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Articles

Dietary Copper, Manganese and Iron Affect the Formation of Aberrant Crypts in Colon of Rats Administered 3,2'-Dimethyl-4-Aminobiphenyl^{1, ,2}

Cindy D. Davis^*,3 and Yi Feng

^* United States Department of Agriculture, Grand Forks Human Nutrition Research Center and Department of Surgery, University of North Dakota, Grand Forks, North Dakota 58202-9034.

³To whom correspondence should be addressed.

	ABSTRACT

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Aberrant crypt foci (ACF) are preneoplastic lesions for colon cancer. Altered amounts of copper-zinc (CuZnSOD) and manganese (MnSOD) superoxide dismutases have been implicated in multistage carcinogesis of both rodents and humans. Dietary factors are potential modulators of both CuZnSOD and MnSOD activity. The purpose of this study was to investigate the interactive effects of dietary copper, manganese, and iron on 3,2'-dimethyl-4-aminobiphenyl (DMABP)-induced ACF and superoxide dismutase activities in weanling rats fed low or adequate copper (0.8 or 5.1 µg Cu/g diet), low or adequate manganese (0.6 or 17 µg Mn/g diet), and adequate or high iron (37 or 140 µg Fe/g diet). Twelve rats were allowed free access to each of these eight diets for 3.5 wk prior to DMABP administration and for an additional 8 wk after the first DMABP injection. Rats fed low dietary copper had 105% (P < 0.0001) higher formation of DMABP-induced ACF than those fed adequate dietary copper. Rats ingesting low rather than adequate dietary manganese had 23% higher formation of ACF, and rats ingesting high rather than adequate dietary iron had 18% higher formation of ACF. Heart total superoxide dismutase activity was significantly correlated with the number of ACF (r = -0.43, P < 0.0001) in rats administered DMABP. These results suggest that dietary alterations that affect superoxide dismutase activity may affect cancer susceptibility.

KEY WORDS: • copper • manganese • iron • aberrant crypt foci • superoxide dismutase • rats

	INTRODUCTION

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Colon cancer is the second leading cause of cancer mortality in the United States and the fourth most common cause of cancer mortality worldwide (Cancer Facts and Figures 1997 ). Dietary factors, including exposure to chemical carcinogens present in cooked meats, are widely accepted as risk factors for colorectal cancer (Lang et al. 1994 ). Because of the great interest in animal models for colorectal cancer that would accurately reflect the disease in humans, the aromatic amine 3,2'-dimethyl-4-aminobiphenyl (DMABP)⁴ was used to study the induction of colon cancer in rodents (Fiala et al. 1981 , Shirai et al. 1990 ). DMABP is often used because of the close chemical similarity between it and certain mutagens isolated from cooked meat or fish. The target cells of colon carcinogens are colonic crypt epithelial cells. Aberrant crypt foci (ACF) are putative preneoplastic lesions that have been detected in human colon resections and in experimental animals treated with chemical carcinogens (Bird 1987 and 1995 , Feng et al. 1996 , Pretlow et al. 1991 ). Studies in humans have shown that colonic ACF are precursor lesions from which adenomas and adenocarcinomas will develop (Konstantakos et al. 1996 , Pretlow et al. 1991 , Siu et al. 1997 ). This characteristic of ACF favors their use as a biomarker for investigating modulation of colon carcinogenesis. A number of natural compounds and nutrients that inhibit ACF development induced by exposure to several colon carcinogens were proven to have chemopreventive activity against colon cancer in rodents (Pereira et al. 1994 ). Thus, scoring of ACF is currently being used as a bioassay to scan potential colon carcinogens in rodent models.

