Toxicology Letters, 62 (1992) 139-i 53 0
139
1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$05.00
TOXLET 02760
Dietary pyrrolizidine (Senecio) alkaloids and tissue distribution of copper and vitamin A in broiler chickens
Jianya Huana, Peter R. Cheeke”, Robert R. Lowryb, Harry S. Nakauea, Stanley P. Snyder” and Philip D. Whangerb ~~epa~t~ent of Animo~ Sciences, ‘department
o~Ag~~~ltura1 Chemistry and “Veterinary diagnostic
Laboratory, Oregon State University, CorvaiIis, OR {USA)
(Received 3 February 1992) (Accepted 3 April 1992) Key words; Senecio jacobaea; Pyrrolizidine alkaloid; Copper; Vitamin A; Chick
--SUMMARY The effect of feeding a diet containing 5% tansy ragwort (TRf (~enec~o jaco~aea~, a poisonous plant containing pyrrolizidine alkaloids (PA), on the blood and liver levels of copper, zinc, iron and vitamin A in broiler chicks was examined. Serum and liver copper and liver iron concentrations were increased in chicks fed a diet with 5% TR, while serum and liver zinc and vitamin A decreased. When PA were removed from the diet, partial restoration of normal serum vitamin A level occurred, indicating that the ability to mobilize liver vitamin A is not irreversibly inhibited by PA. The decline in serum vitamin A occurred by 8 days of TR feeding with a concurrent decline in growth rate. When chicks were fed a diet high in vitamin A (25 000 IUlkg), followed by a basal diet containing TR, serum vitamin A levels were signi~cantly (PcO.01) decreased. while liver vitamin A level increased. This indicates that mobilization of previously stored vitamin A from the liver is impaired by PA. Prior feeding of a high vitamin A level resulted in protective effects against PA toxicity, as assessed by histopathology. This study shows that a dietary source of PA modifies metabolism and tissue distribution of minerals and vitamin A.
Pyrrolizidine alkaloids (PA) occur in a number of plants such as Senecio and CrotaZuria sp., and have caused numerous toxicological problems in humans and livestock
Correspondence to: Dr. P.R. Cheeke, Department of Animal Sciences, Oregon State University, Corvallis,
OR 97331. USA.
140
[1,2]. The PA are metabolized by liver tissue to reactive metabolites such as pyrroles which cross-link DNA strands, causing impaired cell division and protein synthesis, leading to a loss of liver function and irreversible liver necrosis [1,2]. Exposure to dietary PA may modify nutrient metabolism and tissue distribution. Consumption of PA-containing plants by sheep may result in markedly elevated liver copper concentrations [3]. This has been observed in numerous other species as well [4-71. An interaction between PA exposure and vitamin A metabolism has also been observed [S]. Rats fed a source of Senecio PA had reduced plasma and liver vitamin A concentrations. Inhibitory effects of PA on hepatic retinol-binding protein (RBP) synthesis, and impaired biliary excretion resulting in reduced vitamin A absorption were postulated as possible mechanisms for the PA effect [8]. The objectives of this study were: (i) to determine the effects of dietary PA on the tissue distribution of copper, iron, zinc and vitamin A in chicks; (ii) to determine whether dietary PA affected the mobilization of previously stored vitamin A in the liver; and (iii) to determine whether the effects of PA on liver and serum vitamin A levels were reversible after PA were removed from the diet. Because the major metabolic effect of PA is on hepatic protein synthesis [l], the ability to mobilize previously stored vitamin A should provide evidence as to whether inhibition of vitamin Atransport protein synthesis (e.g., hepatic RBP) is involved in the effect of PA on tissue distribution of vitamin A. MATERIALS AND METHODS
