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Effect of dietary selenium on the metabolism of aflatoxin B1 in turkeys
Fd Chem. Toxic. Vol. 22, No. 8, pp. 637-642, 1984
0278-6915/84 $3.00+ 0.00 Copyright © 1984 Pergamon Press Ltd
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EFFECT OF DIETARY SELENIUM ON THE METABOLISM OF AFLATOXIN BI IN TURKEYS J. F. GREGORY III Food Science and Human Nutrition Department, University of Florida and G . T . EDDS
Department of Preventive Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA (Received 29 December 1983)
Abstract--To investigate the biochemical mechanism of the previously reported protective effect of dietary selenium against aflatoxin toxicity, the hepatic metabolism of aflatoxin Bt in turkey poults was examined at various dietary selenium concentrations. Diets were supplemented with 0.2, 2.0 or 4.0 ppm selenium (as sodium selenite) and 500 ng aflatoxin B~/g diet in an 18-day trial. Free and conjugated aflatoxin and metabolites were quantified using high-performance liquid chromatography. The proportion of liver aflatoxins in conjugated forms increased and the ratio of free aflatoxin B~/M] decreased with increasing dietary selenium concentrations. These in vivo results provide evidence of selenium-induced enhancement of aflatoxin detoxification processes. In a similar experiment using 2.0 ppm selenium and 750 ng aflatoxin B~/g diet, the concentration of hepatic reduced glutathione, cytochrome P-450 and the activity of enzymes involved in the metabolism of aflatoxin B~ and glutathione were determined. Although the selenium supplement increased glutathione peroxidase activity, dietary selenium had no effect on reduced glutathione or cytochrome P-450 concentrations or on the activities of glutathione transferase E, glucuronyl transferase and cytochrome c reductase. These data indicate that the protective action of selenium is not mediated by an increase in glutathione availability for aflatoxin conjugation or by effects on the activities of these enzymes as measured in vitro.
INTRODUCTION
Similarly, studies of rats fed diets containing various vegetables and aflatoxin B~ indicate that the hepatoRecent research has shown that a variety of nutrients carcinogenic effects of the toxin are strongly and other dietary components can influence the acute influenced by minor food components (Boyd, Babish and chronic toxicity of aflatoxins. While the general & Stoewsand, 1982; Boyd, Misslebeck & Stoewsand, processes of aflatoxin metabolism have been well 1983; Boyd, Sell & Stoewsand, 1979). elucidated, many of the qualitative and quantitative Several studies have demonstrated that certain dietary effects are largely unknown. dietary metals provide a protective effect against Variations in the macronutrient components of the aflatoxin toxicity in several animal species. Newberne diet have been shown to have pronounced effects on & Conner (1974) first reported that selenium suppleaflatoxin toxicity. Deficiency of dietary lipotropic mentation up to a dietary level of 1.0ppm factors (choline and methionine) may increase or progressively reduced the acute toxicity of aflatoxin decrease the resistance of rats to the acute toxicity Bt in rats, while greater levels enhanced the observed and hepatocarcinogenic effects of aflatoxin (Camp- mortality. This protective effect was confirmed in bell, Hayes & Newberne, 1978; Newberne & Gross, several later studies with swine and turkeys (Bur1977), depending on the overall diet composition. guera, Edds & Osuna, 1983; Davila, Edds, Osuna & These effects are strongly linked to dietary variables Simpson, 1983). A similar protective effect has been such as protein and fat levels and the vitamin B-12 observed with cadmium when added to the diets of and folacin status of the animal (Campbell et al. swine (Osuna & Edds, 1982a,b; Osuna, Edds & 1978; Newberne, Weigert & Kula, 1979; Rogers, Simpson, 1982) although the mechanism of action Lenhart & Morrison, 1980; Temcharoen, An- has not been identified. Protective effects of selenium ukarahanonta & Bhamarapravati, 1978). Various against the hepatocarcinogenesis induced by types of dietary fibre have also been shown to affect 2-acetylaminofluorene also have been reported the susceptibility of animals to the toxicity of the (Wortzman, Besbris & Cohen, 1980). In view of the mycotoxin (Frape, Wayman & Tuck, 1981 & 1982). demonstrated protective effect of selenium against the Minor dietary components may modify the metab- acute toxicity of aflatoxin Bt and the postulated olism and subsequent toxic effects of aflatoxins by anticarcinogenic effect of selenium (Griffin, 1979), induction of microsomal or other enzyme systems. studies were conducted by Chen and associates to Experiments with microsomal enzyme inducers such investigate the possible influence of selenium and as phenobarbital (Farris & Campbell, 1981) and the vitamin E on certain aspects of the metabolism of antioxidant, ethoxyquin (Cabral & Neal, 1983) dem- aflatoxin B~ in rats and chicks (Chen, Goetchius, onstrated protective effects for these compounds. Campbell & Combs, 1982; Chen, Goetchius, Combs 637
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J. F. GREGORYIII and G. T. EDDS
& Campbell, 1982). Their results suggested that combined vitamin E-selenium deficiency enhanced aflatoxin binding to hepatic DNA and RNA in rats, while opposite effects were found for chicks. No clear relationships were detected between supplementation levels of either nutrient and the activity of various enzymes of the mixed function oxidase system, various transferases involved in aflatoxin conjugation, or the extent of covalent binding of the toxin to cellular macromolecules for either species. The purpose of this study was to elucidate further the metabolic basis of the protective effect of dietary selenium by examining the effects of various levels of dietary selenium on the metabolism of aflatoxin B~ in turkey poults. The influence of selenium on the hepatic levels and pattern of distribution of free and conjugated aflatoxin metabolites and on the activity of enzymes involved in the activation or detoxification of the mycotoxin was examined. EXPERIMENTAL
Aflatoxin compounds. A mixed solution of aflatoxins B~, G~, B2 and G2 was purchased from Applied Science Laboratories, Inc. (State College, PA). Aflatoxin Qt was synthesized by enzymatic hydroxylation of aflatoxin B 1 (Hsieh, Dalezios, Krieger et al. 1974). Aflatoxin B~ used in this synthesis, aflatoxin M~, and aflatoxicol were obtained from Sigma Chemical Co. (St Louis, MO). Aflatoxin B~ used in diet fortification was obtained from Calbiochem-Behring Corp. (San Diego, CA). The purity of all compounds was confirmed by high performance liquid chromatography (HPLC). Experimental protocols. The basal diet consisted of Purina Startena Game Bird Chow (Ralston Purina Co., Inc., St Louis, MO), which contained 30~o protein and 0.1 #g selenium/g diet (ppm) from sodium selenite. A sodium selenite premix was used for additional fortification. Aflatoxin fortification was carried out as described previously (Gregory, Goldstein & Edds, 1983). In experiment I, day-old turkey poults (male, Large White; Thaxton Turkey Farm, Watkinsville, GA) were randomly assigned to diet groups of four animals. Poults were kept initially for 15 days in two brooder rings fitted with infra-red lamps to provide constant temperature. During this adaptation period the basal diet was fed ad lib. The poults were then moved to 1.5 x 3.1 m pens for an 18-day experimental period during which they were fed the basal diet supplemented with 0.2, 2.0 or 4.0 ppm selenium (as sodium selenite) containing no added aflatoxin or supplemented with 500 ng aflatoxin B~/g of diet. At the end of the 18-day feeding period, the poults were killed by cervical dislocation and the livers rapidly excised. The livers were frozen on dry ice, then stored at - 2 0 ° C until they were analysed for aflatoxins. In experiment II, an identical protocol was followed except that groups of eight poults were fed diets supplemented with: (a) no added aflatoxin or selenium (control); (b) 2.0 ppm selenium (as sodium selenite); (c) 750 ng aflatoxin B~/g diet; or (d) 2.0 ppm selenium plus 750ng aflatoxin Bi/g diet. After the 18-day experimental period, the poults were killed (two/day from each group over a 4-day period) and