Superoxide dismutases are metalloenzymes that play a vital role in the protection of aerobic cells against oxygen toxicity (Fridovich, 1975 ). In eukaryotic cells, there are two main intracellular forms of this enzyme. The first contains both copper and zinc (CuZnSOD) and is found in the cytosol (McCord and Fridovich 1969 ), and the second contains manganese (MnSOD) and is found mainly in the mitochondria (Weisiger and Fridovich 1973 ). Altered activities of superoxide dismutase were shown to be important in multistage carcinogenesis of both rodents and humans. When compared to their appropriate normal cell counterparts, tumor cells are almost always low in MnSOD and CuZnSOD activity (Sun 1990 ). This observation was made in a number of different cell types and is independent of the mechanism of cell transformation (Marlens et al. 1985 , McCormick et al. 1991 , Oberley et al. 1978 , Sun et al. 1993 ). Furthermore, increased amounts of superoxide dismutase were shown to be protective against cancer. The addition of exogenous superoxide dismutase has led to the inhibition of oncogenic transformation induced by X-rays both in vitro and in vivo (Petkau et al. 1975 , St. Clair et al. 1992 ), and transgenic mice overexpressing MnSOD are resistant to chemically induced skin carcinogenesis (Oberley and Oberley 1997 ).

Dietary factors are potential modulators of both MnSOD and CuZnSOD activity. Heart MnSOD activity in rats is significantly reduced in rats fed deficient dietary manganese compared to those fed adequate or high dietary manganese (Davis et al. 1990 and 1992a ). Ingestion of high amounts of dietary iron significantly decreased heart and colonic mucosa MnSOD activities (Davis et al. 1990 and 1992a , Kuratko 1997 ). Similarly, lymphocyte MnSOD activity in women was significantly affected by dietary manganese and iron (Davis and Greger 1992 , Davis et al. 1992b ).

The relationship between dietary iron and MnSOD activity may have implications for cancer susceptibility. Four epidemiologic studies have shown an increased cancer risk in patients with larger iron stores than in those with small iron stores (Reizenstein 1991 ), and hemochromatosis was associated with iron-induced carcinogenesis (Toyokuni 1996 ). In fact, the major cause of death in hemochromatosis patients is hepatocellular carcinoma (Niedereau et al. 1985 ). A dose-response relationship was also observed between serum ferritin concentrations and colon adenocarcinoma risk (Nelson et al. 1994 ). High amounts of dietary iron were shown to be carcinogenic in experimental animals (Toyokuni 1996 ). However, the literature examining the effect of dietary iron on cancer susceptibility is inconsistent. Two animal studies (Lai et al. 1997 , Soyars and Fischer 1998 ) have shown no increased risk of colon cancer with increased dietary iron, and several epidemiological studies have not found a positive correlation between iron stores and colon cancer risk (Tseng et al. 1996 , Ullen et al. 1997 ).

Dietary iron can catalyze the production of reactive oxygen species, which may be proximate carcinogens (Reizenstein 1991 ). Thus, high concentrations of dietary iron will lead to an increased need for antioxidant protection. However, MnSOD activity is down regulated in hepatic iron overload (Zhao et al. 1995 ). People with iron overload have an increased need for MnSOD activity, but decreased enzyme available. High dietary iron was shown to increase the incidence of colon cancer in human subjects; however, no one has investigated whether this is a result of depressed MnSOD activity and whether high dietary iron would interact with deficient manganese to increase susceptibility.

The relationship between diet, superoxide dismutase activity, and cancer susceptibility offers many promising possibilities that need to be explored. No one has investigated the effect of changes in dietary copper or manganese on chemically induced aberrant crypt formation. The purpose of this study was to investigate the interactive effects of dietary copper, manganese, and iron on DMABP-induced aberrant crypt formation and superoxide dismutase activities.

	MATERIALS AND METHODS

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Chemicals.

DMABP was purchased from Toronto Research Chemicals (Toronto, Canada). Peanut oil was obtained from Sigma Chemical (St. Louis, MO.) Methylene blue was purchased from Eastman Kodak (Rochester, NY). Xylazine was purchased from Rompan Mobay (Shawnee, KS) and ketamine from Ketaset Aveco (Fort Dodge, IA).

Experimental design.

The basic design of this study was a 2 x 2 x 2 factorial. The diets contained low or adequate concentrations of copper (by analysis, 0.8 or 5.1 µg Cu/g diet, respectively), low or adequate concentrations of manganese (by analysis, 0.6 or 17 µg Mn/g diet, respectively), and adequate or high concentrations of iron (by analysis, 37 or 140 µg Fe/g diet, respectively). Twelve rats were allowed free access to each of these eight diets for 3.5 wk prior to DMABP administration and for an additional 8 wk after the first DMABP injection.

