1152
G. F. COMBS, J R . , A. H. CANTOR AND M. L. SCOTT
Sci. 50: 1090-1096. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman and W. G. Hockstra, 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science, 179: 588-590. Siami, G., A. R. Schulbert and R. A. Neal, 1972. A possible role for the mixed function oxidase enzyme system in the requirement for selenium in the rat. J. Nutr. 102: 857-862. Thompson, J. N , and M. L. Scott, 1969. Role of selenium in the nutrition of the chick. J. Nutr. 97: 335-342. Wilbur, K. M., F. Bernheim and O. W. Shapiro, 1949. The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acids by various reagents. Arch. Biochem. 24: 305-313.
Polychlorinated Biphenyl-Stimulated Selenium Deficiency in the Chick1 G. F . COMBS, J R . 2 AND M. L . SCOTT
Department of Poultry Science and Graduate School of Nutrition, Cornell University, Ithaca, New York 14850 (Received for publication October 23, 1974)
ABSTRACT Experiments were conducted to determine individually the effects of dietary PCBs on the physiologic functions of vitamin E and selenium. Results showed that dietary PCBs did not affect the function of vitamin E in protection of biological membranes. However, PCBs did decrease the biological utilization of dietary selenium as measured by glutathione peroxidase activity in plasma and by the protection of biological membranes from peroxidation. These results indicate that dietary PCBs potentiate vitamin E-selenium deficiency in the chick by interference with the biological utilization of dietary selenium. An hypothesis for the mechanism of this effect is offered. POULTRY SCIENCE 54: 1152-1158, 1975
INTRODUCTION
this effect was overcome by feeding higher levels of vitamin E or selenium. Thus, PCBs
OLYCHLORINATED biphenyls (PCBs)
P
were shown to increase the apparent dietary
have been demonstrated to potentiate
requirements of the chick for vitamin E or
vitamin E and selenium deficiency in growing
selenium
chicks (Combs et al., 1975). PCBs increased
However, since the parameters measured in
for
protection
against
ED.
the incidence of exudative diathesis (ED) in
those experiments (growth, efficiency of feed
chicks receiving vitamin E-free diets which
utilization, incidence of ED) responded to
contained sub-optimal levels of selenium, and
either vitamin E or selenium, possible effects of PCBs on the physiologic function of vitamin E could not be discriminated from possi-
1. Supported in part by U.S. Public Health Service Grant NS 05632 and by Hoffmann-La Roche, Inc., Nutley, New Jersey 07110. 2. Present address: Department of Poultry Science, Auburn University, Auburn, Alabama 36830.
ble effects on the physiologic function of selenium. Two important discoveries have provided methods which can be used to discriminate
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
approach to a study of the unduction of rat liver hydroxylase after pretreatment with 3,4-benzopyrene and aflatoxin B , . Biochem. Pharmacol. 19: 1729-1736. Li, J. C , 1964. Statistical Inference, Vol. 1., Edwards Brothers, Ann Arbor, Michigan, P. 658. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Noguchi, T., A. H. Cantor and M. L. Scott, 1973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103: 1502-1511. Rehfield, B. M., R. L. Bradley, Jr. and M. L. Sunde, 1971. Toxicity studies on polychlorinated biphenyls in the chick. 1. Toxicity and symptoms. Poultry
PCB
AND SELENIUM
The present studies were conducted, therefore, to determine individually the effects of dietary PCBs on the physiologic functions of vitamin E and selenium. EXPERIMENTAL Animals and Diets. Vitamin E-depleted day-old Vantress x White Plymouth Rock chicks obtained as described previously (Thompson and Scott, 1969) were used in all experiments. Chicks were housed in temperature-controlled battery brooders with raised wire floors. Feed and water were supplied ad libitum. Equal numbers of males and females were used in each experiment. The basal diet used in these experiments was the torula yeast-casein-soybean proteinglucose diet used previously in this laboratory (Noguchi et al., 1973) to produce exudative diathesis in chicks. The diet was deficient in selenium (less than 0.02 p.p.m.) and was essentially free of tocopherols. In Experiments 2 and 5, 0.5% taurocholic acid and 0.5% oleic acid were added at the expenses of glucose monohydrate and stripped corn oil, respectively, to enhance lipid absorption and, thereby, to promote vitamin E absorption. Determination
of
Glutathione
Peroxidase.
