, the incidence of atypical hyperplasia was significantly higher in rats on the high cholesterol diet (24 versus 4%; P = 0.049). In experiment 2 we also estimated plasma and intra-prostatic oxidative stress by measuring levels of antioxidants (vitamin E, vitamin C and uric acid) and the redox status of coenzyme Q. Coenzyme Q takes either the reduced form (ubiquinol) or the oxidized form (ubiquinone), and the percentage of the oxidized form in the %CoQ is a sensitive marker of oxidative stress (31). We have developed a reliable method for the simultaneous detection of these two forms of coenzyme Q (32). This assay demonstrated an ~2-fold increase (203%) in %CoQ in the rat prostate on the high cholesterol diet. This increase was associated with reciprocal reduction of vitamin C (9.5% of the control) and uric acid (46% of the control) in the target organ. In contrast, tissue levels of vitamin E remained unchanged. This is not surprising, as vitamin E radicals are reduced to vitamin E by ubiquinol and vitamin C (31,38). Plasma levels of these substances were not affected by diet, despite the 4-fold difference in cholesterol level. These observations indicate that a high cholesterol diet promoted carcinogenesis and oxidative stress in the rat prostate. To our knowledge, this is the first report to demonstrate the promotion of prostate carcinogenesis by dietary cholesterol and its association with tissue oxidative stress in vivo.

These results contradict those from a previous study that showed a lack of promotional effects of dietary cholesterol on rat prostate carcinogenesis (25). In that experiment, cancer was induced by 3,2'-dimethyl-4-aminobiphenyl and ethinyl estradiol, with a high cholesterol diet given between 20 and 60 weeks of age. This model may be less sensitive at detecting dietary effects because of a shorter feeding period, earlier death and possible interference by chemical and/or hormonal treatments. Lifelong exposure may be important for dietary intervention in prostate carcinogenesis (21), because prostate cancer is a heavily age-dependent human malignancy (1).

Our results also contradict epidemiologic observations indicating no, or even negative, association between cholesterol and prostate cancer (23,24) and should be connected to oxidative stress rather than dietary cholesterol. In our study, oxidative stress was assessed using the redox status of coenzyme Q, which can detect oxidation at an early stage (31). Although the redox status of coenzyme Q is not a standard marker for oxidative stress, it correlated closely with vitamin C and uric acid content, with vitamin E level remaining unchanged (31,3335).

The observed effects of dietary cholesterol on prostate carcinogenesis can be explained in several ways. First, the diet caused higher caloric ingestion, which was confirmed by the larger body weight in rats on the high cholesterol diet. The high fat diets supplemented with corn oil were, however, certainly high caloric, and exerted no or marginal promotional effects in previous investigations (7,1921). Secondly, the diet changed the endocrine milieu by increasing, albeit non-significantly, plasma testosterone levels in rats on the high cholesterol diet. Testosterone is one of the key factors in prostate carcinogenesis in rats (1820) as well as in humans (39). Other sex hormones, dihydrotestosterone (40) and estrogens (25), are known to exert mixed effects on prostate carcinogenesis, and were possibly involved in the demonstrated effect of dietary cholesterol. Thirdly, cholesterol metabolites could directly act as carcinogens. Cholesterol is oxidized to mutagenic and carcinogenic cholesterol epoxides (30). Last, the high cholesterol diet increased tissue oxidative stress. Increased androgen levels might additionally enhance oxidative stress (41). Oxidative stress accelerates lipid peroxidation, which subsequently could induce DNA damage (12) or synthesis of prostaglandins and leukotrienes that stimulate prostate cancer cell proliferation (13,14). These consequences may be prevented using antioxidants (16,17). It must be admitted that the processes of cancer promotion through oxidative stress are at present speculation only. Further, there are contradictory data (2325) and the redox status of coenzyme Q is not the standard marker of oxidative stress. To test our speculations, we are now examining whether supplementation with antioxidants does or does not reverse oxidative stress in the prostate and suppress cholesterol-promoted carcinogenesis.

In conclusion, for the first time we have demonstrated that a diet containing 1% cholesterol promotes prostate carcinogenesis and elevates intra-prostatic oxidative stress in vivo. Further investigations are needed to elucidate the molecular process of the role of dietary fat in prostate carcinogenesis.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 25, 2003; revised January 20, 2004; accepted January 26, 2004.