Animals and diets.

Weanling male Fischer-344 rats were purchased from Sasco (Omaha, NE). All rats were housed individually in stainless steel, wire-bottomed cages in a room with controlled temperature and light. They were provided free access to demineralized water and purified diet. The formulation of the purified diet was consistent with the AIN-93G diet (Reeves et al. 1993 ). Cupric carbonate, manganese carbonate, and ferric citrate were added to achieve the concentrations described above.

After 24 and 31 d of consuming the experimental diets, 10 rats/diet were administered DMABP dissolved in peanut oil (50 g/L) by subcutaneous injection (100 mg/kg body weight). Two additional animals/diet received the comparable vehicle injection of peanut oil only. Animals were killed by exsanguination 8 wk after the first DMABP or vehicle administration.

The study was approved by the Animal Care Committee of the Grand Forks Human Nutrition Research Center, and the animals were maintained in accordance with the National Research Council guidelines for the care and use of laboratory animals.

Sample collection.

Food was withheld overnight before rats were anesthetized with xylazine (Rompan Mobay) and ketamine (Ketaset Aveco) and killed by exsanguination. Blood was collected by cardiac puncture into syringes containing 1 g EDTA/L blood. Kidneys, spleens, and 1-g samples of liver were cleaned of adhering material; weighed; and frozen in liquid nitrogen. Hearts and liver samples were washed with cold 0.9 g NaCl/L and placed in liquid nitrogen. The colon and rectum were removed, flushed with 0.9 g NaCl/L, opened longitudinally, and fixed flat between paper towels in 70% ethanol. The colon and rectum were stored in 70% ethanol at 4°C prior to analysis.

Analysis of ACF.

The fixed colon and rectum were stained with 0.1% methylene blue in 0.1 mol sodium phosphate buffer/L (pH 7.4). ACF and the total number of aberrant crypts (AC) were scored in a blind fashion by using a dissecting microscope to visualize the ACF and AC as previously described (Feng et al. 1996 ). The length of the colon was measured and divided into thirds with the upper third considered as the ascending colon, the middle third as the transverse colon, and the bottom third as the descending colon.

Laboratory analysis.

Fresh plasma was analyzed for ceruloplasmin activity by the method of Schosinsky et al. (1974) . Plasma and HDL cholesterol concentrations were determined by using a Cobas Fara automated analyzer (Hoffman La Roche, Nutley, NJ). Heart and liver total superoxide dismutase activity were determined by the inhibition of pyrogallol auto-oxidation (Marklund and Marklund, 1974 ). In a second reaction, KCN (0.1 mmol/L) was added to the reaction mixture to inhibit copper-zinc superoxide dismutase activity. Copper-zinc superoxide dismutase activity was calculated as total superoxide dismutase activity minus manganese superoxide dismutase activity. One unit of superoxide dismutase activity was defined as the amount of enzyme needed to obtain 50% inhibition of pyrogallol auto-oxidation.

Samples of liver, kidney, spleen, and colon were analyzed for manganese, iron, copper, and zinc by inductively coupled argon atomic emission spectrometer (Liberty Series II, Varian Associates, Sugarland, TX). Briefly, the tissues were weighed, lyophilized to constant weight, and wet ashed multiple times with nitric acid until most of the organic residue was gone. The charred samples were dissolved in 3 mL nitric acid and 10 mL hydrogen peroxide and heated to dryness on a hotplate. The mineral residue was dissolved in 1 mL of 6 mol HCl/L and diluted appropriately with deionized water. Liver standard reference material (1577b, National Institute of Standards and Technology, Gaithersburg, MD) was analyzed with each batch of tissue samples for quality control. Liver samples (n = 12) were determined to contain 93%, 95%, 101%, and 93% of the certified values for copper, manganese, iron, and zinc, respectively.

Plasma samples were precipitated with 30 g trichloroacetic/L acid and 6 mol HCl/L. The precipitate was analyzed by inductively coupled argon atomic emission spectrometer (Liberty Series II, Varian Associates). Control samples that contained demineralized water, which had been collected through the syringes containing 1 g EDTA/L water and processed in a manner similar to that used for plasma samples, were not found to contain any copper, iron, or zinc contamination.