Blood was obtained by heart puncture using a heparinized syringe. Plasma was prepared according to the method of Noguchi et al. (1973). Glutathione peroxidase was determined in plasma according to the method of Rotruck et al. (1973), as modified by Noguchi et al. (1973). Results were expressed as units of enzyme activity per mg. protein using the mean values of duplicate determinations for each chick as the observation. An enzyme unit represented a decrease in reduced glutathione concentration of 0.1 log unit per minute after subtraction of the non-enzymic rate. Determination of Ascorbic Acid-Stimulated Lipid Peroxidation. Hepatic microsomal suspensions, prepared as described previously (Combs and Scott, 1974) were diluted five-fold with 0.154 M KC1, 0.025 M Tris 3 HC1 buffer, pH 7.4. Ascorbic acid-stimulated lipid peroxidation in microsomes was measured in vitro according to the method of Noguchi et al. (1973). The thiobarbituric acid (TBA) test for lipid peroxide formation was performed according to a modification (Noguchi et al., 1973) of the method of Wilbur et al. (1949). Results were expressed as AOD530 per mg. protein per 60 minutes using the mean value of duplicate determinations for each chick as the observation. Determination of Protein. Total protein was determined by the method of Lowry et al. (1951) modified for automation. 4 Bovine serum albumin was used as a standard. Determination of Plasma Tocopherols. Blood was obtained by anterior heart puncture using a heparinized syringe. Plasma was prepared from whole blood by centrifugation at 1,000 x g for 10 minutes. Total plasma 3. Tris(hydroxymethyl)aminomethane. 4. Auto Analyzer(R) Methodology, Technicon Instruments Corp., Chauncy, N.Y. 1961.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
between vitamin E function and selenium function in the prevention of ED in chicks. First was the finding that selenium is an integral component of glutathione peroxidase in rats (Rotruck et al., 1973), which was later demonstrated in chicks (Noguchi et al., 1973). Second was the finding that both vitamin E and selenium are required in concert to protect biological membranes from oxidative degradation (Noguchi et al., 1973; Combs and Scott, 1974). These methods permit independent investigation of some of the functions of vitamin E and selenium uncomplicated by the sparing effects encountered using parameters such as the development of ED in chicks.
1153
1154
G. F. COMBS, JR. AND M. L. SCOTT
tocopherols were determined according to the method of Martinek (1964). Results were expressed as mg. dl-alpha-tocopherol equivalents per 100 ml. using the mean value of duplicate determinations for each chick as the observation.
Experiment 2. Duplicate lots of 10 chicks each were assigned randomly to dietary treatments which consisted of the basal diet supplemented with 0.15 p.p.m. selenium (as N a 2 S e 0 3 ) , four levels of vitamin E (0, 15, 30 and 60 I.U. dl-alpha-tocopheryl acetate per kg.) and two levels of PCBs (0 and 50 p.p.m. Aroclor (R) 1254) in a 4 x 2 factorial design. At two weeks of age, ascorbic acidstimulated lipid peroxidation in hepatic microsomes, total plasma tocopherols, growth and performance were determined. Experiment 3. In order to confirm the results of Experiment 2 and to examine more closely the effects of dietary PCBs on physiologic function of vitamin E, the experiment was repeated. Single lots of 10 cockerels each were assigned randomly to dietary treatments which consisted of the basal diet supplemented with 0.15 p.p.m. selenium (as N a 2 S e 0 3 ) , six levels of vitamin E (0, 10, 25, 50, 100 and 150 I.U. dl-alpha-tocopheryl acetate per kg.) and two levels of PCBs (0 and 50 p.p.m. Aroclor (R) 1254) in a 6 x 2 factorial design. Ascorbic acid-stimulated lipid peroxidation was determined in hepatic microsomal preparations from five chicks of each treatment at 14 days of age.
Statistical evaluation of data employed analyses of variance and Student's T tests (Li, 1964). Treatment means were ranked according to Duncan's multiple range test (Duncan, 1955) when significant treatment effects were determined by analysis of variance. RESULTS Experiment 1. Dietary PCBs significantly (P < 0.05) increased the incidence of ED after 11 days of age (Fig. 1). The marginal selenium level (0.04 p.p.m. supplemental Se) was apparently adequate for increases in glutathi-
100-,
r 8 10 Days After Hatching
FIG. 1. Effect of dietary PCBs on the incidence of exudative diathesis in vitamin E-deficient chicks receiving 0.04 p.p.m. Se (Exp. 1).
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
Experiment 1. Triplicate lots of 20 chicks each were fed the basal chick diet supplemented with 0.04 p.p.m. selenium as N a 2 S e 0 3 and either 0 or 50 p.p.m. PCBs (Aroclor (R) 1254). Plasma glutathione peroxidase was determined in eight chicks randomly selected from each treatment on days 3, 5, 7, 9 and 11 after hatching. The cumulative incidence of ED was also recorded.