the livers were removed. This staggered experimental design was necessary because of the lengthy centrifugation procedures required in the fractionation of the livers for biochemical analysis. A small portion of each liver was taken for immediate analysis of reduced glutathione, while the remainder of the liver was subjected to differential centrifugation (van der Hoeven & Coon, 1974), as described below, prior to enzymatic analysis. Aflatoxin determination. The concentrations of free and conjugated forms of aflatoxins in livers were determined in Experiment I using the HPLC method of Gregory & Manley (1982). This method is based on a determination of free aflatoxins (chloroformacetone soluble), after treatment with trifluoroacetic acid, using a reverse-phase column and fluorometric detection. For the determination of conjugated aflatoxin metabolites, the aqueous phase remaining after extraction of free toxins is subjected to acid hydrolysis (0.2 N-HCI, 90°C, 2 hr), followed by extraction of the released aflatoxins with chloroform, treatment with trifluoroacetic acid, and HPLC analysis. This procedure permitted accurate quantitation of free and conjugated aflatoxins B1, G1, B2, G2, MI, Q~ and aflatoxicol at limits of sensitivity of at least 0.01 ng B~ equivalents/g tissue. The specificity of this procedure has been supported by fluorescence spectral examination (Gregory & Manley, 1982). Biochemical analyses. The liver analyses of Experiment II were started on the day the animals were killed, on which nonprotein thiols (mainly reduced glutathione) were determined colorimetrically (Sedlack & Lindsay, 1968) and differential centrifugation (van der Hoeven & Coon, 1974) was performed to prepare liver fractions for subsequent enzymatic assays. Post-mitochondrial supernatant (12,000g supernatant; S-12), microsomal (105,000g pellet), and 105,000g supernatant fractions were prepared for each liver. Microsomal fractions were suspended in 0.01M-tris-acetate buffer (pH7.4) containing 0.1 mM-EDTA and 25~ (v/v) glycerol. All liver fractions were stored at - 2 0 ° C until they were analysed for various enzymes potentially involved in aflatoxin and/or glutathione metabolism. Microsomal cytochrome P-450, cytochrome c reductase, and glucuronyl transferase were assayed essentially as described by Omura & Sato (1964), Gigon, Gram & Gillette (1969), and Lucier, McDaniel & Matthews (1971), respectively. Microsomes were activated by treatment with Triton X-100 prior to assay of glucuronyl transferase (Lucier et al. 1971). Glutathione peroxidase activity in the 105,000g supernatant was determined by the method of Lawrence & Burk (1976) was 0.25 mM-hydrogen peroxide as substrate in order to measure selenium-dependent activity. The activity of glutathione S-transferase E, which specifically catalyses the conjugation of epoxide substrates, was determined in the 12,000 g supernatant (S-12) fractions using the method of Fjellstedt, Allen, Duncan & Jakoby (1973). Assays were performed in duplicate or triplicate. Preliminary experiments were performed for each enzyme assay to determine the linear range with respect to enzyme concentration and reaction timecourse. Protein was determined by the biuret method (Gornall, Bardawill & David, 1949).
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Effect of Se on AFB, metabolism in turkeys Statistical methods. Differences between aflatoxin levels in Experiment I were evaluated using one-way analysis of variance and the Tukey procedure for multiple comparisons. The main effects and interactions of selenium and aflatoxin B, in Experiment II were evaluated using two-way analysis of variance. These methods have been described by Neter & Wasserman (1974).
included in calculations of total aflatoxin residue concentrations because of this inconsistency. Previous chromatographic and spectral studies indicated that aflatoxin Q~ was not formed in significant quantities in turkey liver (Gregory & Manley, 1982). As previously reported, the conjugated aflatoxins present in the aqueous tissue fraction comprised a major portion of the total detected residues. Analysis of livers from control groups fed diets without added aflatoxin revealed small amounts of free and conjugated aflatoxin G~, and occasionally detectable B2 and G2 (data not shown), all of which were the result of low concentrations of naturally occurring aflatoxins in the basal diet. Several effects of selenium supplementation on the hepatic metabolism of aflatoxin B~ were apparent from the data of Table 1. Concentrations of free aflatoxins B~ and M~ and total free aflatoxin (B, + M~) in the organic phase were inversely related to the dietary selenium level. In contrast, the concentration of conjugated (aqueous phase) aflatoxins B1, M, and total (B~ + M , ) was higher for 2.0 and 4.0 ppm selenium groups than for the 0.2 ppm group. The ratio of free/conjugated aflatoxins was thus greatly reduced at the 2.0 and 4.0ppm selenium levels. The ratio of free B~/M, decreased with increasing dietary selenium, although large standard deviations were observed in these derived data. Selenium had a weak effect in reducing the total aflatoxin residue concentration.
RESULTS
Toxicity of aflatoxin BI The level of aflatoxin B~ used in Experiment I was selected on the basis of the data of Pier & Heddleston (1970) which indicated that 500ng/g diet induced mild aflatoxicosis in turkeys. Experiment I in this study was run concurrently with the withdrawal study reported in our previous publication (Gregory et al. 1983). As reported previously, this dosage level yielded mild toxicity as evidenced by growth suppression and mild liver hypertrophy. In addition, mild petechial haemorrhages were observed frequently throughout the carcasses, along with occasional tan liver discoloration with sporadic fatty streaks. In order to enhance the effect of aflatoxin B, in Experiment II, the concentration of the toxin was increased to 750 ng/g of diet. The results concerning growth and relative liver mass were similar to those obtained in the first experiment. Haematological, immunological, and histological evidence of aflatoxicosis, and the modulating effect of selenium supplementation were noted in Experiment I (Goldstein, 1981) and Experiment II and will be published separately.
Selenium effects on liver enzymes The influence of aflatoxin B1 (750 ng/g diet) and/or 2.0 ppm dietary selenium was examined using a 2 x 2 factorial design in Experiment II. The observed specific activity values of various liver enzymes potentially involved in aflatoxin metabolism are presented in Table 2, along with values for hepatic reduced glutathione (GSH). Aflatoxin B, tended to increase levels of cytochrome P-450, although the effect was not significant at the 5~o level. Aflatoxin B1 had no significant effect on the activity of cytochrome c (P-450) reductase, glutathione peroxidase, glutathione S-transferase E or glucuronyl transferase.
Distribution of aflatoxin metabolites in livers The pattern of aflatoxins present in the livers of turkey poults fed diets containing 500 ng/g aflatoxin B, and various levels of selenium were qualitatively similar to those reported previously (Gregory et al. 1983). The predominant aflatoxin residues were free and conjugated (water soluble) forms of B, and M, (Table 1). Small and highly variable amounts of aflatoxicol were detected. Aflatoxicol data were not
Table 1. Effect of dietary selenium on the concentration of free and conjugated aflatoxins in livers of turkey poults fed diets containing 500 ng aflatoxin Bi/g for 18 days Aflatoxin concn (ng aflatoxin BI equivalents/g liver) Aflatoxin residue
Free aflatoxins B~ Mt Total (B~ + M 0 Ratio (BI/MI)
Conjugated aflatoxins B~ Mt Total (B~ + M 0 Ratio (Bi/Mt)
Total aflatoxins
Dietary selenium ( p p m ) . . .
0.2 0.211 + 0.096 + 0.307 + 2.20 +
0.075* 0.004* 0.079* 0.79*
0.023 -- 0.003* 0.051 + 0.026* 0.074 __.0.031" 0.45 + 0.22*
2.0
4.0
0.092 __.0.015*t 0.066 + 0.028* 0.158 + 0.024t 1.39 + 0.63*
0.040 __.0.007t 0.049 + 0.005* 0.089 + 0.009t 0.82 + 0.17"
0.088 +_0.022* 0.162 + 0.030t 0.250 __.0.04It 0.54 __.0.12"
0.068 _+ 0.034* 0.114 + 0.018*t 0.182 _+ 0.0461" 0.60 -t- 0.31'
0.383 __.0.085* 0.408 + 0.048* 0.271 -t- 0.047t Total 4.54 _+0.42* 0.646 + 0.178t 0.582 + 0.173~" Free/conjugated Values are means + SEM for four turkey poults/group. Within each horizontal row values followed by the same letter do not differ significantly at the 9570 confidence level.
