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Division of Nutritional Toxicology, National Center for Toxicological Research, Jefferson, AR 72079
ABSTRACT
INTRODUCTION
EXPERIMENTAL METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The objective of this study was to examine effects of food restriction (FR) on the metabolic activation of aflatoxin B1 (AFB1) in rats and mice, which are AFB1-sensitive and -resistant rodent species, respectively. Forty percent FR [60% of ad libitum (AL) food consumption] reduced the metabolic activation of AFB1 in both rats and mice, causing formation of hepatic AFB1-DNA adducts to be 43% and 31% lower, respectively. The AFB1-DNA adduct 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) was predominantly formed in rat liver DNA; the formation of the ring-open analogue of AFB1-N7-Gua, AFB1-formamidopyrimidine (AFB1-FAP), was predominantly found in mouse liver DNA. In contrast to the in vivo results, the in vitro AFB1-DNA adduct formation mediated by the microsomes of liver, kidney or lung from FR-mice was greater than the formation of AFB1-DNA adducts mediated by the tissue microsomes from the AL-mice. Food restriction induced hepatic glutathione S-transferase (GST) activity, as measured by the formation of AFB1-glutathione conjugates (AFB1-SG), in both rats and mice; AFB1-SG was also formed in mouse kidney. Food restriction-induced GST activity assayed in an in vitro system, using [3H]AFB1-8,9-epoxide and glutathione (GSH) as substrates, was also found when mouse kidney and lung cytosolic fractions were used. Food restriction inhibited the AFB1-induced DNA double strand breaks in mouse kidney. The reduction of levels of AFB1-DNA adduct formation in mouse kidney was comparable to the degree of AFB1-induced DNA strand breakages. The results of this study indicate that the metabolic activation of AFB1 can be modulated by FR through the alteration of the formation of AFB1-DNA adducts and AFB1-SG conjugation. However, species and tissue specificities exist regarding the metabolic activation of AFB1.
Key words: rats, mice, food restriction, aflatoxin B1-DNA adducts, aflatoxin B1-glutathione conjugates.INTRODUCTION
Nutritional modification of carcinogenesis has become an active area of research, in part because the growing awareness that dietary excess, deficiencies and imbalances can play a major role in the etiology or modulation of cancer. Food restriction (FR)5 extends life span and decreases the prevalence of spontaneous and chemically induced cancers in laboratory animals (Allaben et al. 1991
, Pariza and Boutwell 1987, Tannenbaum 1942). The effects of FR on chemically induced tumors have been reported for various target tissues, including the aflatoxin B1 (AFB1)-induced liver tumors in rats (Newberne and Rogers 1986). The mechanisms of the reduction of tumor incidence by FR are not known. However, the finding that FR inhibits tumor formation induced by the indirect-acting carcinogen (requiring metabolic activation) methylazomethanol, but not by the direct-acting carcinogen N-methylnitrosourea (Pollard and Luckert 1985), led us to focus our studies on the role FR may play in the modulation of neoplastic disease by inducing alterations in activities of drug-metabolizing enzymes, both phase I and phase II, of animals.