Experiment 4. Single lots of 10 cockerels each were assigned randomly to dietary treatments consisting of the basal diet containing taurocholic and oleic acids and 100 I.U. vitamin E (dl-alpha-tocopheryl acetate) per kg., six levels of supplemental selenium (0, 0.02, 0.04, 0.06, 0.08 and 0.10 p.p.m. as N a 2 S e 0 3 ) and two levels of PCBs (0 and 50p.p.m. Aroclor 0.05) affect the dietary requirement of the chick for vitamin E for inhibition of in vitro ascorbic acid-stimulated lipid peroxidation in hepatic microsomes. This requirement was 30-501.U. kg. diet.
i
10 PCBs Fed
60
45
FIG. 3. Effect of dietary PCBs on utilization of dietary vitamin E as plasma tocopherols in two-week old chicks (Exp. 2). The concentration of total tocopherols in the plasma divided by the actual amount of vitamin E consumed was defined for the purpose of analysis as "dietary vitamin E utilization." The results showed that dietary PCBs significantly (P < 0.05) depressed this parameter (Fig. 3). Experiment 3.
The result
lg. 4) confirmed
TABLE 2.—Effect of dietary PCBs on the efficacy of selenium for growth and maintenance of plasma tocopherols concentration in 5-week old chicks (Exp. 4) Dietary treatments' PCBs 2
Selenium 3
p.p.m. 0
p.p.m. 0 0.02 0.04 0.06 0.08 0.10 0 0.02 0.04 0.06 0.08 0.10
Hepatic Microsomes
50
25
50 75 100 D i e t a r y V i t a m i n E, IU/kg
125
FIG. 4. Effect of dietary PCBs on the vitamin E requirement for inhibition of lipid peroxidation in chicks receiving an adequate level of dietary selenium (Exp. 3).
5-week gain eta
30
DIETARY VITAMIN E, lU/kg
753b4.5
756 b 804 de 836 fs 820 et 853 s 693 a 751 b 778 bcd 770 bc 800 de •7Q3 cde
Plasma tocopherols mg. % 1.60a5-6 1.59a 2.13" 2.77 c 2.80 c 3.96d 1.27a 1.55a 2.15" 2.80 c 2.24 b 3.61 d
'Basal diet contained 100 I.U. dl-alpha-tocopheryl acetate per kg., 0.5% taurocholic acid and 0.5% oleic acid. 2 Aroclor 1254. 3 AddedasNa 2 Se0 3 . 4 Means of 8 chicks per treatment. 'Analysis of variance showed significant treatment effect (P < 0.05); means with like superscripts within a column are not significantly different (P > 0.05). 6 Means of duplicate determinations of 5 chicks each per treatment.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
15
Experiment 4. Dietary PCBs significantly (P < 0.05) depressed growth of chicks receiving an adequate level of vitamin E, however the effect of dietary PCBs on plasma tocopherols concentration was not significant (P > 0.05) (Table 2). Dietary PCBs significantly (P < 0.05) increased lipid peroxidation in hepatic microsomal fractions at levels of supplemental dietary selenium less than 0.08
PCB
AND SELENIUM
Hepatic Microsomes
v* p > 0.05) interactions of PCBs x selenium were observed. DISCUSSION Biochemical techniques have been employed to determine whether dietary PCBs affect the physiologic function of vitamin E, of selenium or of both of these nutrients in the potentiation of exudative diathesis in the chick. The plasma activity of the seleniumcontaining enzyme glutathione peroxidase was depressed when PCBs were fed in a vitamin E deficient diet marginal with respect to selenium. Decreased plasma glutathione peroxidase activity was associated with increased incidence of exudative diathesis, as demonstrated previously in this laboratory (Noguchi et al, 1973). The inhibition of in vitro ascorbic acidstimulated lipid peroxidation of hepatic microsomal preparations also was used to determine the specificity for vitamin E function or for selenium function of the observed effects of PCBs in the chick. Studies demon-
strated that both vitamin E and selenium are required in the diet to protect chick hepatic microsomes, as reported earlier (Noguchi et al, 1973; Combs and Scott, 1974). Dietary PCBs did not affect this function of vitamin E, but PCBs did increase the amount of dietary selenium required to protect microsomal fractions from peroxidation in vitro. Dietary PCBs depressed plasma tocopherols concentrations, however this effect appeared to be functionally unrelated to the in vitro peroxidation of hepatic microsomal fractions. These experiments and those reported previously (Combs et al., 1975) indicate that dietary PCBs interfere with the vitamin Eselenium nutrition of the chick through effects on the physiologic function of selenium. Dietary PCBs interfere with the utilization of dietary selenium, increasing the chick's requirement slightly but critically if the diet contains a marginal level and is low in vitamin E content. PCBs do not affect the physiologic function of vitamin E in