Statistical analyses.

The data were analyzed by a three-way ANOVA (diet copper, manganese, and iron) using a SAS general linear model program (SAS Version 6.12, SAS Institute, Cary, NC). Tukey's contrasts were used to differentiate among means for variables that had been significantly (P < 0.05) affected by the treatments. Pearson correlations were analyzed to determine the association between superoxide dismutase activity and ACF formation. Values are reported as means ± SEM in the text.

	RESULTS

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The mean weight of the rats at the end of the study was 283 ± 16 g. Rats fed low dietary copper had a significantly lower (P < 0.02) final body weight than those fed adequate dietary copper (280 ± 2 vs. 287 ± 2 g, respectively); however, these differences were not apparent when the individual dietary treatments were compared.

Aberrant Crypts.

Aberrant crypt foci were identified in the colon and rectum of rats administered DMABP. However, no ACF were identified in vehicle-treated control animals. Most of the ACF were present in the descending colon; 0, 14.2 ± 2.3, 69.9 ± 3.7, and 17.1 ± 3.0% of the ACF were observed in the ascending colon, transverse colon, descending colon, and rectum, respectively. The frequency of ACF in the colon plus rectum was significantly higher (P < 0.0001) in rats fed low dietary copper compared to those fed adequate dietary copper (1.65 ± 0.29 ACF vs. 3.38 ± 0.29, respectively) (Fig. 1 ). The number of ACF tended to be higher in rats fed low rather than adequate dietary manganese (2.76 ± 0.30 vs. 2.25 ± 0.30 ACF, P = 0.09) and in those fed high rather than adequate dietary iron (2.25 ± 0.30 vs. 1.90 ± 0.30 ACF, P = 0.11). The frequency of aberrant crypts in the colon and rectum was also significantly higher (P < 0.00001) in rats fed low dietary copper compared to those fed adequate dietary copper (Fig. 2 ). Similar to the ACF results, the number of AC in the colon and rectum tended to be higher in rats fed low rather than adequate dietary manganese (4.68 ± 0.64 vs. 3.60 ± 0.64 AC, P = 0.09) and in those fed high rather than adequate dietary iron (5.58 ± 0.64 vs. 4.85 ± 0.64 AC, P = 0.12).

View larger version (32K): [in this window] [in a new window]   Figure 1. Total number of aberrant crypt foci in the colon and rectum of rats treated with 3,2'-dimethyl-4-aminobiphenyl and fed diets containing low or adequate concentrations of copper (by analysis, 0.8 or 5.1 µg Cu/g diet), low (LMn) or adequate (AMn) concentrations of manganese (by analysis, 0.6 or 17 µg Mn/g diet), and adequate (AFe) or high (HFe) concentrations of iron (by analysis, 37 or 140 µg Fe/g diet). Values are means ± SEM, n = 10 Means without common letters are significantly different (P < 0.05) as determined by Tukey's contrasts.

View larger version (34K): [in this window] [in a new window]   Figure 2. Total number of aberrant crypts in the colon and rectum of rats treated with 3,2'-dimethyl-4-aminobiphenyl and fed diets containing low or adequate concentrations of copper (by analysis, 0.8 or 5.1 µg Cu/g diet), low (LMn) or adequate (AMn) concentrations of manganese (by analysis, 0.6 or 17 µg Mn/g diet), and adequate (AFe) or high (HFe) concentrations of iron (by analysis, 37 or 140 µg Fe/g diet). Values are means ± SEM, n = 10 Means without common letters are significantly different (P < 0.05) as determined by Tukey's contrasts.

Hematocrits were 9% lower (0.381 vs. 0.415%, P < 0.0001), hemoglobin was 10.8% lower (12.9 vs. 14.3 g/L, P < 0.0001), and ceruloplasmin activity was 492% lower (6.2 vs. 36.7 U/L, P < 0.0001) in rats fed low copper than in those fed adequate dietary copper (Table 1 ). Rats fed the low manganese diets had significantly depressed hematocrits and hemoglobin concentrations, but the practical importance of such small changes in hematocrit and hemoglobin is questionable. Dietary iron did not significantly affect hematocrit; however, high dietary iron significantly (P < 0.005) increased hemoglobin concentrations. Plasma and high density lipoprotein cholesterol concentrations were significantly higher in rats fed low rather than adequate dietary copper and in rats fed adequate rather than low dietary manganese (Table 1) . Compared to rats fed adequate dietary copper, rats fed low dietary copper had significantly (P < 0.0001) lower plasma copper and iron concentrations and slightly but significantly (P < 0.05) greater plasma zinc concentrations. Dietary manganese and iron did not significantly affect plasma copper, iron, or zinc concentrations.

