Neurotoxicologyand Teratology,Vol. 13, pp. 147-152. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0892-0362/91 $3.00 + .00
Development of Esterase Activities in the Chicken Before and After Hatching M. F A R A G E - E L A W A R 1
Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061 R e c e i v e d 14 A u g u s t 1989
FARAGE-ELAWAR, M. Developmentof esterase activities in the chicken before and after hatching. NEUROTOXICOL TERATOL 13(2) 147-152, 1991.--The embryonic chick has long been a model for developmental biology and has often been recommended as a model system in developmental toxicology. More recently, several investigators have shown that the chick embryo also provides a good model for identifying the neurotoxic effects of environmental pollutants, especially cholinesterase-inhibiting pesticides. Although numerous studies detail the structural development of chick embryos, few describe embryonic levels of enzyme synthesis and their changes during development. In this study, the development of esterase activity in chick embryos was measured from day 9 of incubation until 46 days after hatching. Brain acetylcholinesterase (AChE) activity was detected on day 9 of incubation at a concentration of 0.364 i~moles/min/g tissue. An increase between AChE activity and age of the embryos was observed. In the liver, the nonspecific cholinesterases (ChE) and carboxylesterase activities during incubation were not different from activities after the chicks had hatched. Plasma ChE and carboxylesterase activities did not change with age after hatching. Brain neuropathy target esterase (NTE) activity was not detected on day 9 of incubation and was extremely low (6.12 nmoles/15 min/mg protein) the next day, but increased rapidly with increasing age. This study demonstrates that chick embryos have developed esterase activities in the brain and liver by day 10 of incubation and again confh'ms that the insensitivity of chick embryos and young chicks to organophosphorus ester-induced delayed neurotoxicity is not due to absence of NTE. In addition, the results provide baseline data for evaluating the response of embryonic and immature chicks to neurotoxicants and teratogens. Chick embryos
Chickens
AChE
ChE
NTE
Carboxylesterase
Brain
Liver
Plasma
terase enzyme activities is limited. The enzyme acetylcholinesterase (ACHE) has been extensively studied and several studies have investigated the development of its activity in the developing chick nervous system (7, 17, 26, 30, 42, 45). The development of nonspecific cholinesterases (CHE) has been studied mostly in rodents and invertebrates (3, 9, 40, 43, 46, 48, 49). Levels of NTE during development have not been determined either for chick embryos or for young chicks. Young chicks, below the age of 55-70 days of age, are considered insensitive to OPIDN (24). However, many studies have shown that exposure of chicks in ovo to certain OP insecticides can lead to ataxia and paralysis at hatching (4, 16, 29, 33, 36). The relationship between the paralytic syndrome seen at hatching and delayed neurotoxicity or paralysis is not yet understood. The purpose of this paper is to investigate the developmental pattern of some esterase activities in the chicken during development both before and after hatching. The esterase activities to be measured were selected for their relevance to pesticide neurotoxicity.
TOXICOLOGICAL studies often depend on animal models, particularly when, as with neurotoxic or teratogenic agents, consequences of exposure may be irreversible. The use of the chick embryo has been popular because the eggs are inexpensive and the embryos easy to handle. Moreover, the chick embryo has been used extensively in developmental biology (32, 38, 47) so its embryology is well defined. Capitalizing on this extensive data base, toxicologists have suggested the chick embryo be used in development toxicity screens (20, 21, 41) and have used both chick embryos and young chicks to test a wide range of chemicals (6, 15, 18, 27, 29). In toxicity studies, chemicals have been injected into eggs at various times during incubation, ranging from day 0 to day 15 of incubation (16, 27, 29, 33). Adult chickens are also used in toxicity testing. The hen is the accepted animal model to determine if insecticidal organophosphorus esters (OPs) are capable of causing organophosphate-induced delayed neuropathy (OPIDN) (1,31). The clinical signs of poisoning in the hen most closely resemble those seen in humans, whereas the diverse effects of OPs on the central as well as the peripheral nervous system are not all duplicated in rodent models (2,44). Although the chick embryo and the adult chicken are widely used in testing, information on the sequential development of es-
METHOD
Animals Fertile chicken eggs, hybrids between Peterson strain roosters and Hubbard hens, were obtained from Rocco Chicken Farms,
~Present address: The Procter and Gamble Co., Ivorydale Technical Center, 5299 Spring Grove Ave., Cincinnati, OH 45217.
