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The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 63-69
United States Department of Agriculture, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034 and * University of North Dakota, Departments of Surgery and Pharmacology and Toxicology, Grand Forks, ND 58202
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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There is increasing evidence that selenium can protect against tumorigenesis or preneoplastic lesion development induced by chemical carcinogens. This study examined whether selenite, selenate or selenomethionine would be protective against 3,2'-dimethyl-4-aminobiphenyl (DMABP)-DNA adduct formation in the liver and colon of rats and sought to delineate the mechanism for the protective effects of the different chemical forms of selenium against aberrant crypt formation, a preneoplastic lesion for colon cancer. After injection of DMABP, two DNA adducts were identified in the liver and colon of rats. Supplementation with either 0.1 or 2.0 mg selenium/kg diet as either selenite or selenate but not selenomethionine resulted in significantly fewer (53-70%; P < 0.05) N-(deoxyguanosin-8-yl)-(deoxyguanosin-8-yl)-3,2'-dimethyl-4-aminobiphenyl (C8-DMABP)-DNA adducts in the colon but not the liver than in rats fed a selenium-deficient diet. Rats supplemented with selenomethionine had greater (P < 0.05) plasma and liver selenium concentrations and glutathione peroxidase activity than those supplemented with selenite or selenate; however, they also had more DMABP-DNA adducts. The protective effect of selenite and selenate against DMABP-DNA adduct formation apparently is not a result of alterations in plasma or liver selenium concentrations or altered glutathione peroxidase or glutathione transferase activities but may be related to differences in the metabolism of the different forms of selenium.
KEY WORDS: selenium · rats · DNA adducts · colon cancer
Selenium is an essential trace element for human health; it has received considerable attention for its possible role as an effective, naturally occurring, anticarcinogenic agent. Epidemiologic studies reveal that selenium intake correlates inversely with mortality from various types of cancer (Clark et al. 1996 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). The target cells of colon carcinogens are colonic crypt epithelial cells. Aberrant crypt foci (ACF)3 are putative preneoplastic lesions that have been detected in human colon resections and in experimental animals treated with chemical carcinogens (Bird 1995 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) has been used to study the induction and development of colon cancer in rodents (Shirai et al. 1990 Measurements of DNA adducts can be used to estimate the potential carcinogenicity of chemicals. The 32P-postlabeling assay introduced by Randerath et al. (1985) Several studies suggest that dietary selenium can alter the ability of cells to metabolize carcinogenic compounds (Arciszewska et al. 1982, Marshall et al. 1979 Chemicals.
DMABP was purchased from Toronto Research Chemicals (Toronto, Canada). Calf serum phosphodiesterase, micrococcal endonuclease, potato apyrase (grade I), ribonuclease A (Type IIIA) and ribonuclease T1 were purchased from Sigma Chemical (St. Louis, MO). Dithiothreitol and T4 polynucleotide kinase were purchased from U.S. Biochemical (Cleveland, OH). Adenosine 5'-triphosphate( 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. Animals had free access to demineralized water and purified diet. The basal diet was a selenium-deficient, torula yeast-based diet, which contained 30% torula yeast, 0.3% dl-methionine, 59% sucrose, 5% corn oil, 3.5% selenium-deficient AIN-76A mineral mix, 1.2% calcium carbonate, 1% AIN-76A vitamin mix, 0.1% choline bitartrate and 0.001% menadione sodium bisulfite complex (AIN 1977). By analysis, the basal diet contained <0.005 mg selenium/kg diet. Basal diet supplemented with 0.1 or 2 mg selenium/kg diet as selenite, selenate or selenomethionine was purchased from Harlan Teklad (Madison, WI). After 21 d of consuming the experimental diets, five rats per diet group were administered DMABP dissolved in peanut oil (33.3 g/L) by subcutaneous injection (50 mg/kg body weight). Three additional control animals per diet group received the comparable vehicle injection of peanut oil only. Rats were killed by exsanguination 48 h after DMABP or vehicle administration.
DNA isolation.
Rats were anesthetized with xylazine (Rompon, Mobay, Shawnee, KS) and ketamine (Ketaset, Aveco, Fort Dodge, IA) and killed by exsanguination. Liver and colon were collected, cleaned, rinsed with cold saline, immediately frozen in liquid nitrogen and stored at 32P-Postlabeling.
DNA adducts were measured by 32P-postlabeling using modifications of methods previously published (Feng et al. 1997 TLC analysis.