640
J.F. GREGORYIII and G. T. EDDS Table2. Individualand combinedeffectsof 750ng aflatoxinB1/gdietand 2 ppm dietaryseleniumon hepaticreduced glutathioneconcentrationand selectedenzymeactivitiesin turkey poults Selenium(2 ppm) Selenium Aflatoxin B, and aflatoxinB, Parameter Control (2 ppm) (750ng/g) (750ng/g) Reduced glutathione* 7.42__.0.29 7.33 _+0.31 8.93 + 0.29 9.70 _+0.42 Cytochrome P-450t 213 _+32 240 _+35 279 _ 42 265 ± 35 Cytochrome c reductase~ 0.142__.0.035 0.114+ 0.012 0.130 + 0.016 0.110± 0.18 Glutathioneperoxidase:~ 1.84___0.31 2.18 +__0.18 1.89+__0.16 3.09 + 0.46 Glucuronyltransferase:~ 3.81 + 0.45 2.95 + 0.32 2.79+ 0.41 3.10__.0.42 Glutathionetransferase~ 25.2 + 1.1 26.6 + 3.5 26.5 _ 5.3 19.5 ± 1.4 *nmol/mgliver. tpmol/mg protein. Snmol/min/mgprotein. Values are means+ SEM for groups of eight turkey pours. Analysisof variance for both the individualand interactiveeffects of seleniumand aflatoxinB~ showedonlytwo significanteffectsat the 95~ confidencelevel, the effect of aflatoxinon reduced glutathioneand that of seleniumon glutathioneperoxidase.
The activity of these enzymes was also largely unaffected by the selenium supplement. The only effect of selenium noted was the expected increase in selenium-dependent glutathione peroxidase activity, which, although small was statistically significant (P < 0.05). The activities of glutathione S-transferase E and glucuronyl transferase, both enzymes involved in the formation of conjugated aflatoxin derivatives were not influenced by dietary aflatoxin B1 or selenium. Liver reduced glutathione concentration was significantly increased by aflatoxin B~ (P < 0.05), but unaffected by selenium. No significant statistical interactions between selenium and aflatoxin effects were detected for any of the parameters examined. DISCUSSION These results indicate that dietary selenium supplementation promotes the in vivo formation of watersoluble conjugated aflatoxins. These findings suggest that the protective effect of selenium may be mediated by enhanced conjugation of aflatoxins, which could promote excretion of the toxin. Pharmacokinetic studies would be required to test this hypothesis. The nonspecific nature of the acid hydrolysis step used in the analytical method prevents identification of the types of aflatoxin conjugates present in the aqueous phase of the liver samples. Because of the difficulty in preparing the glutathione-aflatoxin B1 conjugate for use as a chromatographic standard, its behaviour in this assay procedure has not been determined. The recent development of a procedure for the in vitro synthesis of the glutathione-aflatoxin B~ conjugate (Moss, Judah, Przybylski & Neal, 1983) will assist the resolution of this question. The decrease in the ratio of free aflatoxin B~/M~ with increasing selenium supplementation may also be related to enhanced detoxification as a result of the lower toxicity of aflatoxin M~ (Campbell & Hayes, 1976). These metabolic effects of selenium have not been evaluated in other species. Further research comparing the effect of selenium on metabolic patterns in various animal species would facilitate interpretation of these results in relation to the overall protective effect of selenium. In view of the observed changes in aflatoxin metabolite distribution, Experiment II was conducted to determine whether the results could be explained by enhancement or suppression of enzyme activities related to aflatoxin activation or detoxification. Pre-
vious studies have shown that a major mechanism of aflatoxin detoxification is the conjunction of aflatoxin Br2,3-oxide with reduced glutathione to form the 2,3-dihydro-2-(S-glutathionyl)-3-hydroxy-aflatoxinBt derivative (Degen & Neumann, 1978; Lotlikar, Insetta, Lyons & Jhee, 1980; Moss et al. 1983). The results shown in Table 2 indicate that the dietary selenium supplement did not exert its protective effect through an increase in the specific activity of the conjugating enzymes glucuronyl transferase or glutathione S-transferase. The observation that selenium did not affect the concentration of liver glutathione suggests that selenium supplementation does not enhance the formation of glutathione conjugates by increasing the availability of reduced glutathione. The effect of aflatoxin in increasing the concentration of reduced glutathione in the liver has been reported previously (Mainigi & Campbell, 1981). Several previous studies have indicated that selenium supplementation of rats increases the activity of various microsomal enzymes including glucuronyl transferase (Dauod & Grifin, 1978) and certain mixed-function oxidases (Shull, Buckmaster & Cheeke, 1979), and, thus, may influence the metabolism of xenobiotics. Studies by Wortzman et al. (1980), in rats, indicated that the protective effect of selenium against 2-acetylaminofluorene-induced hepatocarcinogenesis is not mediated by any influence on DNA-repair processes or covalent binding to DNA. In contrast, Chen et al. (1982) reported that both selenium deficiency and excess inhibited the covalent binding of aflatoxin BI in rats. These researchers also found that selenium supplementation had no effect on covalent binding of aflatoxin to DNA in chicks (Chen et al. 1982). Indirect studies by Hughes & Bjeldanes (1983) provided evidence that selenium supplementation of rats did not influence the activity of microsomal enzymes involved in the activation or detoxification of aflatoxin B1. In these studies rats were fed basal diets with various levels of added selenium prior to isolation of liver microsomal fractions. When these liver fractions were examined for their ability to activate aflatoxin BI in a bacterial mutagenicity test, no effect of the diet was detected. In general these data strongly suggest a role of selenium in the metabolism of certain toxicants but the mechanism is unclear at present. An important consideration in evaluating the enzymatic data presented in this study is that enzyme
Effect of Se on AFB~ metabolism in turkeys
641
activities measured in vitro under optimal conditions S-175, and the Florida Agricultural Experiment Station. to yield maximal activities do not accurately reflect in The technical assistance of D. B. Sartain is gratefully acknowledged. Florida Agricultural Experiment Stations vivo activities. In vivo variables such as substrate or cofactor concentration and reversible inhibition or Journal Series No. 5399. stimulation by selenium or other compounds could not be evaluated with this methodology. Another potentially important factor which could REFERENCES not be assessed in these experiments is the distribuBoyd J. N., Babish J. G. & Stoewsand G. S. (1982). tion of the various forms of hepatic cytochrome Modification by beet and cabbage diets of aflatoxin P-450 which is influenced by diet. Selective induction Brinduced rat plasma ~t-fetoprotein elevation, hepatic of P-450 isozymes differing in substrate and product tumorigenesis, and mutagenicity of urine. Fd Chem. specificity has been reported in several species (DanToxic. 20, 47. nan, Guengerich, Kaminsky & Aust, 1983; Guen- Boyd J. N., Misslbeck N. & Stoewsand G. S. (1983). gerich, Wang & Davidson, 1982; Pickett, Jeter, Changes in preneoplastic response to aflatoxin Bj in rats fed green beans, beets or squash. Fd Chem. Toxic. 21, 37. Morin & Lu, 1981; Yoshizawa, Uchimaru, Kamataki, Kato & Ueno, 1982). An influence of selenium Boyd J. N., Sell S. & Stoewsand G. S. (1979). Inhibition of aflatoxin-induced serum ~-fetoprotein in rats fed on the distribution of cytochrome P-450 isozymes cauliflower. Proc. Soc. exp. Biol. Med. 161, 473. could yield an alteration in aflatoxin activation versus Burguera J. A., Edds G. T. & Osuna O. (1983). Influence of detoxification by the microsomal mixed-function oxselenium on aflatoxin Bt or crotolaria toxicity in turkey idase system; such a change is suggested by the poults. Am. J. vet. Res. 44, 1714. aflatoxin BI/M~ ratios observed in this study (Table Cabral J. R. P. & Neal G. E. (1983). The inhibitory effects 1). Examination ofisozyme proportions as a function of ethoxyquin on the carcinogenic action of aflatoxin B~ in rats. Cancer Lett. 19, 125. of dietary selenium supplementation would be an important aspect of future studies. Potential effects of Campbell T. C. & Hayes J. R. (1976). The role of aflatoxin metabolism in its toxic lesion. Toxic. appl. Pharmac. 