The metabolic activation of xenobiotics and drugs is catalyzed by cytochrome P450 (CYP)-dependent xenobiotic or drug-metabolizing enzymes. Any factor altering the activity of these enzymes may affect the metabolic activation of certain carcinogens. Such alterations may influence both the pharmacokinetics and the efficacy of various drugs as well as the metabolic activation of toxic xenobiotics. Drug-metabolizing enzymes are involved not only in the initiation of neoplastic disease but also in the acute cellular activation and detoxification of many chemicals and drugs. Furthermore, the alteration of activities of drug-metabolizing enzymes by FR may affect the metabolism, utilization, and efficacy of drugs needed. The effect of FR on the metabolic activation of xenobiotics in laboratory animals can be estimated by measuring either the specific enzyme activities (activation or detoxification) or changes in carcinogen-DNA adduct formation. Previous studies demonstrated that FR alters xenobiotic metabolizing enzyme activities (Chou et al. 1993a, Koizumi et al. 1987, Leakey et al. 1989, Sachan and Das 1982), decreases 7,12-dimethylbenz[a]anthracene (DMBA) binding to dermal DNA in mice (Pashko and Schwartz 1983) and reduces the binding of AFB1 to hepatic nuclear DNA in rats (Chou et al. 1993a). Results indicate that the effect of FR on the initiation stage of carcinogenesis could differ in different species of animals and that FR may play an important role in the modulation of neoplastic diseases. Here, we report results of a study on the effects of FR on AFB1 activation in mice, a species less sensitive to AFB1-induced carcinogenesis than rats because of the high glutathione S-transferase (GST) activity in mouse liver. A species comparison between mice and rats, in terms of the formation of the major AFB1-DNA adducts and AFB1-glutathione conjugates (AFB1-SG), was also conducted.
Aflatoxin B1 is a potent food carcinogen and has been implicated epidemiologically as a causative agent in human liver cancer (Busby and Wogan 1984, Wogan 1992). Aflatoxin B1 requires metabolic activation to exert its genotoxicity. Metabolic activation of AFB1 (Fig. 1) by microsomal xenobiotic metabolizing enzymes, both in vivo and in vitro, results in the formation of AFB1-8,9-epoxide, which binds to cellular macromolecules such as DNA, RNA or proteins (Essigmann et al. 1982, Swenson et al. 1974) and exhibits mutagenic and carcinogenic activities. The major AFB1-DNA adducts are identified as 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) and AFB1-formamidopyrimidine (AFB1-FAP) (Fig 1). The latter is a ring-open analogue of AFB1-N7-Gua (Croy et al. 1978, Lin et al. 1977, Shaulsky et al. 1990). Published data suggest that the extent of AFB1-induced covalent modification in DNA correlates well with the degree of carcinogenic susceptibility of testing animals (Croy et al. 1983, Groopman et al. 1991). Indeed, the AFB1-DNA adduct has been considered as a useful biomarker for AFB1-induced mutagenesis and carcinogenesis. Aflatoxin B1-induced mutagenic or carcinogenic activities are altered by various nutritional factors, including changes in fat (Newberne et al. 1979), protein (Dunaif and Campbell 1987), essential vitamins (Bhattacharya et al. 1987) and minerals (Francis et al. 1988) and by restriction of total food consumption or energy intake (Newberne and Rogers 1986). In this study, the hypothesis that FR may reduce AFB1 activation in both sensitive and resistant species of rodents by modulating AFB1-metabolizing enzyme activities (both phase I and phase II) is tested.
EXPERIMENTAL METHODS
-32P]dCTP (specific radioactivity 111 GBq/µmol) was purchased from Amersham (Arlington Height, IL). Dimethyldioxirane was synthesized according to the method of Adam et al. (1989). Other reagents required for DNA purification and HPLC analyses were HPLC grade.
Animals and diets. Weanling male B6C3F1 mice and Fischer 344 rats from the breeding colony of the National Center for Toxicological Research were housed individually. Free access (AL) was given to a standard NIH-31 open formula diet (Purina Mills, Richmond, IN) with the following composition (g/kg): fish meal (60% protein), 90; soybean meal (48.59% protein), 50; alfalfa meal (17% protein), 20; corn gluten meal (60% protein), 20; ground whole hard wheat, 355; ground #2 yellow shelled corn, 210; ground whole oat, 100; wheat middlings, 100; brewer dried yeast, 10; soybean oil, 15; sodium chloride, 0.5; decalcium phosphate, 15; ground limestone, 5; vitamin premixes, 5. The FR animals were fed 60% of the AL diet using a vitamin-supplemented NIH-31 ration (containing 8.35 g/kg of vitamin premixes) at AL concentrations (Witt et al. 1991
). Rats began FR at the age of 10 wk; mice started FR at the age of 16 wk. At 16 wk of age, groups of AL and FR rats (four per group) received a single dose of [3H]AFB1 [0.1 mg AFB1/kg body wt, specific radioactivity, 7.7 GBq/mmol, dissolved in dimethylsulfoxide (DMSO), 10.5 mol/L]. The control groups of rats were treated with 10.5 mol/L DMSO only. At 20 wk of age, AL- and FR-mice (four animals per group) were treated with a single dose (1, 2, 5, 10 mg/kg body wt, respectively) of [3H]AFB1 (specific radioactivity, 5.5 GBq/mmol, dissolved in 10.5 mol/L DMSO) injected intraperitoneally. Control groups were treated with vehicle only. At various time points, animals were killed by hypoxia with CO2 followed by exsanguination. Livers, kidney, lungs and other tissues were removed immediately, rinsed with cold saline, and stored at 
70°C for further analysis.