147
148
FARAGE-ELAWAR
40-
----D---
Body welght
2010, 1680 •
30.
~ |
1350' ~ .
+
:
~ +I
.
.
.
7
.
.
.
.
13 15 Days of Incubation
.
.
.
21
.
.
0
10
20 30 Days after Hatching
40
50
FIG. 1. Body weight of chickens versus days of incubation and days after hatching. Results are expressed in g ___SE, n = 6 for unhatched and 12 for hatched chicks.
for the determination of ACHE. Plasma and liver carboxylesterase were determined as described by Levine and Murphy (28). For the NTE assay, 1% brain homogenate was prepared in ice-cold Tris-EDTA buffer and measured as described by Sprague et al. (39). The protein content of tissue samples was determined by the dye-binding method of Bradford (5). In addition, the body, brain and liver weights were recorded on each day of the biochemical assays.
Harrisonburg, VA. They were incubated at 38°C and candled on days 4 and 14 of incubation to discard infertile and dead eggs. After hatching, chicks were fed a corn-soybean diet, without antibiotics or other additives, designed for chicks at Virginia Tech. Both food and water were provided ad lib throughout the study. Chicks were kept in thermostatically controlled starter batteries for 2.5 weeks, then moved to cages containing 4-6 birds each.
Esterase Activity Measurements Statistics
Activities of brain and liver enzymes were determined at different times during incubation and after hatching; plasma activities were only determined after hatching. Brains and livers were quickly removed and put on ice and all esterase activities were determined on the day of sacrifice. Acetylthiocholine (purchased from Sigma, St. Louis, MO) was used as the substrate for both AChE and nonspecific ChE. Acetylthiocholine can be used as the substrate to measure chicken's ChE (34). et-Naphthyl acetate (purchased from Sigma, St. Louis, MO) was used as the substrate to measure carboxylesterase. Activities of plasma and liver cholinesterases (ChE) and brain acetylcholinesterase (ACHE) were measured using the method of Ellman et al. (12). The tissues were homogenized in cold phosphate buffer (pH 8.0, 0.1 M). A 1% brain homogenate was used
Analysis of variance was used to compare activities during incubation and after hatching, using the statistical package "Epistar" (written by T. L. Gustafson, 1984). A p-value of less than 0.05 (two-sided) was considered statistically significant. RESULTS As expected, body, liver and brain weights increased steadily with increase in age (Fig. 1). The body weight on day 9 of incubation was 1.84---0.007 g ( m e a n - S E , N = 6 ) and reached 1567---252 g ( m e a n _ S E , N = 12) on day 46 after hatching (Fig. 1). The liver was extremely small on day 10 of incubation (0.035±0.004 g, m e a n - S E , n = 6 ) ; its size increased to
brain weight liver weight
• 1.0 -
~ ~
40 "
0.8
30
0.6
~ ~
~° 0.4 10
0.2
0.0
,
7
9
11
13
15
17
Days of Incubation
19
21
•
0
!
0
I0
!
2'0 310 Days alter Halchlng
40
50
FIG. 2. Brain and liver weights versus days of incubation and days after hatching. Results are expressed in g __.SE, n = 6 for unhatched and 12 for hatched chicks.
DEVELOPMENT OF ESTERASES IN CHICKENS
149
14.
Brain AChE 12t
Liver ChE
10.
u ~
1.4
1.2 ¢ l.U
1.0
0.8 0.6' 0.4' 0.2' 0.0
Days after Hatching
FIG. 6. Plasma carboxylesterase activity in chickens versus days after hatching. Results are expressed as ~moles ct-naphthyl acetate hydrolyzed/ min/ml _+SE, n = 6 for unhatched and 12 for hatched chicks.