35, selenium on the activity of epoxide hydrolase should 199. also be investigated. Campbell T. C., Hayes J. R. & Newberne P. M. (1978). The known nutritional role of selenium is as a Dietary lipotropes, hepatic microsomal mixed-function cofactor for glutathione peroxidase, which is imoxidase activities,and in vivo covalent binding of aflatoxin portant in the detoxification of hydrogen peroxide B~ in rats. Cancer Res. 38, 4569. and lipid hydroperoxides. Extensive studies of Chen J., Goetchius M. P., Campbell T. C. & Combs G. F. aflatoxin metabolism have failed to yield evidence of (1982). Effects of dietary sele.nium and vitamin E on hepatic mixed-function oxidase activities and in vivo lipid peroxidation-mediated toxic effects. The results covalent binding of aflatoxin B1 in rats. J. Nutr. 112, 324. of the present study strongly suggest that the protective effect of selenium does not involve glutathione Chen J., Goetchius M. P., Combs G. F. & Campbell T. C. (1982). Effects of dietary selenium and vitamin E on peroxidase. The similar protective effects of selenium covalent binding of aflatoxin to chick liver cell macroand cadmium at nontoxic levels suggests that the molecules. J. Nutr. 112, 350. protection may involve a nonspecific inhibitory effect Dannan G. A., Guengerich F. P., Kaminsky L. S. & Aust of these elements on certain enzymatic processes in S. D. (1983). Regulation of cytochrome P-450. Immuaflatoxin activation. Cadmium has been found to nochemical quantitation of eight isozymes in liver microsomes of rats treated with polybrominated biphenyl coninterfere with hepatic microsomal drug metabolism in geners. J. biol. Chem. 258, 1282. rats, although this effect is due to a reduction in cytochrome P-450 concentration (Schnell, Means, Daoud A. H. & Griffin A. C. (1978). Effects of selenium and retinoic acid on the metabolism of N-acetylaminofluorene Roberts & Pence, 1979). The metabolic similarities and N-hydroxyacetylaminofluorene. Cancer Lett. 5, 231. and interactions of selenium and cadmium have been Davila J. C., Edds G. T., Osuna O. & Simpson C. F. (1983). recently reviewed as they relate to the nutritional and Modification of the effects of aflatoxin B~ and warfarin in toxicological roles of these elements (Whanger, Ridyoung pigs given selenium. Am. J. vet. Res. 44, 1877. lington & Holcomb, 1980). Comparative studies with Degen G. H. & Neumann H.-G. (1978). The major metabother nutritionally and environmentally important olite of aflatoxin B~ in the rat is a glutathione conjugate. Chemico-BioL Interactions 22, 239. elements such as zinc, mercury, and lead may provide Faris R. A. & Campbell T. C. (1981). Exposure of newborn more relevant information. rats to pharmacologically active compounds may perIn summary, the results of this study show that manently alter carcinogen metabolism. Science, N. Y. 211, selenium supplementation of diets affects the metab719. olism of aflatoxin B1 in turkey poults. Selenium Fjellstedt T. A., Allen R. H., Duncan B. K. & Jakoby enhanced the formation of water-soluble conjugated W. B. (1973). Enzymatic conjugation of epoxides with forms of aflatoxins, which may promote the clearance glutathione. J. biol. Chem. 248, 3702. of the toxin. Enzymatic data indicate that this effect Frape D, L., Wayman B. J. & Tuck M. G. (1981). The effect is not a result of increases in the specific activity of of dietary fibre of aflatoxicosis in the weanling male rat. Br. J. Nutr. 46, 315. glucuronyl transferase or glutathione S-transferase E or in the concentration of reduced glutathione in Frape D. L., Wayman B. J. & Tuck M. G. (1982). The effects of gum arabic, wheat offal and various of its liver. The results of this study support the contention fractions on the metabolism of ~4C-labelledaflatoxin B~in that selenium supplementation at nutritional levels the male weanling rat. Br. J. Nutr. 48, 97. would enhance the resistance of animals to the effects Gigon P. L., Gram T. E. & Gillette J. R. (1969). Studies on of aflatoxin B~. the rate of reduction of hepatic microsomal cytochrome P-450 by reduced nicotinamide adenine dinucleotide phosphate: Effect of drug substrates. Molec. PharmacoL Acknowledgements--This research was supported in part by 5, 109. funds from NSF Grant INT 7922086, Regional Project
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J. F. GREGORY III and G. T. EDOS
Goldstein S. L. (1981). Aflatoxicosis in young turkey poults as modified by dietary selenium. M.S. Thesis, University of Florida, Gainesville, FL, Gornall A. G., Bardawill C. J. & David M. M. (1949). Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751. Gregory J. F., Goldstein S. L. & Edds G. T. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B~. Fd Chem. Toxic. 21, 463. Gregory J. F. & Manley D. B. (1982). High performance liquid chromatographic quantitation of aflatoxin metabolites in animal tissues. J. Ass. off. analyt. Chem. 65, 869. Griffin A. C. (1979). Role of selenium in the chemoprevention of cancer. Adv. Cancer Res. 29, 419. Guengerich F. P., Wang P. & Davidson N. K. (1982). Estimation of isozymes of microsomal cytochrome P-450 in rats, rabbits, and humans using immunochemical staining coupled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry, N.Y. 21, 1698. Hsieh D. P. H., Dalezios J. I., Krieger R. I., Masri M. S. & Haddon W. F. (1974). Use of monkey liver microsomes in production of aflatoxin QI. J. agric. Fd Chem. 22, 515. Hughes D. A. & Bjeldanes L. F. (1983). Influence of selenium-supplemented Torula yeast diets on livermediated mutagenicity of aflatoxin B~. J. Fd Sci. 48, 759. Lawrence R. A. & Burk R. F. (1976). Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. biophys. Res. Commun. 71, 952. Lotlikar P. D., Insetta S. M., Lyons P. A. & Jhee E.-C. (1980). Inhibition of microsome-mediated binding of aflatoxin B~ to DNA by glutathione S-transferase. Cancer Lett. 9, 143. Lucier G. W., McDaniel O. S. & Matthews H. B. (1971). Microsomal rat liver UDP glucuronyltransferase: Effects of piperonyl butoxide and other factors on enzyme activity. Archs Biochem. Biophys. 145, 520. Mainigi K. D. & Campbell T. C. (1981). Effects of !ow dietary protein and dietary aflatoxin on hepatic glutathione levels in F-344 rats. Toxic. appl. Pharmac. 59, 196. Moss E. J,, Judah D. J., Przybylski M. & Neal G. E. (1983). Some mass-spectral and n.m.r, analytical studies of a glutathione conjugate of aflatoxin B~. Biochem. J. 210, 227. Neter J. & Wasserman W. (1974). Applied Linear Statistical Models. Richard D. Irwin, Inc., Homewood, IL. Newberne P. M. & Conner M. W. (1974). Effect of selenium on acute response to aflatoxin B~. In Trace Substances in Environmental Health-VIL Edited by D. D. Hemphill. p. 323. University of Missouri, Columbia, MO. Newberne P. M. & Gross R. L. (1977). The role of nutrition in aftatoxin injury. In Mycotoxins in Human and Animal Health. Edited by J. V. Rodricks, C. W. Hesseltine & M. A. Mehlman. p. 51. Pathotox Publishers, Inc., Park Forest South, IL. Newberne P. M., Weigert J. & Kula N. (1979). Effects of dietary fat on hepatic mixed-function oxidases and hepa-