Preparation of subcellular fractions.
Tissues were homogenized with cold sucrose-Tris buffer, pH 7.4, containing 0.25 mol/L sucrose, 25 mmol/L KCl, 10 mmol/L MgCl2 and 50 mmol/L Tris-HCl. Nuclei prepared by washing with 0.5% Triton X-100 in buffer were used for DNA isolation and AFB1-DNA adduct analysis. DNA was purified with RNase A and protease K treatment, followed by phenol and chloroform-isoamyl alcohol (24:1) extractions, and assayed according to the method of Beland et al. (1984). Microsomal and cytosolic fraction were prepared by differential centrifugation (Chou et al. 1987) and stored at 70°C prior to use. Protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin as standard.
Preparation of aflatoxin B1-8,9-epoxide.
Aflatoxin B1-epoxide was prepared from the peroxidation of AFB1 by dimethyldioxirane, which was prepared as previously described (Baertschi et al. 1989, Chen et al. 1995). Briefly, approximately 1.5 equivalent weight of freshly prepared dimethyldioxirane (concentration range from 0.05 to 0.12 µmol/L) in anhydrous acetone was added to AFB1 solution (dissolved in anhydrous acetone). The reaction mixture was stirred at room temperature for 20 min, and the resulting AFB1-8,9-epoxide was obtained. Solvent and excess dimethyldioxirane were removed by vacuum evaporation. The residue was redissolved in anhydrous acetone used for the preparation of AFB1-DNA adducts and AFB1-SG conjugation products. [3H]Aflatoxin B1-8,9-epoxide was similarly prepared as described using [3H]AFB1 as the starting material.
Analysis of aflatoxin B1-DNA adduct.
Standard AFB1-N7-Gua adduct was prepared by a reaction of AFB1-8,9-epoxide with calf-thymus DNA (1 g/L in 50 mmol/L phosphate buffer solution, pH 7.4) with vigorous stirring. After 30 min of incubation, DNA was precipitated by adding two volumes of ethanol. The ethanol phase contained the AFB1-8,9-dihydrodiol. The purified DNA was hydrolyzed with 0.15 mol/L HCl at 90°C for 30 min. The ring-open form AFB1-FAP adducts were prepared by incubating AFB1-N7-Gua solution with 15 mmol/L sodium carbonate and 30 mmol/L sodium bicarbonate, pH 9.5, for 30 min. After incubation, the pH was adjusted to 1.0 with 1 mol/L HCl. The DNA solution was hydrolyzed at 70°C for 20 min, and 1 mol/L NaOH was added to adjust to pH 5 prior to the HPLC analysis. The AFB1-N7-Gua and AFB1-FAP adducts were separated by a reversed-phase HPLC equipped with a µBondapak C-18 column (3.9 × 300 mm) eluted with 3.5 mol/L acetonitrile in water. The structural confirmation was accomplished by mass and NMR spectrometry (Baertschi et al. 1989, Chen et al. 1995, Shaulsky et al. 1990). The total [3H]AFB1-DNA binding of in vivo samples was determined by measuring the radioactivity in the modified DNA. The [3H]AFB1-N7-Gua adducts obtained from in vivo samples were identified by co-chromatography with the synthesized standard on a reversed-phase HPLC column (Waters, µBondapak C18, 39 × 300 mm) eluted with a linear gradient of 1.75 to 5.25 mol/L of acetonitrile for 40 min and quantified by measuring the radioactivity of bound [3H]AFB1. In vitro microsome-mediated binding of [3H]AFB1 to calf thymus DNA was determined by using a 1.0-mL reaction mixture containing 1.0 mg of DNA, 20 nmol of [3H]AFB1 (specific radioactivity 7.4 GBq/mmol), 0.1 mol/L sodium phosphate, pH 7.4, 0.65 mmol/L NADPH, 3 mmol/L MgCl2, and 1 mg of liver (or other tissue) microsomal protein from AL or FR rats. After 30 min of incubation, DNA was extracted and purified. The total binding and specific AFB1-DNA adducts were measured as described.