DEVELOPMENT OF ESTERASES IN CHICKENS
151
800 -
700
600
o
~.
500
w
o
E
300
200 ,
100
O
11
13
15
17
19
2
0
10
2°
30
40
Hatch
Days o! Incubation
Days after Hatching
FIG. 7. Brain neuropathy target esterase activity in chickens versus days of incubation and days after hatching. Results are expressed as nmoles phenylvalerate hydrolyzed/15 mirgmg protein- SE, n = 6 for unhatched and 12 for hatched chicks.
viously (10, 11, 14, 16, 25). One interesting observation was that the activity of liver carboxylesterase in the 10-day-old embryo was similar to that of the adult chicken. Liver carboxylesterase is a very important enzyme that detoxifies many xenobiotics and usually serves as a nonspecific binding site for organophosphate and carbamate insecticides (8,10). Therefore, the high activity of the enzyme early during embryogenesis may provide protection to the embryo from early exposure to foreign materials. The physiological and biochemical functions of the brain enzyme known as neuropathy target esterase (NTE) are still unknown. However, this enzyme is of great importance in the delayed neurotoxicity associated with organophosphorus esters (OPs). Johnson (22,23) considers the phosphorylation and aging of >75% of this enzyme in adult hens to be a prerequisite for the delayed neuropathy. The activity of NTE was not detectable in the chick embryo before day 10 of incubation. Like the brain ACHE, NTE activities increased with the age of the embryo, mainly from day 10 of incubation until hatching. The most significant increase (5-fold) was noticeable between day 16 of incubation and hatching. A similar pattern of increase in the brain NTE was seen in mallard embryos (19). Many studies using pesticides have demonstrated the inhibition of esterases in chick embryos (16, 19, 33). Young chicks are nonetheless relatively
insensitive to OPIDN, and the age for the development of the paralytic symptoms lies between 55-70 days of age (24). However, chick embryos and very young chicks have been reported to show paralysis and alterations in muscle morphology after in ovo exposure to certain OPs (4, 16, 29, 33, 36). Even though these studies showed that in ovo exposure of chicks to OPs caused an ataxic syndrome that bears some resemblance to OPIDN, the relationship between the paralytic syndrome seen in chicks at hatching to OPIDN is still not well understood. It is accepted that the period of organogenesis of the chick embryo ends on day 10 of incubation when all the major structures except feathers have differentiated. This study is important because it demonstrates the presence of enzyme activities in the chick embryos before day 10 of incubation. In addition, it demonstrates that the chick embryo could be sensitive to chemicals that inhibit esterases in the young or adult chickens. Therefore, the chick embryo could be as or more susceptible to some pesticide neurotoxicity than originally thought. ACKNOWLEDGEMENTS The author would like to express her appreciation to Drs. M. Ehrich and B. Magnus Francis for the critical review of this manuscript and their valuable advice.
REFERENCES
1. Anonymous. Health effects testing guidelines for neurotoxicity. Fed. Regist. V.50:39458-39470; 1985. 2. Baron, R. L. Delayed neurotoxicity and other consequences of orga-
nophosphate esters. Annu. Rev. Entomol. 26:29--48; 1981. 3. Bhattacharjee, J.; Sanyal, S. Developmental changes of esterases in the retina of the mouse. Histochemical study. Histochemie 46: 53-
152