tocellular carcinoma induced by aflatoxin B t in rats. Cancer Res. 39, 3986. Omura T. & Sato R. (1964). The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. biol. Chem. 239, 2370. Osuna O. & Edds G. T, (1982a). Toxicology of aflatoxin Bl, warfarin, and cadmium in young pigs: Performance and hematology. Am. J. vet. Res. 43, 1380. Osuna O. & Edds G. T. (1982b). Toxicology of aflatoxin B~, warfarin, and cadmium in young pigs: Clinical chemistry and blood coagulation. Am. J. vet, Res. 43, 1387. Osuna O., Edds G. T. & Simpson C. F. (1982). Toxicology of aflatoxin B l, warfarin, and cadmium in young pigs: Metal residues and pathology. Am. J. vet. Res. 43, 1395. Pickett C. B., Jeter R. L., Morin J. & Lu A. Y. H. (1981). Electroimmunochemical quantitation of cytochrome P450, cytochrome P-448, and epoxide hydrolase in rat liver microsomes. J. biol. Chem. 256, 8815. Pier A. C. & Heddleston K. L. (1970). The effect of aflatoxin on immunity in turkeys. I. Impairment of activity acquired resistance to bacterial challenge. Avian Dis. 14, 797. Rogers A. E., Lenhart G. & Morrison G. (1980). Influence of dietary lipotrope and lipid content on aflatoxin B~, N-2-fluorenylacetamide, and 1,2-dimethylhydrazine carcinogenesis in rats. Cancer Res. 40, 2802. Schnell R. C., Means J. R., Roberts S. A. & Pence D. H. (1979). Studies on cadmium-induced inhibition of hepatic microsomal drug biotransformation in the rat. Envir. Hlth Perspect. 28, 273. Sedlak J. & Lindsay R. H. (1968), Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analyt. Biochem. 25, 192. Shull L. R., Buckmaster G. W. & Cheeke P. R. (1979). Effect of dietary selenium status on in vitro hepatic mixed-function oxidase enzymes of rats. J. envh'. Path. Toxicol. 2, 1127. Temcharoen P., Anukarahanonta T. & Bhamarapravati N. (1978). Influence of dietary protein and vitamin BI2 on the toxicity and carcinogenicity of aflatoxins in rat liver, Cancer Res. 38, 2185. van der Hoeven T. A. & Coon M. J. (1974). Preparation and properties of partially purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphatecytochrome P-450 reductase from rabbit liver microsomes. J. biol. Chem. 249, 6302. Whanger P. D., Ridlington J. W. & Holcomb C. L. (1980). Interactions of zinc and selenium on the binding of cadmium to rat tissue proteins. Ann. N.Y. Acad. Sci. 355, 333. Wortzman M. S., Besbris H. J. & Cohen A. M. (1980). Effect of dietary selenium on the interaction between 2-acetylaminofiuorene and rat liver DNA in vivo. Cancer Res. 40, 2670. Yoshizawa H., Uchimaru R., Kamataki T., Kato R. & Ueno Y. (1982). Metabolism and activation of aflatoxin B~ by reconstituted cytochrome P-450 system of rat liver. Cancer Res. 42, 1120.
0278-6915/84 $3.00+ 0.00 Copyright © 1984 Pergamon Press Ltd
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EFFECT OF DIETARY SELENIUM ON THE METABOLISM OF AFLATOXIN BI IN TURKEYS J. F. GREGORY III Food Science and Human Nutrition Department, University of Florida and G . T . EDDS
Department of Preventive Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA (Received 29 December 1983)
Abstract--To investigate the biochemical mechanism of the previously reported protective effect of dietary selenium against aflatoxin toxicity, the hepatic metabolism of aflatoxin Bt in turkey poults was examined at various dietary selenium concentrations. Diets were supplemented with 0.2, 2.0 or 4.0 ppm selenium (as sodium selenite) and 500 ng aflatoxin B~/g diet in an 18-day trial. Free and conjugated aflatoxin and metabolites were quantified using high-performance liquid chromatography. The proportion of liver aflatoxins in conjugated forms increased and the ratio of free aflatoxin B~/M] decreased with increasing dietary selenium concentrations. These in vivo results provide evidence of selenium-induced enhancement of aflatoxin detoxification processes. In a similar experiment using 2.0 ppm selenium and 750 ng aflatoxin B~/g diet, the concentration of hepatic reduced glutathione, cytochrome P-450 and the activity of enzymes involved in the metabolism of aflatoxin B~ and glutathione were determined. Although the selenium supplement increased glutathione peroxidase activity, dietary selenium had no effect on reduced glutathione or cytochrome P-450 concentrations or on the activities of glutathione transferase E, glucuronyl transferase and cytochrome c reductase. These data indicate that the protective action of selenium is not mediated by an increase in glutathione availability for aflatoxin conjugation or by effects on the activities of these enzymes as measured in vitro.
INTRODUCTION
Similarly, studies of rats fed diets containing various vegetables and aflatoxin B~ indicate that the hepatoRecent research has shown that a variety of nutrients carcinogenic effects of the toxin are strongly and other dietary components can influence the acute influenced by minor food components (Boyd, Babish and chronic toxicity of aflatoxins. While the general & Stoewsand, 1982; Boyd, Misslebeck & Stoewsand, processes of aflatoxin metabolism have been well 1983; Boyd, Sell & Stoewsand, 1979). elucidated, many of the qualitative and quantitative Several studies have demonstrated that certain dietary effects are largely unknown. dietary metals provide a protective effect against Variations in the macronutrient components of the aflatoxin toxicity in several animal species. Newberne diet have been shown to have pronounced effects on & Conner (1974) first reported that selenium suppleaflatoxin toxicity. Deficiency of dietary lipotropic mentation up to a dietary level of 1.0ppm factors (choline and methionine) may increase or progressively reduced the acute toxicity of aflatoxin decrease the resistance of rats to the acute toxicity Bt in rats, while greater levels enhanced the observed and hepatocarcinogenic effects of aflatoxin (Camp- mortality. This protective effect was confirmed in bell, Hayes & Newberne, 1978; Newberne & Gross, several later studies with swine and turkeys (Bur1977), depending on the overall diet composition. guera, Edds & Osuna, 1983; Davila, Edds, Osuna & These effects are strongly linked to dietary variables Simpson, 1983). A similar protective effect has been such as protein and fat levels and the vitamin B-12 observed with cadmium when added to the diets of and folacin status of the animal (Campbell et al. swine (Osuna & Edds, 1982a,b; Osuna, Edds & 1978; Newberne, Weigert & Kula, 1979; Rogers, Simpson, 1982) although the mechanism of action Lenhart & Morrison, 1980; Temcharoen, An- has not been identified. Protective effects of selenium ukarahanonta & Bhamarapravati, 1978). Various against the hepatocarcinogenesis induced by types of dietary fibre have also been shown to affect 2-acetylaminofluorene also have been reported the susceptibility of animals to the toxicity of the (Wortzman, Besbris & Cohen, 1980). In view of the mycotoxin (Frape, Wayman & Tuck, 1981 & 1982). demonstrated protective effect of selenium against the Minor dietary components may modify the metab- acute toxicity of aflatoxin Bt and the postulated olism and subsequent toxic effects of aflatoxins by anticarcinogenic effect of selenium (Griffin, 1979), induction of microsomal or other enzyme systems. studies were conducted by Chen and associates to Experiments with microsomal enzyme inducers such investigate the possible influence of selenium and as phenobarbital (Farris & Campbell, 1981) and the vitamin E on certain aspects of the metabolism of antioxidant, ethoxyquin (Cabral & Neal, 1983) dem- aflatoxin B~ in rats and chicks (Chen, Goetchius, onstrated protective effects for these compounds. Campbell & Combs, 1982; Chen, Goetchius, Combs 637
638
J. F. GREGORYIII and G. T. EDDS