All three dietary factors (copper, manganese, and iron) significantly affected liver CuZnSOD, MnSOD, and total superoxide dismutase activities (Table 2 ). Copper deprivation reduced liver CuZnSOD activity 54% (3.07 vs. 6.69 U/mg protein, P < 0.001) and liver MnSOD activity by 21% (1.56 vs. 1.97 U/mg protein, P < 0.0001). In contrast, manganese deprivation significantly (P < 0.0001) increased liver CuZnSOD activity, but significantly (P < 0.0001) decreased liver MnSOD activity. An interaction between dietary copper and manganese significantly (P < 0.0001) affected both liver CuZnSOD and total superoxide dismutase activities. Similar to low dietary manganese, high dietary iron caused a significant (P < 0.0001) increase in liver CuZnSOD activity, but a significant (P < 0.0001) decrease in liver MnSOD activity.

The association between superoxide dismutase activity in the liver and heart and DMABP-induced ACF and AC in the colon and rectum of rats is shown in Table 3. Heart CuZnSOD and total superoxide dismutase activity were significantly (P < 0.0008) correlated with both ACF and AC formation. In contrast, in the liver CuZnSOD activity was not significantly correlated with ACF and AC formation; however, hepatic MnSOD and total superoxide dismutase activity were significantly (P < 0.05) correlated with AC formation.

Liver, kidney, spleen, and colon copper concentrations reflected copper intakes (Table 4 ). Ingestion of low amounts of dietary manganese resulted in lower (ANOVA, P < 0.01) liver, spleen and colon copper concentrations. This effect of manganese was not apparent when Tukey's contrasts were used to compare means. Kidney copper concentrations were significantly (P < 0.0001) higher in rats fed low dietary manganese compared to those fed adequate dietary manganese and in those fed adequate dietary iron compared to high dietary iron. An interaction between dietary copper and iron affected kidney copper concentrations; dietary iron affected kidney copper concentrations only when dietary copper was low. Spleen and colon copper concentrations were also significantly (P < 0.005) lower in rats fed high dietary iron compared to those fed adequate dietary iron.

(Table 5 )

(Table 6 )

(Table 7 )

	DISCUSSION

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In the current study, dietary iron did not significantly affect the formation of DMABP-induced ACF. Similarly, a recent study by Soyars and Fischer (1998) observed that dietary iron concentrations that are ~5 and 10 times adequate did not enhance ACF development in the colon of rats administered azoxymethane. The higher level of ACF observed in the study by Soyars and Fischer compared to the current study is a result of the carcinogen being administered. DMABP is a much less potent carcinogen than dimethylhydrazine or its metabolite, azoxymethane. In the current study, we utilized DMABP, rather than dimethylhydrazine or azoxymethane, because of the close similarity between it and certain mutagens isolated from cooked meat or fish.

One potential mechanism for the protective effect of dietary copper against DMABP-induced aberrant crypt formation is alterations in antioxidant enzymes. Two copper containing enzymes, CuZnSOD and ceruloplasmin, that may help protect against oxygen radical-mediated injury were significantly reduced in rats fed the low copper diets. CuZnSOD functions to eliminate superoxide radicals, and ceruloplasmin is hypothesized to inhibit iron-catalyzed radical formation (Fridovich 1975 , Gutteridge and Halliwell 1988 ). Substantial evidence has suggested that free radicals, particularly oxygen radicals, are involved in both the initiation and promotion stages of carcinogenesis (Sun 1990 ). Much of the evidence has come from the fact that antioxidants that scavenge free radicals directly, or that interfere with the generation of free radical-mediated events, inhibit the neoplastic process and that the activities of antioxidant enzymes are changed during tumor formation (Sun 1990 ). For example, when compared to their normal cell counterparts, tumor cells are always low in MnSOD and usually low in CuZnSOD activity (Marlens et al. 1985 , McCormick et al. 1991 , Oberley et al. 1978 , Sun 1990 , Sun et al. 1993 ).