Animals and diets
Broiler chicks (Peterson X Arbor Acres) hatched from eggs obtained from Keith Smith Farm, Hot Springs, AR, were used. They were housed under controlled conditions of 18-22°C and continuous artificial light, in electrically-heated and ventilated wood-shaving litter floor pens. Birds were wing-banded and individual body weights obtained. Tansy ragwort (Senecio jacobaea) flowers were hand harvested, sun dried and used as the dietary source of PA. The dried material contained approx. 0.18% total PA [8]. In Expt. 1, eight different diets were used, based on a corn-soybean meal basal diet with 23% crude protein (Table I) The distribution of treatments is shown in Table II. Treatment variables were 0 and 5% tansy ragwort (TR), 0 and 250 mg/kg diet copper (anhydrous copper sulfate), and 0 and 25 000 IU/kg diet vitamin A (all-trans retinol palmitate). The birds (12 male l-week-old chicks per treatment) were fed the experimental diets for 28 days, at which time six birds from each treatment were killed and tissues obtained for mineral and vitamin A analyses. The remaining chicks were then given a PA-free diet (corn-soybean meal broiler grower diet) containing a high level of added vitamin A (25 000 IU/kg diet) for 14 days. They were then killed by injection with T-61 (Hoechst-Roussel Agri-Vet) euthanasia solution and tissues obtained. In Expt. 2, 50 female 2-week-old chicks were randomly assigned to two groups of 25. In the first 2-week period, both groups were fed the same broiler grower diet
141
TABLE I COMPOSITION OF THE BROILER STARTER AND GROWER BASAL DIETS (g/l00 g) Ingredients
Starter diet
Grower diet
Yellow corn Fat Soybean meal Meat and bone meal Dehydrated alfalfa meal Dicalcium phosphate (32% Ca, 18% P) Limestone flour Salt Trace mineral (mix-65) Vitamin premix (I-75)b o,L-Methionine
58.35 2.00 32.25 5.00 1.oo 0.42 0.35 0.25 0.05 0.20 0.13
63.52 2.00 27.50 5.00 1.00 0.25 0.13 0.25 0.05 0.20 0.10
a Supplied the following quantities per kg feed: vitamin A, 3300 IU; vitamin D, 1100 IU; vitamin E, 1.1 IU; vitamin K, 0.55 mg; vitamin B-12,0.0055 mg; riboflavin, 3.3 mg; pantothenic acid, 5.5 mg; niacin, 22 mg; choline chloride, 220 mg; folic acid, 0.22 mg; ethoxyquin, 64.43 mg. b Supplied the following quantities per kg feed: calcium, 107.5 mg; manganese, 60 mg; iron, 20 mg; zinc, 28 mg; copper, 2 mg; iodine, 1.2 mg; cobalt, 0.205 mg.
supplemented with 25 000 IU/kg of all-trans retinol palmitate. After this period, the vitamin A supplement was discontinued. One group was fed a basal diet of 95% broiler grower diet and 5% ground corn, and the other group was fed 95% broiler diet and 5% TR. At the end of the first 14-day period, three birds from each group were killed and tissue samples taken. Six additional birds in each group were identified, and blood samples obtained from the wing vein every 4 days for a total of 24 days. After 24 days, these birds were killed and tissue samples taken. The remaining birds in each group were monitored for weight gain over the experimental period. Analytical methods Vitamin A was determined by HPLC based on the method of Bieri et al. [9], Chow and Omaye [IO,111.Copper, zinc and iron were analyzed by AOAC [ 121and selenium by the method of Brown and Watkinson [ 131.Calpain activity was measured according to the methnd of Gopalakrishna and Barsky [14]. Statistics Statistical analyses were performed using the statistical software base SAS (SAS Institute, Inc.). Data were assessed for homogeneity of variance using Analysis of Variance procedure with means compared by Student-Neuman-Keuls (SNK) test at PcO.05; uneven number of replications were analyzed by using the General Linear Models Procedure with SNK test from SAS.