& Campbell, 1982). Their results suggested that combined vitamin E-selenium deficiency enhanced aflatoxin binding to hepatic DNA and RNA in rats, while opposite effects were found for chicks. No clear relationships were detected between supplementation levels of either nutrient and the activity of various enzymes of the mixed function oxidase system, various transferases involved in aflatoxin conjugation, or the extent of covalent binding of the toxin to cellular macromolecules for either species. The purpose of this study was to elucidate further the metabolic basis of the protective effect of dietary selenium by examining the effects of various levels of dietary selenium on the metabolism of aflatoxin B~ in turkey poults. The influence of selenium on the hepatic levels and pattern of distribution of free and conjugated aflatoxin metabolites and on the activity of enzymes involved in the activation or detoxification of the mycotoxin was examined. EXPERIMENTAL
Aflatoxin compounds. A mixed solution of aflatoxins B~, G~, B2 and G2 was purchased from Applied Science Laboratories, Inc. (State College, PA). Aflatoxin Qt was synthesized by enzymatic hydroxylation of aflatoxin B 1 (Hsieh, Dalezios, Krieger et al. 1974). Aflatoxin B~ used in this synthesis, aflatoxin M~, and aflatoxicol were obtained from Sigma Chemical Co. (St Louis, MO). Aflatoxin B~ used in diet fortification was obtained from Calbiochem-Behring Corp. (San Diego, CA). The purity of all compounds was confirmed by high performance liquid chromatography (HPLC). Experimental protocols. The basal diet consisted of Purina Startena Game Bird Chow (Ralston Purina Co., Inc., St Louis, MO), which contained 30~o protein and 0.1 #g selenium/g diet (ppm) from sodium selenite. A sodium selenite premix was used for additional fortification. Aflatoxin fortification was carried out as described previously (Gregory, Goldstein & Edds, 1983). In experiment I, day-old turkey poults (male, Large White; Thaxton Turkey Farm, Watkinsville, GA) were randomly assigned to diet groups of four animals. Poults were kept initially for 15 days in two brooder rings fitted with infra-red lamps to provide constant temperature. During this adaptation period the basal diet was fed ad lib. The poults were then moved to 1.5 x 3.1 m pens for an 18-day experimental period during which they were fed the basal diet supplemented with 0.2, 2.0 or 4.0 ppm selenium (as sodium selenite) containing no added aflatoxin or supplemented with 500 ng aflatoxin B~/g of diet. At the end of the 18-day feeding period, the poults were killed by cervical dislocation and the livers rapidly excised. The livers were frozen on dry ice, then stored at - 2 0 ° C until they were analysed for aflatoxins. In experiment II, an identical protocol was followed except that groups of eight poults were fed diets supplemented with: (a) no added aflatoxin or selenium (control); (b) 2.0 ppm selenium (as sodium selenite); (c) 750 ng aflatoxin B~/g diet; or (d) 2.0 ppm selenium plus 750ng aflatoxin Bi/g diet. After the 18-day experimental period, the poults were killed (two/day from each group over a 4-day period) and
the livers were removed. This staggered experimental design was necessary because of the lengthy centrifugation procedures required in the fractionation of the livers for biochemical analysis. A small portion of each liver was taken for immediate analysis of reduced glutathione, while the remainder of the liver was subjected to differential centrifugation (van der Hoeven & Coon, 1974), as described below, prior to enzymatic analysis. Aflatoxin determination. The concentrations of free and conjugated forms of aflatoxins in livers were determined in Experiment I using the HPLC method of Gregory & Manley (1982). This method is based on a determination of free aflatoxins (chloroformacetone soluble), after treatment with trifluoroacetic acid, using a reverse-phase column and fluorometric detection. For the determination of conjugated aflatoxin metabolites, the aqueous phase remaining after extraction of free toxins is subjected to acid hydrolysis (0.2 N-HCI, 90°C, 2 hr), followed by extraction of the released aflatoxins with chloroform, treatment with trifluoroacetic acid, and HPLC analysis. This procedure permitted accurate quantitation of free and conjugated aflatoxins B1, G1, B2, G2, MI, Q~ and aflatoxicol at limits of sensitivity of at least 0.01 ng B~ equivalents/g tissue. The specificity of this procedure has been supported by fluorescence spectral examination (Gregory & Manley, 1982). Biochemical analyses. The liver analyses of Experiment II were started on the day the animals were killed, on which nonprotein thiols (mainly reduced glutathione) were determined colorimetrically (Sedlack & Lindsay, 1968) and differential centrifugation (van der Hoeven & Coon, 1974) was performed to prepare liver fractions for subsequent enzymatic assays. Post-mitochondrial supernatant (12,000g supernatant; S-12), microsomal (105,000g pellet), and 105,000g supernatant fractions were prepared for each liver. Microsomal fractions were suspended in 0.01M-tris-acetate buffer (pH7.4) containing 0.1 mM-EDTA and 25~ (v/v) glycerol. All liver fractions were stored at - 2 0 ° C until they were analysed for various enzymes potentially involved in aflatoxin and/or glutathione metabolism. Microsomal cytochrome P-450, cytochrome c reductase, and glucuronyl transferase were assayed essentially as described by Omura & Sato (1964), Gigon, Gram & Gillette (1969), and Lucier, McDaniel & Matthews (1971), respectively. Microsomes were activated by treatment with Triton X-100 prior to assay of glucuronyl transferase (Lucier et al. 1971). Glutathione peroxidase activity in the 105,000g supernatant was determined by the method of Lawrence & Burk (1976) was 0.25 mM-hydrogen peroxide as substrate in order to measure selenium-dependent activity. The activity of glutathione S-transferase E, which specifically catalyses the conjugation of epoxide substrates, was determined in the 12,000 g supernatant (S-12) fractions using the method of Fjellstedt, Allen, Duncan & Jakoby (1973). Assays were performed in duplicate or triplicate. Preliminary experiments were performed for each enzyme assay to determine the linear range with respect to enzyme concentration and reaction timecourse. Protein was determined by the biuret method (Gornall, Bardawill & David, 1949).
639
Effect of Se on AFB, metabolism in turkeys Statistical methods. Differences between aflatoxin levels in Experiment I were evaluated using one-way analysis of variance and the Tukey procedure for multiple comparisons. The main effects and interactions of selenium and aflatoxin B, in Experiment II were evaluated using two-way analysis of variance. These methods have been described by Neter & Wasserman (1974).
included in calculations of total aflatoxin residue concentrations because of this inconsistency. Previous chromatographic and spectral studies indicated that aflatoxin Q~ was not formed in significant quantities in turkey liver (Gregory & Manley, 1982). As previously reported, the conjugated aflatoxins present in the aqueous tissue fraction comprised a major portion of the total detected residues. Analysis of livers from control groups fed diets without added aflatoxin revealed small amounts of free and conjugated aflatoxin G~, and occasionally detectable B2 and G2 (data not shown), all of which were the result of low concentrations of naturally occurring aflatoxins in the basal diet. Several effects of selenium supplementation on the hepatic metabolism of aflatoxin B~ were apparent from the data of Table 1. Concentrations of free aflatoxins B~ and M~ and total free aflatoxin (B, + M~) in the organic phase were inversely related to the dietary selenium level. In contrast, the concentration of conjugated (aqueous phase) aflatoxins B1, M, and total (B~ + M , ) was higher for 2.0 and 4.0 ppm selenium groups than for the 0.2 ppm group. The ratio of free/conjugated aflatoxins was thus greatly reduced at the 2.0 and 4.0ppm selenium levels. The ratio of free B~/M, decreased with increasing dietary selenium, although large standard deviations were observed in these derived data. Selenium had a weak effect in reducing the total aflatoxin residue concentration.