protection of biological membranes, but PCBs may interfere with the absorption-retention of the vitamin. These studies have demonstrated the interference of dietary PCBs with selenium nutrition in the chick; however, no biochemical basis for this effect has been shown. Dietary PCBs may promote exudative diathesis in the chick in several different ways. First, PCBs may act directly as pro-oxidants or they may induce radical-generating enzyme systems such as NADPH oxidase-coupled drug hydroxylations. Although the available evidence does not preclude this possibility, such a PCB effect would not provide specificity for selenium function. Second, PCBs may affect inhibited selenium absorption. However, the authors believe it more likely that PCBs may induce hepatic microsomal enzymes which alter the biological utilization of absorbed selenium. Siami et al. (1972) proposed that the utilization of dietary selenium in the rat is influenced by certain inducible hepatic mixed-function
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
.02
1157
1158
G. F. COMBS, JR. AND M. L. SCOTT
oxidases. Diplock et al. (1971) proposed that selenide may be the form of tissue selenium with greatest biological activity. PCBs have been shown to be powerful inducers of hepatic microsomal cytochrome P 450 -dependent oxidases (Alvares et al., 1973). Therefore, the results of
these
studies support
the
hypothesis that PCBs induce hepatic microsomal enzymes which alter the utilization of absorbed selenium, by changing its oxida-
corporation into selenium compounds of limited biological usefulness. Further investigation of this hypothesis may elucidate the mechanism of action of selenium in protection of biological membranes. REFERENCES Alveres, A. P., D. R. Bickers and A. Kappas, 1973. Polychlorinated biphenyls: a new type of inducer of cytochrome P-448 in the liver. U.S. Nat. Acad. Sci. Proc. 70: 1321-1325. Combs, G. F., A. H. Cantor and M. L. Scott, 1975. Effects of dietary polychlorinated biphenyls on vitamin E and selenium nutrition in the chick. Poultry Sci. 54: 1143-1152. Combs, Jr., G. F., and M. L. Scott, 1974. Dietary requirements for vitamin E and selenium measured
NEWS AND NOTES (Continued from page 1133) plishments of the winners are featured in the 1975 Ford Almanac which is distributed through Ford car, truck and tractor dealers and through book stores. HUBBARD NOTES Russell A. Mease has joined the field marketing team of Hubbard Farms, Walpole, New Hampshire, and will be concentrating on sales and service for the new Hubbard Leghorn. Prior to joining the Hubbard organization, he spent 18 years with Babcock Poultry Farm, Inc., most recently as Vice President and General Manager of the Lititz, Pennsylvania, branch.
PURDUE NOTES At the recent Indiana-Michigan-Ohio Poultry Conference, Dr. Roland Winterfield, Purdue University, received Indiana's 1974 Golden Egg Award, and Ned Kirby, Kirby Farms Inc., Urbana, Ohio, received Ohio's 11974 Golden Egg Award. Dr. Winterfield is an Associate Editor of Poultry Science. A.E.B. NOTES Television commercials for "The Incredible Edible Egg" of the American Egg Board won the top prize
(Continued on page 1163)
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
tion state thus reducing its incorporation into glutathione peroxidase and increasing its in-
at the cellular level in the chick. J. Nutr. 104: 1292-1296. Diplock, A. T., H. Baum and J. A. Lucy, 1971. The effect of vitamin Eon the oxidation state of selenium in rat liver. Biochem. J. 123: 721-729. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 2-42. Li, J. C , 1964. Statistical Inference, Vol. I., Edwards Bros., Ann Arbor, Michigan, 658 pp. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Martinek, R. G., 1964. Method for the determination of vitamin E (total tocopherols) in serum. Clin. Chem. 10: 1078-1086. Noguchi, T., A. H. Cantor and M. L. Scott, 1973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103: 1502-1511. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Haferman and H. G. Hoekstra, 1973. Selenium: peroxidase. Science, 179: 588-590. Siami, G., A. R. Schulbert and R. A. Neal, 1972. A possible role for the mixed function oxidase enzyme system in the requirement for selenium in the rat. J. Nutr. 102: 857-862. Thompson, J. N„ and M. L. Scott, 1969. Role of selenium in the nutrition of the chick. J. Nutr. 97: 335-342. Wilbur, K. M., F. Bernheim and O. W. Shapiro, 1949. The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acids by various reagents. Arch. Biochem. 24: 305-313.