Although altered antioxidant enzymes were found in many tumors, it is unknown whether this abnormality is one of the causes of cancer or if it is just one of the consequences of the carcinogenic process. Van Driel et al. (1997) found that colorectal carcinomas were characterized immunohistochemically by decreased amounts of CuZnSOD and MnSOD and total superoxide dismutase activity when compared to adjacent normal mucosa. However, quantitative differences in expression of CuZnSOD and MnSOD or superoxide dismutase activity were not detected between adenomas of the colon and normal adjacent mucosa (Van Driel et al. 1997 ). This suggests that decreased superoxide dismutase expression may occur at a later stage of the carcinogenic process. In contrast, overexpression of superoxide dismutase was shown to inhibit malignant transformation (Oberley and Oberley 1997 , Petkau et al. 1975 , St. Clair et al. 1992 ). This suggests that decreased superoxide dismutase expression may occur prior to initiation.

In the current study, we investigated whether alterations in dietary copper, manganese, and iron, which would affect both CuZnSOD and MnSOD activities, would be associated with the formation of ACF, an early step in the carcinogenic process. Heart CuZnSOD and total superoxide dismutase activity were significantly correlated with both DMABP-induced ACF and AC formation. The lowest number of aberrant crypt foci and total aberrant crypts were observed in the rats fed adequate dietary copper and adequate dietary manganese, and the highest number of aberrant crypt foci and total aberrant crypts were observed in those fed low dietary copper and low dietary manganese. Similarly, the highest heart total superoxide dismutase activity was observed in the rats fed adequate dietary copper and manganese, and the lowest heart superoxide dismutase activity was observed in those fed low dietary copper and manganese. These findings suggest that dietary alterations that affect superoxide dismutase activity will affect cancer susceptibility. However, heart and liver superoxide dismutase activities responded differently to the dietary changes. In contrast to the results observed in the heart, the highest liver total superoxide dismutase activity was observed in the rats fed adequate dietary copper and low dietary manganese, and the lowest liver total superoxide dismutase activity was observed in those fed low dietary copper and adequate dietary manganese. Therefore, in future studies, superoxide dismutase activity should be measured in the colon, particularly the epithelial cells, the target organ for DMABP-induced carcinogenesis because measurement of antioxidant enzyme biochemical activities in whole organs or tissues does not provide an estimate of enzyme activities of individual cell types within these organs or tissues from which the tumors develop.

In conclusion, ACF, which are a preneoplastic lesion of colon cancer, were used as a biomarker to measure the effect of dietary copper, manganese, and iron on aromatic amine-induced colon carcinogenesis. Low dietary copper significantly increased and low dietary manganese tended to increase the formation of DMABP-induced ACF and AC. Similarly, the highest heart total superoxide dismutase activity was observed in the rats fed adequate dietary copper and manganese, and the lowest heart superoxide dismutase activity was observed in those fed low dietary copper and manganese. These findings suggest that dietary alterations that affect superoxide dismutase activity may affect cancer susceptibility. Furthermore, the effect of dietary copper and manganese on ACF formation may have practical implications because diets in the United States often contain less copper and manganese than the estimated safe and adequate daily dietary intake for copper and manganese (Davis et al. 1992b , Klevay and Medeiros 1996 ).

	ACKNOWLEDGMENTS

 
The authors would like to thank Tiffany Carbaugh for help with the care of the animals and for help with the ashing of the samples, and Terry Schuler and Deb Johnson for the mineral analysis.

	FOOTNOTES

 
¹ Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

² The US Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer, and all agency services are available without discrimination.

⁴ Abbreviations used: AC, aberrant crypts; ACF, aberrant crypt foci; CuZnSOD, copper-zinc superoxide dismutase; DMABP, 3,2'-dimethyl-4-aminobiphenyl; MnSOD, manganese superoxide dismutase.

Manuscript received November 9, 1998. Initial review completed December 10, 1998. Revision accepted January 12, 1999.

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