RESULTS
Toxicity of aflatoxin BI The level of aflatoxin B~ used in Experiment I was selected on the basis of the data of Pier & Heddleston (1970) which indicated that 500ng/g diet induced mild aflatoxicosis in turkeys. Experiment I in this study was run concurrently with the withdrawal study reported in our previous publication (Gregory et al. 1983). As reported previously, this dosage level yielded mild toxicity as evidenced by growth suppression and mild liver hypertrophy. In addition, mild petechial haemorrhages were observed frequently throughout the carcasses, along with occasional tan liver discoloration with sporadic fatty streaks. In order to enhance the effect of aflatoxin B, in Experiment II, the concentration of the toxin was increased to 750 ng/g of diet. The results concerning growth and relative liver mass were similar to those obtained in the first experiment. Haematological, immunological, and histological evidence of aflatoxicosis, and the modulating effect of selenium supplementation were noted in Experiment I (Goldstein, 1981) and Experiment II and will be published separately.
Selenium effects on liver enzymes The influence of aflatoxin B1 (750 ng/g diet) and/or 2.0 ppm dietary selenium was examined using a 2 x 2 factorial design in Experiment II. The observed specific activity values of various liver enzymes potentially involved in aflatoxin metabolism are presented in Table 2, along with values for hepatic reduced glutathione (GSH). Aflatoxin B, tended to increase levels of cytochrome P-450, although the effect was not significant at the 5~o level. Aflatoxin B1 had no significant effect on the activity of cytochrome c (P-450) reductase, glutathione peroxidase, glutathione S-transferase E or glucuronyl transferase.
Distribution of aflatoxin metabolites in livers The pattern of aflatoxins present in the livers of turkey poults fed diets containing 500 ng/g aflatoxin B, and various levels of selenium were qualitatively similar to those reported previously (Gregory et al. 1983). The predominant aflatoxin residues were free and conjugated (water soluble) forms of B, and M, (Table 1). Small and highly variable amounts of aflatoxicol were detected. Aflatoxicol data were not
Table 1. Effect of dietary selenium on the concentration of free and conjugated aflatoxins in livers of turkey poults fed diets containing 500 ng aflatoxin Bi/g for 18 days Aflatoxin concn (ng aflatoxin BI equivalents/g liver) Aflatoxin residue
Free aflatoxins B~ Mt Total (B~ + M 0 Ratio (BI/MI)
Conjugated aflatoxins B~ Mt Total (B~ + M 0 Ratio (Bi/Mt)
Total aflatoxins
Dietary selenium ( p p m ) . . .
0.2 0.211 + 0.096 + 0.307 + 2.20 +
0.075* 0.004* 0.079* 0.79*
0.023 -- 0.003* 0.051 + 0.026* 0.074 __.0.031" 0.45 + 0.22*
2.0
4.0
0.092 __.0.015*t 0.066 + 0.028* 0.158 + 0.024t 1.39 + 0.63*
0.040 __.0.007t 0.049 + 0.005* 0.089 + 0.009t 0.82 + 0.17"
0.088 +_0.022* 0.162 + 0.030t 0.250 __.0.04It 0.54 __.0.12"
0.068 _+ 0.034* 0.114 + 0.018*t 0.182 _+ 0.0461" 0.60 -t- 0.31'
0.383 __.0.085* 0.408 + 0.048* 0.271 -t- 0.047t Total 4.54 _+0.42* 0.646 + 0.178t 0.582 + 0.173~" Free/conjugated Values are means + SEM for four turkey poults/group. Within each horizontal row values followed by the same letter do not differ significantly at the 9570 confidence level.
640
J.F. GREGORYIII and G. T. EDDS Table2. Individualand combinedeffectsof 750ng aflatoxinB1/gdietand 2 ppm dietaryseleniumon hepaticreduced glutathioneconcentrationand selectedenzymeactivitiesin turkey poults Selenium(2 ppm) Selenium Aflatoxin B, and aflatoxinB, Parameter Control (2 ppm) (750ng/g) (750ng/g) Reduced glutathione* 7.42__.0.29 7.33 _+0.31 8.93 + 0.29 9.70 _+0.42 Cytochrome P-450t 213 _+32 240 _+35 279 _ 42 265 ± 35 Cytochrome c reductase~ 0.142__.0.035 0.114+ 0.012 0.130 + 0.016 0.110± 0.18 Glutathioneperoxidase:~ 1.84___0.31 2.18 +__0.18 1.89+__0.16 3.09 + 0.46 Glucuronyltransferase:~ 3.81 + 0.45 2.95 + 0.32 2.79+ 0.41 3.10__.0.42 Glutathionetransferase~ 25.2 + 1.1 26.6 + 3.5 26.5 _ 5.3 19.5 ± 1.4 *nmol/mgliver. tpmol/mg protein. Snmol/min/mgprotein. Values are means+ SEM for groups of eight turkey pours. Analysisof variance for both the individualand interactiveeffects of seleniumand aflatoxinB~ showedonlytwo significanteffectsat the 95~ confidencelevel, the effect of aflatoxinon reduced glutathioneand that of seleniumon glutathioneperoxidase.
The activity of these enzymes was also largely unaffected by the selenium supplement. The only effect of selenium noted was the expected increase in selenium-dependent glutathione peroxidase activity, which, although small was statistically significant (P < 0.05). The activities of glutathione S-transferase E and glucuronyl transferase, both enzymes involved in the formation of conjugated aflatoxin derivatives were not influenced by dietary aflatoxin B1 or selenium. Liver reduced glutathione concentration was significantly increased by aflatoxin B~ (P < 0.05), but unaffected by selenium. No significant statistical interactions between selenium and aflatoxin effects were detected for any of the parameters examined. DISCUSSION These results indicate that dietary selenium supplementation promotes the in vivo formation of watersoluble conjugated aflatoxins. These findings suggest that the protective effect of selenium may be mediated by enhanced conjugation of aflatoxins, which could promote excretion of the toxin. Pharmacokinetic studies would be required to test this hypothesis. The nonspecific nature of the acid hydrolysis step used in the analytical method prevents identification of the types of aflatoxin conjugates present in the aqueous phase of the liver samples. Because of the difficulty in preparing the glutathione-aflatoxin B1 conjugate for use as a chromatographic standard, its behaviour in this assay procedure has not been determined. The recent development of a procedure for the in vitro synthesis of the glutathione-aflatoxin B~ conjugate (Moss, Judah, Przybylski & Neal, 1983) will assist the resolution of this question. The decrease in the ratio of free aflatoxin B~/M~ with increasing selenium supplementation may also be related to enhanced detoxification as a result of the lower toxicity of aflatoxin M~ (Campbell & Hayes, 1976). These metabolic effects of selenium have not been evaluated in other species. Further research comparing the effect of selenium on metabolic patterns in various animal species would facilitate interpretation of these results in relation to the overall protective effect of selenium. In view of the observed changes in aflatoxin metabolite distribution, Experiment II was conducted to determine whether the results could be explained by enhancement or suppression of enzyme activities related to aflatoxin activation or detoxification. Pre-
vious studies have shown that a major mechanism of aflatoxin detoxification is the conjunction of aflatoxin Br2,3-oxide with reduced glutathione to form the 2,3-dihydro-2-(S-glutathionyl)-3-hydroxy-aflatoxinBt derivative (Degen & Neumann, 1978; Lotlikar, Insetta, Lyons & Jhee, 1980; Moss et al. 1983). The results shown in Table 2 indicate that the dietary selenium supplement did not exert its protective effect through an increase in the specific activity of the conjugating enzymes glucuronyl transferase or glutathione S-transferase. The observation that selenium did not affect the concentration of liver glutathione suggests that selenium supplementation does not enhance the formation of glutathione conjugates by increasing the availability of reduced glutathione. The effect of aflatoxin in increasing the concentration of reduced glutathione in the liver has been reported previously (Mainigi & Campbell, 1981). Several previous studies have indicated that selenium supplementation of rats increases the activity of various microsomal enzymes including glucuronyl transferase (Dauod & Grifin, 1978) and certain mixed-function oxidases (Shull, Buckmaster & Cheeke, 1979), and, thus, may influence the metabolism of xenobiotics. Studies by Wortzman et al. (1980), in rats, indicated that the protective effect of selenium against 2-acetylaminofluorene-induced hepatocarcinogenesis is not mediated by any influence on DNA-repair processes or covalent binding to DNA. In contrast, Chen et al. (1982) reported that both selenium deficiency and excess inhibited the covalent binding of aflatoxin BI in rats. These researchers also found that selenium supplementation had no effect on covalent binding of aflatoxin to DNA in chicks (Chen et al. 1982). Indirect studies by Hughes & Bjeldanes (1983) provided evidence that selenium supplementation of rats did not influence the activity of microsomal enzymes involved in the activation or detoxification of aflatoxin B1. In these studies rats were fed basal diets with various levels of added selenium prior to isolation of liver microsomal fractions. When these liver fractions were examined for their ability to activate aflatoxin BI in a bacterial mutagenicity test, no effect of the diet was detected. In general these data strongly suggest a role of selenium in the metabolism of certain toxicants but the mechanism is unclear at present. An important consideration in evaluating the enzymatic data presented in this study is that enzyme