G. F. COMBS, J R . , A. H. CANTOR AND M. L. SCOTT
Sci. 50: 1090-1096. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman and W. G. Hockstra, 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science, 179: 588-590. Siami, G., A. R. Schulbert and R. A. Neal, 1972. A possible role for the mixed function oxidase enzyme system in the requirement for selenium in the rat. J. Nutr. 102: 857-862. Thompson, J. N , and M. L. Scott, 1969. Role of selenium in the nutrition of the chick. J. Nutr. 97: 335-342. Wilbur, K. M., F. Bernheim and O. W. Shapiro, 1949. The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acids by various reagents. Arch. Biochem. 24: 305-313.
Polychlorinated Biphenyl-Stimulated Selenium Deficiency in the Chick1 G. F . COMBS, J R . 2 AND M. L . SCOTT
Department of Poultry Science and Graduate School of Nutrition, Cornell University, Ithaca, New York 14850 (Received for publication October 23, 1974)
ABSTRACT Experiments were conducted to determine individually the effects of dietary PCBs on the physiologic functions of vitamin E and selenium. Results showed that dietary PCBs did not affect the function of vitamin E in protection of biological membranes. However, PCBs did decrease the biological utilization of dietary selenium as measured by glutathione peroxidase activity in plasma and by the protection of biological membranes from peroxidation. These results indicate that dietary PCBs potentiate vitamin E-selenium deficiency in the chick by interference with the biological utilization of dietary selenium. An hypothesis for the mechanism of this effect is offered. POULTRY SCIENCE 54: 1152-1158, 1975
INTRODUCTION
this effect was overcome by feeding higher levels of vitamin E or selenium. Thus, PCBs
OLYCHLORINATED biphenyls (PCBs)
P
were shown to increase the apparent dietary
have been demonstrated to potentiate
requirements of the chick for vitamin E or
vitamin E and selenium deficiency in growing
selenium
chicks (Combs et al., 1975). PCBs increased
However, since the parameters measured in
for
protection
against
ED.
the incidence of exudative diathesis (ED) in
those experiments (growth, efficiency of feed
chicks receiving vitamin E-free diets which
utilization, incidence of ED) responded to
contained sub-optimal levels of selenium, and
either vitamin E or selenium, possible effects of PCBs on the physiologic function of vitamin E could not be discriminated from possi-
1. Supported in part by U.S. Public Health Service Grant NS 05632 and by Hoffmann-La Roche, Inc., Nutley, New Jersey 07110. 2. Present address: Department of Poultry Science, Auburn University, Auburn, Alabama 36830.
ble effects on the physiologic function of selenium. Two important discoveries have provided methods which can be used to discriminate
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
approach to a study of the unduction of rat liver hydroxylase after pretreatment with 3,4-benzopyrene and aflatoxin B , . Biochem. Pharmacol. 19: 1729-1736. Li, J. C , 1964. Statistical Inference, Vol. 1., Edwards Brothers, Ann Arbor, Michigan, P. 658. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Noguchi, T., A. H. Cantor and M. L. Scott, 1973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103: 1502-1511. Rehfield, B. M., R. L. Bradley, Jr. and M. L. Sunde, 1971. Toxicity studies on polychlorinated biphenyls in the chick. 1. Toxicity and symptoms. Poultry
PCB
AND SELENIUM
The present studies were conducted, therefore, to determine individually the effects of dietary PCBs on the physiologic functions of vitamin E and selenium. EXPERIMENTAL Animals and Diets. Vitamin E-depleted day-old Vantress x White Plymouth Rock chicks obtained as described previously (Thompson and Scott, 1969) were used in all experiments. Chicks were housed in temperature-controlled battery brooders with raised wire floors. Feed and water were supplied ad libitum. Equal numbers of males and females were used in each experiment. The basal diet used in these experiments was the torula yeast-casein-soybean proteinglucose diet used previously in this laboratory (Noguchi et al., 1973) to produce exudative diathesis in chicks. The diet was deficient in selenium (less than 0.02 p.p.m.) and was essentially free of tocopherols. In Experiments 2 and 5, 0.5% taurocholic acid and 0.5% oleic acid were added at the expenses of glucose monohydrate and stripped corn oil, respectively, to enhance lipid absorption and, thereby, to promote vitamin E absorption. Determination
of
Glutathione
Peroxidase.