Each labeled sample was spotted on the D1 TLC plate (MN Polygram Cell 300 PEI) with a Whatman 1 wick on the top and developed overnight in D1 buffer (2.4 mol/L sodium phosphate buffer, pH 6.8). After the upper portion of the D1 plate and wick were discarded, each individual origin was cut out according to a radiogram template and transferred to the D3 plate (EM PEI Cellulose-F) with the magnet mediated transfer technique (Lu et al. 1986 Enzyme analyses.
Livers were thawed on ice, homogenized in 0.25 mol/L sucrose and centrifuged at 4°C (105,000 × g for 1 h, Beckman TL-100 ultracentrifuge). Glutathione peroxidase activity in the cytosol was assayed spectrophotometrically by the coupled enzyme procedure using hydrogen peroxide as the substrate (Paglia and Valentine 1967 Mineral analyses.
Samples were analyzed as reported previously (Finley et al. 1996 Statistical analyses.
The data were analyzed by a one-way ANOVA using an 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 affected by the treatments. Values are reported as means ± SEM in the text.
The concentration and chemical form of selenium in the diet did not significantly influence food intake or body weight gain. The mean weight of the rats at the end of the study was 130.8 ± 1.3 g.
Selenium status.
Red blood cell and liver cytosolic glutathione peroxidase activities were significantly (P < 0.0001) less in the selenium-deficient rats than in those supplemented with selenium, regardless of the chemical form (Table 1). The activity of both enzymes increased with dietary selenium concentration. However, both red blood cell and liver glutathione peroxidase activities were significantly greater in rats fed 0.1 mg selenomethionine/kg diet than in those fed 0.1 mg selenite or selenate/kg diet. Similarly, plasma selenium concentrations were significantly (P < 0.0001) less in the deficient rats than in those supplemented with selenium, regardless of the chemical form. Liver cytosolic selenium was significantly greater in rats fed 2.0 mg selenomethionine/kg diet than in those fed 2.0 mg selenite or selenate/kg diet.
DMABP-DNA adducts.
Autoradiograms of DMABP-DNA adducts in hepatic and colonic DNA obtained by the 32P-postlabeling method are shown in Figure 1. After a single DMABP injection of 50 mg/kg body weight, two DMABP-DNA adducts were identified in rats. The two adducts co-chromatogrammed with authentic C8-DMABP and N2-DMABP standards.
Glutathione transferase activity.
The dietary concentration but not the chemical form of selenium affected glutathione transferase activity in the liver (Table 2). Glutathione transferase activity was significantly (P < 0.05) higher in rats fed either the selenium-deficient or the 2 mg selenium/kg diet than in those fed the 0.1 mg selenium/kg diet. Glutathione transferase activity did not differ between the deficient rats and those fed 2 mg selenium/kg diet or as a result of the different chemical forms of selenium.
Two DNA adducts, C8-DMABP and N2-DMABP were present in the liver and the colon of rats after injection of DMABP. Supplementation with either selenite or selenate but not selenomethionine significantly reduced the amount of C-8-DMABP-DNA adducts in the colon but not in the liver compared with levels in rats fed a selenium-deficient diet. Although the liver is not a target organ for DMABP-induced carcinogenesis, the levels of DMABP-DNA adducts in the liver were measured because metabolic N-oxidation of DMABP catalyzed by hepatic cytochromes P450 is regarded as an initial activation step leading to carcinogenesis. The protective effect of selenite and selenate against DMABP-DNA adduct formation in the colon correlates with our previous observation that the frequency of aberrant crypt foci was significantly decreased in groups of rats treated with DMABP and supplemented with either selenite or selenate but not with selenomethionine (Feng et al. 1998
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Kneck et al. 1990) and suggest an increased risk of colon cancer in humans in geographic areas in which selenium is low in the soil (Clark et al. 1991). In a recent study by Clark et al. (1996 ), selenium supplementation reduced the incidence of, and mortality from carcinomas at several sites in the body including the colon. Diets high in selenium have been shown to suppress carcinogenesis in many animal tumor models (Chae et al. 1997, El-Bayoumy et al. 1996, Ip and Ganther 1996, Jao et al. 1996, Milner 1986, Reddy et al. 1996). However, most of these studies have used either synthetic organoselenium compounds (Chae et al. 1997, El-Bayoumy et al. 1996, Ip 1985, 1988 and 1996, Reddy et al. 1996) or inorganic selenium supplemented in the drinking water or in the diet at a concentration ranging from 0.5 to 8 mg selenium/kg diet (Ip 1985, Jao et al. 1996). These amounts are considerably greater than the nutritional requirement of rats (~0.15 mg selenium/kg diet) established by the National Research Council (1995). Furthermore, in humans, the ingestion of selenium is mainly in the form of selenomethionine through the consumption of cereals, grains, fruits and vegetables. Previous reports (Ip and Hayes 1989,Thompson et al. 1984) have shown that high dietary concentrations (5 mg selenium/kg diet) of selenite but not selenomethionine can inhibit mammary carcinogenesis in rats.