60; 1975. 4. Bishoff, A. Tri-ortho-cresyl-phosphate neurotoxicity. In: Roizin, L.; Shiraki, H.; Grecevic, N., eds. Neurotoxicology. New York: Raven Press; 1977:431-441. 5. Bradford, M. A. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72:248-254; 1976. 6. Bursian, S.; Flaga, C.; Ringer, R. The injection of the delayed neurotoxic tri-o-tolyl phosphate into embryonating chicken eggs and its effects on subsequent chick development. J. Toxicol. Environ. Health 10:101-109; 1982. 7. Burt, A. M. Acetylcholinesterase and choline acetyl-transferase activity in the developing chick spinal cord. J. Exp. Zool. 169:107112; 1968. 8. Cohen, S. D.; Murphy, S. D. Inactivation of malaoxon by mouse liver. Proc. Soc. Exp. Biol. Med. 139:1385-1389; 1972. 9. Ecobichon, D. J.; Dykeman, R. W.; Hansell, M. M. The development of hepatic drug-metabolizing enzymes in perinatal guinea-pigs: A biochemical and morphological study. Can. J. Biochem. 56(7): 738-745; 1978. 10. Ehtich, M.; Driscoll, C.; Gross, W. B. Effect of dietary exposure to aflatoxin B1 on resistance of young chickens to organophosphate pesticide challenge. Avian Dis. 29:715-720; 1985. 11. Ehrich, M.; Jortner, B. S.; Gross, W. B. Dose-related beneficial and adverse effects of dietary corticosterone on organophosphorus-induced delayed neuropathy in chickens. Toxicol. Appl. Pharmacol. 83:250-260; 1986. 12. Ellman, G. L.; Courtney, K.; Featherstone, R. A new and rapid determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88-95; 1961. 13. Ensminger, M. E. Poultry sciences. 2nd ed. Danville, IL:: The Interstate Printers and Publishers Inc.; 1980. 14. Farage-Elawar, M. Effects of in ovo injection of carbamates on chick embryo hatchability, esterase enzyme activities and locomotion of chicks. J. Appl. Toxicol. 10(3):197-201; 1990. 15. Farage-Elawar, M.; Francis, B. M. Acute and delayed effects of fenthion in young chicks. J. Toxicol. Environ. Health 21:455-469; 1987. 16. Farage-Elawar, M.; Francis, B. M. Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholineterase, and neurotoxic esterase in young chicks after in ovo exposure. Toxicology 49:253261; 1988. 17. Giacobini, G.; Marchisio, C.; Giacobini, E.; Koslow, H. Developmental changes of cholinesterases and monoamine oxidase in chick embryo spinal and sympathetic ganglia. J. Neurochem. 17:11771185; 1970. 18. Hoffman, D.; Eastin, W. C., Jr. Effects of malathion, diazinon and parathion on mallard embryo development and cholinesterase activity. Environ. Res. 26:472-485; 1981. 19. Hoffman, D.; Sileo, L. Neurotoxic and teratogenic effects of an organophosphorus insecticide (phenyl phosphonothioic acid-O-methylO-[4-nitrophenyl]ester) on mallard development. Toxicol. Appl. Pharmacol. 73:284-294; 1984. 20. Jelinek, R. The chick embryotoxicity screening test (CHEST). In: Neubert, D.; Merker, H.-J.; Kwasigroch, T. E., eds. Methods in prenatal toxicology. Stuttgart: Thieme; 1977:381-386. 21. Jelinek, R. Embryotoxicity assay on morphogenic systems. In: Benesova, O.; Rychter, Z.; Jelinek, R., eds. Evaluation of embryotoxicity, mutagenicity and carcinogenicity risks in new drugs. Praha: Univerzita Karlova; 1979:195-205. 22. Johnson, M. K. A phosphorylation site in brain and the delayed neurotoxic effects of some organophosphorus compounds. Biochem. J. 111:487--495; 1969. 23. Johnson, M. K. Delayed neurotoxicity induced by organophosphorus compounds--areas of understanding and ignorance. In: Holmstedt, B.; Lauwerys, R.; Mercier, M.; Roberfroid, R., eds. Mechanisms of toxicity and hazard evaluation. Elsevier: North Holland Biomedical Press; 1980:27-38. 24. Johnson, M. K.; Barnes, J. Age and sensitivity of chicks to delayed neurotoxic effects of some organophosphorus compounds. Biochem. Pharmacol. 19:3045-3047; 1970. 25. Jortner, B. S.; Ehrich, M. Neuropathological effects of phenyl sali-