Analysis of aflatoxin B1-glutathione conjugate.
The authentic standard of AFB1-SG (or [3H]AFB1-SG) was prepared by the reaction of AFB1-8,9-epoxide (or [3H]AFB1-8,9-epoxide) with GSH catalyzed using GST of rat liver cytosol as enzyme source (Chen et al. 1995). An aliquot of rat liver cytosol was dialyzed overnight against 3 L of 10 mmol/L sodium phosphate buffer, pH 7.4. Aflatoxin B1-epoxide in acetone was gradually added with a microsyringe into 1 mL of AFB1-8,9-epoxide reaction mixture with vigorous stirring. The reaction mixture (pH 7.4) contained 50 µmol of sodium phosphate, 5 µmol of GSH and 1 g/L liver cytosolic protein. After 5 min of incubation at room temperature, 50 µL of 2 mol/L acetic acid was added, and the mixture was centrifuged to remove the precipitated cytosolic proteins. The supernatant was filtered through a 0.45-µm filter, and the filtrates were stored at 70°C until analyzed by HPLC. The [3H]AFB1-SG samples were analyzed by HPLC eluted with a linear gradient of 0-7 mol/L of acetonitrile-H2O over a period of 40 min (1 mL/min). The HPLC was monitored by a photodiode-array detector (Waters model 996) at 362 nm wavelength. The fractions containing [3H]AFB1-SG were collected, and the radioactivity was measured by a liquid scintillation counter. The analysis of [3H]AFB1-SG of in vivo samples was performed by applying 30 µL of respective cytosolic samples on HPLC, and the radioactivity of the fractions containing [3H]AFB1-SG was measured as described. The cytosolic GST activity was expressed as micromoles of [3H]AFB1-SG per milligram of protein per minute.
Assay of DNA strand breaks.
To determine the effect of FR on AFB1-induced DNA strand breaks, random oligonucleotide-primed synthesis DNA fragmentation assay was employed (Basnakian and James 1994). Mouse kidney (200 mg) was powdered with liquid nitrogen and digested with proteinase K (0.1 g/L) at 50°C for 12 h. The genomic DNA was isolated according to the method of Ausubel et al. (1989). DNA was dialyzed against 100 volumes of 10 mmol/L Tris-HCl-1 mmol/L EDTA buffer (TE buffer), pH 7.4, overnight. The DNA concentration was diluted to 25 g/L with TE buffer. A 50-µL DNA solution was incubated at 100°C for 5 min to denature the DNA and then cooled on ice. Ten microliters of each DNA sample was placed in a 96-well polypropylene plate on ice, and a 15-µL reaction mixture (containing 2.5 µL of 0.5 mmol/L dGTP, dATP and dTTP mixture, 0.5 unit of Klenow fragment, 2.5 µL of Klenow buffer, 0.45 µL of 33 µmol/L dCTP, and 18.5 kBq of [32P]dCTP) was added to each sample well. The plate was incubated at 16°C for 30 min. The reaction was stopped by cooling the sample on ice, and 25 µL of 12.5 mmol/L EDTA, pH 8.0, was added. An aliquot (5 µL) from the incubation mixture was applied on DE81 filter paper. The filter paper was rinsed five times in 100 mL of 0.5 mol/L sodium phosphate buffer and twice with water. After drying, the radioactivity on the paper was counted in a scintillation counter.