Cu (mg/kg)
25 000 25 000 25 000 25 000
0
0 0
0
vitamin A @U/kg) 1667 F 632 + 1604 + 668 f 1643 * 615 + 1751 2 700 rt
51db 23 37b 33’ 39ab 26 48” 29”
Average weight at 4 wks
54 I3 rt 1f7”b 17.4 i: 0.8’ 52.0 + 1.3b 18.5 rt 1.2” 53 4 f: 1 3”” 18:8 rt 0:9c 51.3 rt 1.6” 19.7 t 1.0”
Average daily gain (g) 04 wks
Means i SE in the same column followed by different superscripts are different (P
139
1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$05.00
TOXLET 02760
Dietary pyrrolizidine (Senecio) alkaloids and tissue distribution of copper and vitamin A in broiler chickens
Jianya Huana, Peter R. Cheeke”, Robert R. Lowryb, Harry S. Nakauea, Stanley P. Snyder” and Philip D. Whangerb ~~epa~t~ent of Animo~ Sciences, ‘department
o~Ag~~~ltura1 Chemistry and “Veterinary diagnostic
Laboratory, Oregon State University, CorvaiIis, OR {USA)
(Received 3 February 1992) (Accepted 3 April 1992) Key words; Senecio jacobaea; Pyrrolizidine alkaloid; Copper; Vitamin A; Chick
--SUMMARY The effect of feeding a diet containing 5% tansy ragwort (TRf (~enec~o jaco~aea~, a poisonous plant containing pyrrolizidine alkaloids (PA), on the blood and liver levels of copper, zinc, iron and vitamin A in broiler chicks was examined. Serum and liver copper and liver iron concentrations were increased in chicks fed a diet with 5% TR, while serum and liver zinc and vitamin A decreased. When PA were removed from the diet, partial restoration of normal serum vitamin A level occurred, indicating that the ability to mobilize liver vitamin A is not irreversibly inhibited by PA. The decline in serum vitamin A occurred by 8 days of TR feeding with a concurrent decline in growth rate. When chicks were fed a diet high in vitamin A (25 000 IUlkg), followed by a basal diet containing TR, serum vitamin A levels were signi~cantly (PcO.01) decreased. while liver vitamin A level increased. This indicates that mobilization of previously stored vitamin A from the liver is impaired by PA. Prior feeding of a high vitamin A level resulted in protective effects against PA toxicity, as assessed by histopathology. This study shows that a dietary source of PA modifies metabolism and tissue distribution of minerals and vitamin A.
Pyrrolizidine alkaloids (PA) occur in a number of plants such as Senecio and CrotaZuria sp., and have caused numerous toxicological problems in humans and livestock
Correspondence to: Dr. P.R. Cheeke, Department of Animal Sciences, Oregon State University, Corvallis,
OR 97331. USA.
140
[1,2]. The PA are metabolized by liver tissue to reactive metabolites such as pyrroles which cross-link DNA strands, causing impaired cell division and protein synthesis, leading to a loss of liver function and irreversible liver necrosis [1,2]. Exposure to dietary PA may modify nutrient metabolism and tissue distribution. Consumption of PA-containing plants by sheep may result in markedly elevated liver copper concentrations [3]. This has been observed in numerous other species as well [4-71. An interaction between PA exposure and vitamin A metabolism has also been observed [S]. Rats fed a source of Senecio PA had reduced plasma and liver vitamin A concentrations. Inhibitory effects of PA on hepatic retinol-binding protein (RBP) synthesis, and impaired biliary excretion resulting in reduced vitamin A absorption were postulated as possible mechanisms for the PA effect [8]. The objectives of this study were: (i) to determine the effects of dietary PA on the tissue distribution of copper, iron, zinc and vitamin A in chicks; (ii) to determine whether dietary PA affected the mobilization of previously stored vitamin A in the liver; and (iii) to determine whether the effects of PA on liver and serum vitamin A levels were reversible after PA were removed from the diet. Because the major metabolic effect of PA is on hepatic protein synthesis [l], the ability to mobilize previously stored vitamin A should provide evidence as to whether inhibition of vitamin Atransport protein synthesis (e.g., hepatic RBP) is involved in the effect of PA on tissue distribution of vitamin A. MATERIALS AND METHODS