, Caderni et al. 1995, Feng et al. 1997, Pretlow et al. 1991 and 1994). Studies in humans have suggested 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). We recently observed that the frequency of ACF was significantly (P < 0.05) decreased in groups of Fisher-344 rats treated with an aromatic amine carcinogen and supplemented with selenite or selenate but not selenomethionine (Feng et al. 1998). These results indicate that selenium, depending on chemical form, can be efficacious against aromatic amine-induced ACF formation.
). DMABP is often used because of the close chemical similarity between it and certain mutagens isolated from cooked meat or fish. Similar to other aromatic amine carcinogens, DMABP must be metabolically activated to exert its genotoxicity. This is accomplished via a multistep process involving N-hydroxylation of the exocyclic amino group by cytochrome P450, followed by Phase II esterification thought to be mediated by cytosolic acetyltransferase (Boobis et al. 1994, Flammang et al. 1985, Westra et al. 1985). N-Acetoxy-DMABP is unstable and hydrolyzes to electrophilic arylnitrenium ions which bind covalently to DNA. The formation of covalent adducts between chemical carcinogens and DNA can have deleterious effects and is thought to be a prerequisite for the initiation of the carcinogenic process. DNA adducts can distort the shape of the DNA molecule, potentially causing mistranslations or, when the DNA replicates, an adducted base can be misread which causes a mutation in the new strand. Furthermore, repair of bulky adducts can result in breakages of the DNA strand, which can result in turn in mutations or deletions of genetic material (World Cancer Research Fund 1997).
is a highly sensitive tool for the detection and quantification of carcinogen-DNA adducts. By this method, adducts are resolved as fingerprints on TLC sheets after ion-exchange chromatography and autoradiography (Beach and Gupta 1992, Gupta et al. 1982).
). Thus, alterations in carcinogen metabolism may explain the ability of selenium to inhibit the initiation phase of carcinogenesis. This study assessed whether selenium supplementation would inhibit the formation of DMABP-DNA adducts in the colon and liver. Furthermore, we determined whether the chemical form of selenium, namely, selenite, selenate or selenomethionine, would affect DMABP-DNA binding.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
-32P) (purity >90%) was purchased from ICN Biomedicals (Costa Mesa, CA). Polyethyleneimine-cellulose TLC plates, MN polygram CEL 300 PEI and EM PEI-cellulose-F were purchased from Alltech Associates, (Deerfield, MI).
70°C until analyzed. Tissues were homogenized in a buffer of 1% SDS, 1 mmol/L EDTA and 25 mmol/L Tris, pH 7.4 (1 mL/100 mg tissue) with a mechanical homogenizer at a low speed for 10 s. Homogenate (1 mL) was incubated with 0.25 mg of RNAse A (10 g/L) in 10 mmol/L Tris, pH 7.4) and 50 units of RNAse T1 (5 × 106 U/L in 25 mmol/L Tris, pH 7.4) for 1 h at 37°C . Proteinase K (0.5 mg) was added and the mixture was incubated for an additional 1 h. After enzyme digestion, the mixture was extracted with equal volumes of phenol, phenol/sevag (1:1) and sevag (chloroform/isoamyl alcohol, 24:1, v/v). The DNA was washed twice with 70% ethanol, dried and dissolved in 5 mmol/L Bistris, pH 7.0. The purity of DNA was measured by A260/A280 and the concentration (mg/L) of DNA was determined by absorbance at 260 nm (Flammang et al. 1992, Gupta 1984).
). DNA (1.5-3 µg) from each sample and standards was digested to deoxyribonucleotide 3'monophosphates by incubation with 2-3 µg of micrococcal nuclease and 2-3 µg of phosphodiesterase at 37°C. After 3 h, 10 mmol/L tetrabutylammonium chloride and 10 mmol/L ammonium formate (pH 3.5) were added and the mixture was extracted twice with water-saturated 1-butanol. The combined 1-butanol phase was neutralized with 50 mmol/L bicine (pH 9.5) and 100 mmol/L dithiothreitol and evaporated to dryness in a Speed Vac. The adduct residues were redissolved in 25 µL of distilled water and vortexed for ~10 min. Each sample was labeled with 5 µL of PNK mix containing 925 kBq [-32P]ATP, 2 U of T4 polynucleotide kinase, 3 µL of 10 × PNK buffer (50 mmol/L bicine, 100 mg MgCl2, 10 mmol/L spermidine and 100 mmol/L dithiothreitol, pH 9.5) and distilled water. The reaction mixture was incubated at 37°C for 40 min. After each sample was labeled, 1.5 µL of potato apyrase (20 mU/µL) was added and the sample was incubated at 37°C for an additional 30 min.