Statistical analysis.
Data were expressed as means ± SEM. Differences between the AL and FR groups were evaluated using one-way ANOVA. Differences were considered statistically significant when P < 0.05.
RESULTS
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Table 1. Effects of 6 wk of food restriction (FR) on the body weights, relative liver weights, hepatic and renal aflatoxin B1 (AFB1)-DNA adduct formation, and AFB1-glutathione (AFB1-SG) conjugation of male B6C3F1 mice and F344 rats1,2 |
Table 2.
Effects of food restriction (FR) on in vitro aflatoxin B1 (AFB1)-DNA adduct formation and on AFB1-glutathione (AFB1-SG) conjugation mediated by microsomes obtained from liver, kidney and lung of male B6C3F1 mice and F344 rats1,2
Fig. 2.
Dose-response curves of the formation of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) and its ring-open form, AFB1-formamidopyrimidine (AFB1-FAP), in DNA from livers and kidneys of mice that consumed food ad libitum (AL) or were 40% food restricted (FR). The mice were treated with tritiated aflatoxin B1 ([3H]AFB1, 7.7 GBq/mmol) and killed 3 h after dosing. Values are means ± SEM, n = 4. *FR significantly different from AL (P < 0.05, A vs. B, and C vs. D).
[View Larger Version of this Image (30K GIF file)]
Fig. 3.
Time courses of the removal of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) and its ring-open form, AFB1-formamidopyrimidine (AFB1), from livers and kidneys of mice that consumed food ad libitum (AL) or were 40% food restricted (FR). The mice were treated with a single dose of tritiated aflatoxin B1 ([3H]AFB1, 7.7 GBq/mmol) and killed 3 h after dosing. Values are means ± SEM, n = 4. *FR significantly different from AL (P < 0.05, A vs. B and C vs. D).
[View Larger Version of this Image (28K GIF file)]
). Contrary to the results in FR rats, the AFB1 metabolic activation in a microsome-mediated system using liver microsomes from FR mice was greater than that for AL mice (Table 2). This enhancement of adduct formation was probably due to an increase of mouse liver AFB1-metabolizing enzyme activity, such as CYP1A-dependent 7-ethoxyresorufin O-dealkylase and CYP2B-dependent ethoxyresorufin O-deethylase (Chou et al. 1993b). The in vitro formation of AFB1-DNA adducts mediated by microsomal fractions from mouse kidney and lung was also induced by FR (Table 2). The order of the rates of in vitro AFB1-DNA adduct formation in these mouse tissues was liver > kidney > lung (Table 2), comparable to the order of tissue susceptibility toward the microsomal metabolic activation of AFB1.
Fig. 4.
The effect of food restriction (FR) on the dose-response curve of the formation of aflatoxin B1-glutathione (AFB1-SG) conjugation in mouse livers (A) and kidneys (B). Mice were treated with a single dose of [3H]AFB1 (7.7 GBq/mmol) and were killed 3 h after dosing. Values are means ± SEM, n = 4. *FR significantly different from AL (P < 0.05).
[View Larger Version of this Image (20K GIF file)]
0.05). The result was comparable to the data for the reduction of in vivo adduct formation and the greater detoxification of AFB1 by FR mice.