Blood was obtained by heart puncture using a heparinized syringe. Plasma was prepared according to the method of Noguchi et al. (1973). Glutathione peroxidase was determined in plasma according to the method of Rotruck et al. (1973), as modified by Noguchi et al. (1973). Results were expressed as units of enzyme activity per mg. protein using the mean values of duplicate determinations for each chick as the observation. An enzyme unit represented a decrease in reduced glutathione concentration of 0.1 log unit per minute after subtraction of the non-enzymic rate. Determination of Ascorbic Acid-Stimulated Lipid Peroxidation. Hepatic microsomal suspensions, prepared as described previously (Combs and Scott, 1974) were diluted five-fold with 0.154 M KC1, 0.025 M Tris 3 HC1 buffer, pH 7.4. Ascorbic acid-stimulated lipid peroxidation in microsomes was measured in vitro according to the method of Noguchi et al. (1973). The thiobarbituric acid (TBA) test for lipid peroxide formation was performed according to a modification (Noguchi et al., 1973) of the method of Wilbur et al. (1949). Results were expressed as AOD530 per mg. protein per 60 minutes using the mean value of duplicate determinations for each chick as the observation. Determination of Protein. Total protein was determined by the method of Lowry et al. (1951) modified for automation. 4 Bovine serum albumin was used as a standard. Determination of Plasma Tocopherols. Blood was obtained by anterior heart puncture using a heparinized syringe. Plasma was prepared from whole blood by centrifugation at 1,000 x g for 10 minutes. Total plasma 3. Tris(hydroxymethyl)aminomethane. 4. Auto Analyzer(R) Methodology, Technicon Instruments Corp., Chauncy, N.Y. 1961.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
between vitamin E function and selenium function in the prevention of ED in chicks. First was the finding that selenium is an integral component of glutathione peroxidase in rats (Rotruck et al., 1973), which was later demonstrated in chicks (Noguchi et al., 1973). Second was the finding that both vitamin E and selenium are required in concert to protect biological membranes from oxidative degradation (Noguchi et al., 1973; Combs and Scott, 1974). These methods permit independent investigation of some of the functions of vitamin E and selenium uncomplicated by the sparing effects encountered using parameters such as the development of ED in chicks.
1153
1154
G. F. COMBS, JR. AND M. L. SCOTT
tocopherols were determined according to the method of Martinek (1964). Results were expressed as mg. dl-alpha-tocopherol equivalents per 100 ml. using the mean value of duplicate determinations for each chick as the observation.
Experiment 2. Duplicate lots of 10 chicks each were assigned randomly to dietary treatments which consisted of the basal diet supplemented with 0.15 p.p.m. selenium (as N a 2 S e 0 3 ) , four levels of vitamin E (0, 15, 30 and 60 I.U. dl-alpha-tocopheryl acetate per kg.) and two levels of PCBs (0 and 50 p.p.m. Aroclor (R) 1254) in a 4 x 2 factorial design. At two weeks of age, ascorbic acidstimulated lipid peroxidation in hepatic microsomes, total plasma tocopherols, growth and performance were determined. Experiment 3. In order to confirm the results of Experiment 2 and to examine more closely the effects of dietary PCBs on physiologic function of vitamin E, the experiment was repeated. Single lots of 10 cockerels each were assigned randomly to dietary treatments which consisted of the basal diet supplemented with 0.15 p.p.m. selenium (as N a 2 S e 0 3 ) , six levels of vitamin E (0, 10, 25, 50, 100 and 150 I.U. dl-alpha-tocopheryl acetate per kg.) and two levels of PCBs (0 and 50 p.p.m. Aroclor (R) 1254) in a 6 x 2 factorial design. Ascorbic acid-stimulated lipid peroxidation was determined in hepatic microsomal preparations from five chicks of each treatment at 14 days of age.
Statistical evaluation of data employed analyses of variance and Student's T tests (Li, 1964). Treatment means were ranked according to Duncan's multiple range test (Duncan, 1955) when significant treatment effects were determined by analysis of variance. RESULTS Experiment 1. Dietary PCBs significantly (P < 0.05) increased the incidence of ED after 11 days of age (Fig. 1). The marginal selenium level (0.04 p.p.m. supplemental Se) was apparently adequate for increases in glutathi-
100-,
r 8 10 Days After Hatching
FIG. 1. Effect of dietary PCBs on the incidence of exudative diathesis in vitamin E-deficient chicks receiving 0.04 p.p.m. Se (Exp. 1).
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on April 19, 2015
Experiment 1. Triplicate lots of 20 chicks each were fed the basal chick diet supplemented with 0.04 p.p.m. selenium as N a 2 S e 0 3 and either 0 or 50 p.p.m. PCBs (Aroclor (R) 1254). Plasma glutathione peroxidase was determined in eight chicks randomly selected from each treatment on days 3, 5, 7, 9 and 11 after hatching. The cumulative incidence of ED was also recorded.