). The D3 plate was developed in a 72% dilution of D3 buffer (3.6 mol/L lithium formate and 8.5 mol/L urea, pH 3.5). The D4 plate was developed perpendicular to the D3 plate. The bottom edge of the D4 plate was dipped quickly into distilled water and then developed in a 45% dilution of D4 buffer (1.2 mol/L lithium chloride, 8 mol/L urea and 0.5 mol/L Tris-base, pH 8.0). The D4 plates were exposed to a cassette screen, which was analyzed with a Phosphorimager. Individual adduct spots and background spots from each plate were quantitated.
).
. The reaction mixture contained 100 mmol/L phosphate buffer (pH 6.5), 1 mmol/L glutathione and 1 mmol/L 1-chloro-2,4-dinitrobenzene. The reaction was started by the addition of cytosol. Protein concentration was determined by the Bio-Rad protein assay (Hercules, CA).
). Briefly, plasma and liver cytosol samples were digested in concentrated nitric acid followed by overnight dry ashing (MgNO3 was added as an ashing aide to prevent volatilization of the Se). Samples were analyzed by hydride generation atomic absorption spectrometry. Quality control was maintained by analyzing a normal serum toxicology control (Utak Laboratories, Valencia, CA) with each assay. Serum control samples were determined to contain 97% of their certified values for selenium.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Effect of chemical form and concentration of selenium on indicators of selenium status in rats fed a selenium-deficient torula yeast-based diet supplemented with 0, 0.1 or 2 mg selenium/kg diet as selenite, selenate or selenomethionine1,2
View larger version (69K):
[in a new window]
Fig 1.
32P-Postlabeling profile of 3,2'-dimethyl-4-aminobiphenyl (DMABP)-DNA adducts in liver and colon of rats injected with DMABP.
View larger version (52K):
[in a new window]
Fig 2.
Levels of N-(deoxyguanosin-8-yl)-3,2'-dimethyl-4-aminobiphenyl (C8-DMABP) (A) and 5-(deoxyguanosin-N2-yl)-3,2'-dimethyl-4-aminobiphenyl (N2-DMABP) (B) DNA adducts in the colon of rats fed a selenium-deficient torula yeast-based diet supplemented with 0, 0.1 or 2 mg Se/kg diet as selenite, selenate or selenomethionine. Values are means ± SEM, n = 5. Bars without common superscript letters are significantly different (P < 0.05).
View larger version (53K):
[in a new window]
Fig 3.
Levels of N-(deoxyguanosin-8-yl)-3,2'-dimethyl-4-aminobiphenyl (C8-DMABP) (A) and 5-(deoxyguanosin-N2-yl)-3,2'-dimethyl-4-aminobiphenyl (N2-DMABP) (B) DNA adducts in the liver of rats fed a selenium-deficient torula yeast-based diet supplemented with 0, 0.1 or 2 mg Se/kg diet as selenite, selenate or selenomethionine. Values are means ± SEM, n = 5.
View this table:
Table 2.
Effect of Chemical Form and Concentration of Selenium
on Liver Glutathione Transferase Activity in Rats Fed a
Selenium-Deficient Torula Yeast-Based Diet Supplemented
with 0, 0.1 or 2 mg Selenium/kg Diet as Selnite,
Selenate or Selenomethonine1,2
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
).
, El-Bayoumy et al. 1996, Ip and Ganther 1996, Jao et al. 1996, Milner et al. 1986, Reddy et al. 1996 ). Most animal experiments studying the relationship of selenium to colon cancer have utilized dietary supplements of selenium greatly in excess, possibly in toxic concentrations, of what humans are exposed to. In contrast, the literature investigating the relationship between colon cancer susceptibility and dietary selenium deficiency is limited and controversial. Selenium deficiency increased mammary tumorigenesis only in animals fed a diet high in polyunsaturated fat (25% corn oil) but not in those fed a diet high in saturated fat or low in dietary fat (Ip 1985). Selenium-deficient rats have also been shown to have a significantly increased karryhoctic index (a measure of acute carcinogen-induced nuclear toxicity) in the colonic mucosa when they were treated with azoxymethane (Nelson et al. 1996), suggesting that deficiency increases the risk of cancer. In contrast, however, in a study by Pence and Buddingh (1985), selenium deficiency did not result in increased tumor incidence or tumor burden compared with a physiologically adequate selenium (0.1 mg/kg diet) concentration in the diet in animals administered dimethylhydrazine.