DISCUSSION
studied AFB1-induced hepatocarcinoma in male F344 rats fed 25% restricted diets and found that the liver tumor incidence was reduced significantly. Recently, we found that FR decreased spontaneous and AFB1-induced mutations in rat lymphocytes at the hypothanthine guanine phosphoribosyl transferase (hprt) locus (Casciano et al. 1996). This finding agrees with our results that FR may decrease AFB1-induced carcinogenic initiation by reducing rat liver AFB1 metabolizing enzyme activity and subsequently decreasing the AFB1-DNA binding (Chou et al. 1993a). The in vivo formation of AFB1-DNA adducts involves metabolic activation (AFB1 epoxidation) and detoxification (AFB1-SG formation). Thus, the objective of this study was to generalize our hypothesis that FR reduces metabolic activation of AFB1 in both AFB1-sensitive (male F344 rats) and AFB1-resistant animals (male B6C3F1 mice) and to compare the mechanisms of the effect of FR on the metabolic activation of AFB1 in these two species. Our results demonstrate a reduction of metabolic activation of AFB1 in both FR rats and mice; however, species and tissue specificities exist regarding AFB1 epoxidation, AFB1-DNA adduct formation and AFB1-SG conjugation. Mice are less sensitive to AFB1-induced hepatocarcinogenesis, probably because of their high levels of cytosolic GST activity. The GST activities in livers of both rats and mice can be induced by FR. The FR-induced GST activity and FR-reduced CYP2C11 in rat liver (Leakey et al. 1989) resulted in the decrease of AFB1-DNA adduct formation in rats. The FR-induced mouse liver GST activity and subsequently increased formation of AFB1-SG. FR also reduced in vivo AFB1-DNA adduct formation in mouse liver, in spite of the increase in mouse liver microsomal AFB1-epoxidation induced by FR. Removal of the AFB1-DNA adducts in rat and mouse liver and kidney showed biphasic slopes (Gao and Chou 1992). The resulting AFB1-DNA adducts detected were dependent upon the levels of the reactive metabolite (AFB1-8,9-epoxide) formed and the levels of cellular GST that catalyzed the formation of the detoxification product, AFB1-SG. In addition, the total AFB1-DNA adducts may also be affected by DNA repair enzymes and the depurination of AFB1-bound purine moiety. Our results demonstrate that the reduction of the hepatic AFB1-metabolizing enzyme activities by FR resulted in the decrease of AFB1-DNA adduct formation and AFB1-induced DNA strand breaks in both mouse kidney and rat liver (Gao and Chou 1992). Random oligonucleotide-primed synthesis, the method employed in this study to measure the mouse DNA strand breaks, was more sensitive than the alkaline unwinding technique (Gao and Chou 1992) used previously to assay rat liver DNA strand breaks. The tissue specificity of the effect of FR on the formation of the AFB1-DNA adduct formation agreed well with the susceptibility of the tissue to AFB1 carcinogenesis.
, Eaton and Gallagher 1994) and is relatively resistant to DNA repair processes (Martin and Garner 1977). It has been proposed that the initial DNA adduct formation may be more important to the ultimate tumor formation than the persistent DNA adducts, such as AFB1-FAP (Bailey 1994, Eaton and Gallagher 1994, Hoseyni 1993). Thus, our finding that AFB1-N7-Gua was formed predominantly in rat liver and that the ring-open form (AFB1-N7-FAP) was predominantly generated in mouse liver may support the hypothesis. The data suggest that the rate of formation of the ring-open form from AFB1-N7-Gua under our experimental conditions may be different in these two species. The ring-open DNA adducts may occur non-enzymatically either in situ after the DNA was modified by AFB1 or during the sample preparation. However, the possibility that the formation of ring-open adducts was catalyzed by enzymes could not be excluded. Metabolites of AFB1, such as aflatoxin M1, P1 and Q1 can also be metabolically activated and bind to DNA to form minor AFB1-DNA adducts (Bailey 1994, Lutz et al. 1980, Raney et al. 1992). However, under our HPLC conditions, only two AFB1-DNA adduct peaks were identified.
FOOTNOTES
Manuscript received 29 January 1996. Initial reviews completed 19 March 1996. Revision accepted 3 October 1996.
LITERATURE CITED
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