Experiment 4. Single lots of 10 cockerels each were assigned randomly to dietary treatments consisting of the basal diet containing taurocholic and oleic acids and 100 I.U. vitamin E (dl-alpha-tocopheryl acetate) per kg., six levels of supplemental selenium (0, 0.02, 0.04, 0.06, 0.08 and 0.10 p.p.m. as N a 2 S e 0 3 ) and two levels of PCBs (0 and 50p.p.m. Aroclor 0.05) affect the dietary requirement of the chick for vitamin E for inhibition of in vitro ascorbic acid-stimulated lipid peroxidation in hepatic microsomes. This requirement was 30-501.U. kg. diet.
i
10 PCBs Fed
60
45
FIG. 3. Effect of dietary PCBs on utilization of dietary vitamin E as plasma tocopherols in two-week old chicks (Exp. 2). The concentration of total tocopherols in the plasma divided by the actual amount of vitamin E consumed was defined for the purpose of analysis as "dietary vitamin E utilization." The results showed that dietary PCBs significantly (P < 0.05) depressed this parameter (Fig. 3). Experiment 3.
The result
lg. 4) confirmed
TABLE 2.—Effect of dietary PCBs on the efficacy of selenium for growth and maintenance of plasma tocopherols concentration in 5-week old chicks (Exp. 4) Dietary treatments' PCBs 2
Selenium 3
p.p.m. 0
p.p.m. 0 0.02 0.04 0.06 0.08 0.10 0 0.02 0.04 0.06 0.08 0.10
Hepatic Microsomes
50
25
50 75 100 D i e t a r y V i t a m i n E, IU/kg
125
FIG. 4. Effect of dietary PCBs on the vitamin E requirement for inhibition of lipid peroxidation in chicks receiving an adequate level of dietary selenium (Exp. 3).
5-week gain eta
30
DIETARY VITAMIN E, lU/kg
753b4.5
756 b 804 de 836 fs 820 et 853 s 693 a 751 b 778 bcd 770 bc 800 de •7Q3 cde
Plasma tocopherols mg. % 1.60a5-6 1.59a 2.13" 2.77 c 2.80 c 3.96d 1.27a 1.55a 2.15" 2.80 c 2.24 b 3.61 d
'Basal diet contained 100 I.U. dl-alpha-tocopheryl acetate per kg., 0.5% taurocholic acid and 0.5% oleic acid. 2 Aroclor 1254. 3 AddedasNa 2 Se0 3 . 4 Means of 8 chicks per treatment. 'Analysis of variance showed significant treatment effect (P < 0.05); means with like superscripts within a column are not significantly different (P > 0.05). 6 Means of duplicate determinations of 5 chicks each per treatment.
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15
Experiment 4. Dietary PCBs significantly (P < 0.05) depressed growth of chicks receiving an adequate level of vitamin E, however the effect of dietary PCBs on plasma tocopherols concentration was not significant (P > 0.05) (Table 2). Dietary PCBs significantly (P < 0.05) increased lipid peroxidation in hepatic microsomal fractions at levels of supplemental dietary selenium less than 0.08
PCB
AND SELENIUM
Hepatic Microsomes
v* p > 0.05) interactions of PCBs x selenium were observed. DISCUSSION Biochemical techniques have been employed to determine whether dietary PCBs affect the physiologic function of vitamin E, of selenium or of both of these nutrients in the potentiation of exudative diathesis in the chick. The plasma activity of the seleniumcontaining enzyme glutathione peroxidase was depressed when PCBs were fed in a vitamin E deficient diet marginal with respect to selenium. Decreased plasma glutathione peroxidase activity was associated with increased incidence of exudative diathesis, as demonstrated previously in this laboratory (Noguchi et al, 1973). The inhibition of in vitro ascorbic acidstimulated lipid peroxidation of hepatic microsomal preparations also was used to determine the specificity for vitamin E function or for selenium function of the observed effects of PCBs in the chick. Studies demon-
strated that both vitamin E and selenium are required in the diet to protect chick hepatic microsomes, as reported earlier (Noguchi et al, 1973; Combs and Scott, 1974). Dietary PCBs did not affect this function of vitamin E, but PCBs did increase the amount of dietary selenium required to protect microsomal fractions from peroxidation in vitro. Dietary PCBs depressed plasma tocopherols concentrations, however this effect appeared to be functionally unrelated to the in vitro peroxidation of hepatic microsomal fractions. These experiments and those reported previously (Combs et al., 1975) indicate that dietary PCBs interfere with the vitamin Eselenium nutrition of the chick through effects on the physiologic function of selenium. Dietary PCBs interfere with the utilization of dietary selenium, increasing the chick's requirement slightly but critically if the diet contains a marginal level and is low in vitamin E content. PCBs do not affect the physiologic function of vitamin