Animals and diets
Broiler chicks (Peterson X Arbor Acres) hatched from eggs obtained from Keith Smith Farm, Hot Springs, AR, were used. They were housed under controlled conditions of 18-22°C and continuous artificial light, in electrically-heated and ventilated wood-shaving litter floor pens. Birds were wing-banded and individual body weights obtained. Tansy ragwort (Senecio jacobaea) flowers were hand harvested, sun dried and used as the dietary source of PA. The dried material contained approx. 0.18% total PA [8]. In Expt. 1, eight different diets were used, based on a corn-soybean meal basal diet with 23% crude protein (Table I) The distribution of treatments is shown in Table II. Treatment variables were 0 and 5% tansy ragwort (TR), 0 and 250 mg/kg diet copper (anhydrous copper sulfate), and 0 and 25 000 IU/kg diet vitamin A (all-trans retinol palmitate). The birds (12 male l-week-old chicks per treatment) were fed the experimental diets for 28 days, at which time six birds from each treatment were killed and tissues obtained for mineral and vitamin A analyses. The remaining chicks were then given a PA-free diet (corn-soybean meal broiler grower diet) containing a high level of added vitamin A (25 000 IU/kg diet) for 14 days. They were then killed by injection with T-61 (Hoechst-Roussel Agri-Vet) euthanasia solution and tissues obtained. In Expt. 2, 50 female 2-week-old chicks were randomly assigned to two groups of 25. In the first 2-week period, both groups were fed the same broiler grower diet
141
TABLE I COMPOSITION OF THE BROILER STARTER AND GROWER BASAL DIETS (g/l00 g) Ingredients
Starter diet
Grower diet
Yellow corn Fat Soybean meal Meat and bone meal Dehydrated alfalfa meal Dicalcium phosphate (32% Ca, 18% P) Limestone flour Salt Trace mineral (mix-65) Vitamin premix (I-75)b o,L-Methionine
58.35 2.00 32.25 5.00 1.oo 0.42 0.35 0.25 0.05 0.20 0.13
63.52 2.00 27.50 5.00 1.00 0.25 0.13 0.25 0.05 0.20 0.10
a Supplied the following quantities per kg feed: vitamin A, 3300 IU; vitamin D, 1100 IU; vitamin E, 1.1 IU; vitamin K, 0.55 mg; vitamin B-12,0.0055 mg; riboflavin, 3.3 mg; pantothenic acid, 5.5 mg; niacin, 22 mg; choline chloride, 220 mg; folic acid, 0.22 mg; ethoxyquin, 64.43 mg. b Supplied the following quantities per kg feed: calcium, 107.5 mg; manganese, 60 mg; iron, 20 mg; zinc, 28 mg; copper, 2 mg; iodine, 1.2 mg; cobalt, 0.205 mg.
supplemented with 25 000 IU/kg of all-trans retinol palmitate. After this period, the vitamin A supplement was discontinued. One group was fed a basal diet of 95% broiler grower diet and 5% ground corn, and the other group was fed 95% broiler diet and 5% TR. At the end of the first 14-day period, three birds from each group were killed and tissue samples taken. Six additional birds in each group were identified, and blood samples obtained from the wing vein every 4 days for a total of 24 days. After 24 days, these birds were killed and tissue samples taken. The remaining birds in each group were monitored for weight gain over the experimental period. Analytical methods Vitamin A was determined by HPLC based on the method of Bieri et al. [9], Chow and Omaye [IO,111.Copper, zinc and iron were analyzed by AOAC [ 121and selenium by the method of Brown and Watkinson [ 131.Calpain activity was measured according to the methnd of Gopalakrishna and Barsky [14]. Statistics Statistical analyses were performed using the statistical software base SAS (SAS Institute, Inc.). Data were assessed for homogeneity of variance using Analysis of Variance procedure with means compared by Student-Neuman-Keuls (SNK) test at PcO.05; uneven number of replications were analyzed by using the General Linear Models Procedure with SNK test from SAS.
Cu (mg/kg)
25 000 25 000 25 000 25 000
0
0 0
0
vitamin A @U/kg) 1667 F 632 + 1604 + 668 f 1643 * 615 + 1751 2 700 rt
51db 23 37b 33’ 39ab 26 48” 29”
Average weight at 4 wks
54 I3 rt 1f7”b 17.4 i: 0.8’ 52.0 + 1.3b 18.5 rt 1.2” 53 4 f: 1 3”” 18:8 rt 0:9c 51.3 rt 1.6” 19.7 t 1.0”
Average daily gain (g) 04 wks
Means i SE in the same column followed by different superscripts are different (P