), there was a decrease in the formation of aberrant crypt foci when rats were fed 2.0 mg selenite or selenate/kg diet compared with those fed 0.1 mg selenite or selenate/kg diet. Furthermore, the absence of an effect on DNA adduct levels when the concentration of selenomethionine was increased from 0 to 0.1 mg/kg diet suggests that supplementation with selenite or selenate but not selenomethionine is decreasing DMABP-DNA adduct formation in the colon of rats. Future work needs to investigate whether selenium deficiency will increase tumor incidence.
, Liu et al. 1991). Although the mechanism through which selenium depresses DMBA-DNA binding remains unclear, it has been suggested that selenium may alter biliary excretion of DMBA metabolites (Liu and Milner 1992). The reduction in DMBA-DNA adducts by dietary selenium correlated directly with the ability of dietary selenium to inhibit the incidence of DMBA-induced mammary tumors (Liu et al. 1991, Liu and Milner 1992).
, Shimada et al. 1997). Metabolic N-oxidation of DMABP catalyzed by hepatic cytochromes P4501A2 is regarded as an initial activation step leading to carcinogenesis (Kadlubar and Hammons 1987). N-Hydroxy-DMABP that is formed in the liver may serve as a substrate for hepatic conjugation by 
-glucuronyltransferases; the conjugated form can then be transported to target sites (Kadlubar et al. 1977, Nussbaum et al. 1983). Nussbaum et al. (1983) have identified the N-hydroxy-N-glucuronide conjugate in the bile of rats given DMABP. Once in the intestine, the glucuronide conjugate could be hydrolyzed by bacterial and mucosal -glucuronidases and the active metabolite released. Subsequent reabsorption and metabolic activation of the N-hydroxy-DMABP in the colonic mucosa could occur and serve to initiate the neoplastic process (Davis et al. 1993, Kadlubar et al. 1981 and 1991, Turesky et al. 1991). However, alteration in cytochrome P450 activity apparently is not the explanation for the protective effect of selenite and selenate against DMABP-DNA adduct formation in the colon because there were no significant effects of the different selenium sources on DMABP-DNA adducts in the liver where most of the cytochrome P4501A2 is located (Kadlubar and Hammons 1987). Future studies should investigate whether the different chemical forms of selenium will affect hepatic -glucuronyltransferase, mucosal -glucuronidase or other colonic Phase 2 enzyme activities. Future studies should also investigate the effects of dietary selenium on repair of DMABP-DNA adducts to determine whether selenium inadequacy somehow delays the repair process.
, Lawrence et al. 1978). The elevation of glutathione transferase activity by selenium deficiency has been postulated to be a compensatory response that helps correct for the decreased selenium-dependent glutathione peroxidase activity by providing nonselenium-dependent glutathione peroxidase activity (Lawrence et al. 1978). Previous studies have also shown that colonic glutathione transferase activity is not influenced by the selenium content of the diet (Pence 1991). Thus, alterations in glutathione transferase activity apparently are not important for the protective effect of selenite and selenate against DMABP-DNA adduct formation.
). Selenium in selenomethionine may also be converted to hydrogen selenide, but the transformation involves the transulfuration pathway followed by a specific lyase to cleave the selenium (Deagan et al. 1987). In addition to this pathway, selenomethionine may also be incorporated into proteins as an analog of methionine (Butler et al. 1989). All of these forms of selenium may enter the methylation pathway in preparation for excretion through the urine or breath (Foster et al. 1986).
). The pathway that results in cancer prevention is unclear; however, cancer protection apparently is not related to formation of selenoproteins. Ip and Ganther (1996) hypothesized that it is selenium in the excretory pathway, especially the monomethylated form, that is effective against cancer. Thus, differences in the ability of the different chemical forms of selenium to protect against DNA adduct formation may be related to differences in the metabolic fates of the various compounds with the end result that more of the selenite and selenate are metabolized into the methylated pool.
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FOOTNOTES |
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Manuscript received 28 May 1998. Initial reviews completed 31 July 1998. Revision accepted 14 October 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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