Effect of Se on AFB~ metabolism in turkeys
641
activities measured in vitro under optimal conditions S-175, and the Florida Agricultural Experiment Station. to yield maximal activities do not accurately reflect in The technical assistance of D. B. Sartain is gratefully acknowledged. Florida Agricultural Experiment Stations vivo activities. In vivo variables such as substrate or cofactor concentration and reversible inhibition or Journal Series No. 5399. stimulation by selenium or other compounds could not be evaluated with this methodology. Another potentially important factor which could REFERENCES not be assessed in these experiments is the distribuBoyd J. N., Babish J. G. & Stoewsand G. S. (1982). tion of the various forms of hepatic cytochrome Modification by beet and cabbage diets of aflatoxin P-450 which is influenced by diet. Selective induction Brinduced rat plasma ~t-fetoprotein elevation, hepatic of P-450 isozymes differing in substrate and product tumorigenesis, and mutagenicity of urine. Fd Chem. specificity has been reported in several species (DanToxic. 20, 47. nan, Guengerich, Kaminsky & Aust, 1983; Guen- Boyd J. N., Misslbeck N. & Stoewsand G. S. (1983). gerich, Wang & Davidson, 1982; Pickett, Jeter, Changes in preneoplastic response to aflatoxin Bj in rats fed green beans, beets or squash. Fd Chem. Toxic. 21, 37. Morin & Lu, 1981; Yoshizawa, Uchimaru, Kamataki, Kato & Ueno, 1982). An influence of selenium Boyd J. N., Sell S. & Stoewsand G. S. (1979). Inhibition of aflatoxin-induced serum ~-fetoprotein in rats fed on the distribution of cytochrome P-450 isozymes cauliflower. Proc. Soc. exp. Biol. Med. 161, 473. could yield an alteration in aflatoxin activation versus Burguera J. A., Edds G. T. & Osuna O. (1983). Influence of detoxification by the microsomal mixed-function oxselenium on aflatoxin Bt or crotolaria toxicity in turkey idase system; such a change is suggested by the poults. Am. J. vet. Res. 44, 1714. aflatoxin BI/M~ ratios observed in this study (Table Cabral J. R. P. & Neal G. E. (1983). The inhibitory effects 1). Examination ofisozyme proportions as a function of ethoxyquin on the carcinogenic action of aflatoxin B~ in rats. Cancer Lett. 19, 125. of dietary selenium supplementation would be an important aspect of future studies. Potential effects of Campbell T. C. & Hayes J. R. (1976). The role of aflatoxin metabolism in its toxic lesion. Toxic. appl. Pharmac. 35, selenium on the activity of epoxide hydrolase should 199. also be investigated. Campbell T. C., Hayes J. R. & Newberne P. M. (1978). The known nutritional role of selenium is as a Dietary lipotropes, hepatic microsomal mixed-function cofactor for glutathione peroxidase, which is imoxidase activities,and in vivo covalent binding of aflatoxin portant in the detoxification of hydrogen peroxide B~ in rats. Cancer Res. 38, 4569. and lipid hydroperoxides. Extensive studies of Chen J., Goetchius M. P., Campbell T. C. & Combs G. F. aflatoxin metabolism have failed to yield evidence of (1982). Effects of dietary sele.nium and vitamin E on hepatic mixed-function oxidase activities and in vivo lipid peroxidation-mediated toxic effects. The results covalent binding of aflatoxin B1 in rats. J. Nutr. 112, 324. of the present study strongly suggest that the protective effect of selenium does not involve glutathione Chen J., Goetchius M. P., Combs G. F. & Campbell T. C. (1982). Effects of dietary selenium and vitamin E on peroxidase. The similar protective effects of selenium covalent binding of aflatoxin to chick liver cell macroand cadmium at nontoxic levels suggests that the molecules. J. Nutr. 112, 350. protection may involve a nonspecific inhibitory effect Dannan G. A., Guengerich F. P., Kaminsky L. S. & Aust of these elements on certain enzymatic processes in S. D. (1983). Regulation of cytochrome P-450. Immuaflatoxin activation. Cadmium has been found to nochemical quantitation of eight isozymes in liver microsomes of rats treated with polybrominated biphenyl coninterfere with hepatic microsomal drug metabolism in geners. J. biol. Chem. 258, 1282. rats, although this effect is due to a reduction in cytochrome P-450 concentration (Schnell, Means, Daoud A. H. & Griffin A. C. (1978). Effects of selenium and retinoic acid on the metabolism of N-acetylaminofluorene Roberts & Pence, 1979). The metabolic similarities and N-hydroxyacetylaminofluorene. Cancer Lett. 5, 231. and interactions of selenium and cadmium have been Davila J. C., Edds G. T., Osuna O. & Simpson C. F. (1983). recently reviewed as they relate to the nutritional and Modification of the effects of aflatoxin B~ and warfarin in toxicological roles of these elements (Whanger, Ridyoung pigs given selenium. Am. J. vet. Res. 44, 1877. lington & Holcomb, 1980). Comparative studies with Degen G. H. & Neumann H.-G. (1978). The major metabother nutritionally and environmentally important olite of aflatoxin B~ in the rat is a glutathione conjugate. Chemico-BioL Interactions 22, 239. elements such as zinc, mercury, and lead may provide Faris R. A. & Campbell T. C. (1981). Exposure of newborn more relevant information. rats to pharmacologically active compounds may perIn summary, the results of this study show that manently alter carcinogen metabolism. Science, N. Y. 211, selenium supplementation of diets affects the metab719. olism of aflatoxin B1 in turkey poults. Selenium Fjellstedt T. A., Allen R. H., Duncan B. K. & Jakoby enhanced the formation of water-soluble conjugated W. B. (1973). Enzymatic conjugation of epoxides with forms of aflatoxins, which may promote the clearance glutathione. J. biol. Chem. 248, 3702. of the toxin. Enzymatic data indicate that this effect Frape D, L., Wayman B. J. & Tuck M. G. (1981). The effect is not a result of increases in the specific activity of of dietary fibre of aflatoxicosis in the weanling male rat. Br. J. Nutr. 46, 315. glucuronyl transferase or glutathione S-transferase E or in the concentration of reduced glutathione in Frape D. L., Wayman B. J. & Tuck M. G. (1982). The effects of gum arabic, wheat offal and various of its liver. The results of this study support the contention fractions on the metabolism of ~4C-labelledaflatoxin B~in that selenium supplementation at nutritional levels the male weanling rat. Br. J. Nutr. 48, 97. would enhance the resistance of animals to the effects Gigon P. L., Gram T. E. & Gillette J. R. (1969). Studies on of aflatoxin B~. the rate of reduction of hepatic microsomal cytochrome P-450 by reduced nicotinamide adenine dinucleotide phosphate: Effect of drug substrates. Molec. PharmacoL Acknowledgements--This research was supported in part by 5, 109. funds from NSF Grant INT 7922086, Regional Project
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J. F. GREGORY III and G. T. EDOS
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