E in protection of biological membranes, but PCBs may interfere with the absorption-retention of the vitamin. These studies have demonstrated the interference of dietary PCBs with selenium nutrition in the chick; however, no biochemical basis for this effect has been shown. Dietary PCBs may promote exudative diathesis in the chick in several different ways. First, PCBs may act directly as pro-oxidants or they may induce radical-generating enzyme systems such as NADPH oxidase-coupled drug hydroxylations. Although the available evidence does not preclude this possibility, such a PCB effect would not provide specificity for selenium function. Second, PCBs may affect inhibited selenium absorption. However, the authors believe it more likely that PCBs may induce hepatic microsomal enzymes which alter the biological utilization of absorbed selenium. Siami et al. (1972) proposed that the utilization of dietary selenium in the rat is influenced by certain inducible hepatic mixed-function
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G. F. COMBS, JR. AND M. L. SCOTT
oxidases. Diplock et al. (1971) proposed that selenide may be the form of tissue selenium with greatest biological activity. PCBs have been shown to be powerful inducers of hepatic microsomal cytochrome P 450 -dependent oxidases (Alvares et al., 1973). Therefore, the results of
these
studies support
the
hypothesis that PCBs induce hepatic microsomal enzymes which alter the utilization of absorbed selenium, by changing its oxida-
corporation into selenium compounds of limited biological usefulness. Further investigation of this hypothesis may elucidate the mechanism of action of selenium in protection of biological membranes. REFERENCES Alveres, A. P., D. R. Bickers and A. Kappas, 1973. Polychlorinated biphenyls: a new type of inducer of cytochrome P-448 in the liver. U.S. Nat. Acad. Sci. Proc. 70: 1321-1325. Combs, G. F., A. H. Cantor and M. L. Scott, 1975. Effects of dietary polychlorinated biphenyls on vitamin E and selenium nutrition in the chick. Poultry Sci. 54: 1143-1152. Combs, Jr., G. F., and M. L. Scott, 1974. Dietary requirements for vitamin E and selenium measured
NEWS AND NOTES (Continued from page 1133) plishments of the winners are featured in the 1975 Ford Almanac which is distributed through Ford car, truck and tractor dealers and through book stores. HUBBARD NOTES Russell A. Mease has joined the field marketing team of Hubbard Farms, Walpole, New Hampshire, and will be concentrating on sales and service for the new Hubbard Leghorn. Prior to joining the Hubbard organization, he spent 18 years with Babcock Poultry Farm, Inc., most recently as Vice President and General Manager of the Lititz, Pennsylvania, branch.
PURDUE NOTES At the recent Indiana-Michigan-Ohio Poultry Conference, Dr. Roland Winterfield, Purdue University, received Indiana's 1974 Golden Egg Award, and Ned Kirby, Kirby Farms Inc., Urbana, Ohio, received Ohio's 11974 Golden Egg Award. Dr. Winterfield is an Associate Editor of Poultry Science. A.E.B. NOTES Television commercials for "The Incredible Edible Egg" of the American Egg Board won the top prize
(Continued on page 1163)
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tion state thus reducing its incorporation into glutathione peroxidase and increasing its in-
at the cellular level in the chick. J. Nutr. 104: 1292-1296. Diplock, A. T., H. Baum and J. A. Lucy, 1971. The effect of vitamin Eon the oxidation state of selenium in rat liver. Biochem. J. 123: 721-729. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 2-42. Li, J. C , 1964. Statistical Inference, Vol. I., Edwards Bros., Ann Arbor, Michigan, 658 pp. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Martinek, R. G., 1964. Method for the determination of vitamin E (total tocopherols) in serum. Clin. Chem. 10: 1078-1086. Noguchi, T., A. H. Cantor and M. L. Scott, 1973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103: 1502-1511. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Haferman and H. G. Hoekstra, 1973. Selenium: peroxidase. Science, 179: 588-590. Siami, G., A. R. Schulbert and R. A. Neal, 1972. A possible role for the mixed function oxidase enzyme system in the requirement for selenium in the rat. J. Nutr. 102: 857-862. Thompson, J. N„ and M. L. Scott, 1969. Role of selenium in the nutrition of the chick. J. Nutr. 97: 335-342. Wilbur, K. M., F. Bernheim and O. W. Shapiro, 1949. The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acids by various reagents. Arch. Biochem. 24: 305-313.