FARAGE-ELAWAR
genin phosphate in chickens. Neurotoxicology 8:303-314; 1987. 26. Kato, A. C.; Vrachliotis, A.; Fulpius, B.; Dunant, Y. Molecular forms of acetylcholinesterase in chick muscle and ciliary ganglion: Embryonic tissues and cultured cells. Dev. Biol. 76:222-228; 1980. 27. Khera, K. S.; Lyon, D. A. Chick and duck embryos in the evaluation of pesticide toxicity. Toxicol. Appl. Pharmacol. 13:1-15; 1968. 28. Levine, B. S.; Murphy, S. D. Esterase inhibition and reactivation in relation to piperonyl butoxide-phosphorothionate interactions. Toxicol. Appl. Pharmacol. 40:379-391; 1977. 29. McLaughlin, J., Jr.; Marliac, J. P.; Verrett, M. J.; Mutchler, M. K.; Fitzhugh, O. G. The injection of chemicals into the yolk sac of fertile eggs prior to incubation as a toxicity test. Toxicol. Appl. Pharmacol. 5:760-771; 1963. 30. Marchand, A.; Chapouthier, G.; Massoulie, J. Developmental aspects of acetylcholinesterase activity in chick brain. FEBS Lett. 78: 233-236; 1977. 31. Metcalf, R. L. Historical perspective of organophosphorus ester-induced delayed neurotoxicity. Neurotoxicology 3(4):269-284; 1982. 32. Newman, S. A. Developing systems: lineage and pattern in the development vertebrate limb. Trends Genet. 4:329-332; 1988. 33. Norton, S.; Sheets, L. Neuropathy in the chick from embryonic exposure to organophosphorus compounds. Neurotoxicology 4:137142; 1983. 34. Picketing, C. E.; Picketing, R. G. The interference by erythrocyte "acetylthiocholinesterase" in the estimation of blood cholinesterase activity of the chicken. Toxicol. Pharmacol. 39:229-237; 1977. 35. Romanoff, A. L. A quantitative analysis of prenatal development. In: Biochemistry of the avian embryo. New York: John Wiley & Sons; 1967:69-79. 36. Sheets, L.; Norton, S. Morphological alterations in leg muscles of chicks treated with tri-ortho-cresyl phosphate in ovo. Toxicol. Appl. Pharmacol. 79:39--46; 1985. 37. Shepard, T. H. In: Catalog of teratogenic agents. 3rd ed. Baltimore: The Johns Hopkins University Press; 1980. 38. Smith, S. M.; Pang, K.; Sundin, O.; Wedden, S. E.; Thaller, C.; Eichele, G. Molecular approaches to vertebrate limb morphogenesis. Development (Suppl.):121-131; 1989. 39. Sprague, G. L.; Sandvick, L. L.; Brookins-Hendricks, M. J.; Bickford, A. A. Neurotoxicity of two organophosphoms ester flame retardants in hens. J. Toxicol. Environ. Health 8:507-518; 1981. 40. Sterri, S. H.; Berge, G.; Fonnum, F. Esterase activities and soman toxicity in developing rat. Acta Pharmacol. Toxicol. 57:136-140; 1985. 41. Summerbell, D.; Hornbruch, A. The chick embryo: A standard against which to judge in vitro systems. In: Neubert, D.; Merker, H-J., eds. Culture techniques. Bedim Walter de Gruyfer & Co.; 1981:529-538. 42. Taylor, P.; Rieger, F.; Greene, L. A. Development of multiple molecular forms of acetylcholinesterase in chick paravertebral sympathetic ganglia: an in vivo and in vitro study. Brain Res. 182:383-396; 1980. 43. Uddin, D. E.; Titchener, E. B. Rat liver carboxylesterase: influence of age and sex on activity and kinetics of ester hydrolysis. Comp. Biochem. Physiol. 26: 985-990; 1968. 44. Veronesi, B. A rodent model of organophosphorus-induced delayed neuropathy: Distribution of central (spinal cord) and peripheral nerve damage. Neuropathol. Appl. Neurobiol. 10:357-368; 1984. 45. Villafruela, M. J.; Barat, A.; Manrique, E.; Villa, S.; Ramirez, G. Molecular forms of acetylcholinesterase in the developing chick visual system. Dev. Neurosci. 4:25-36; 1981. 46. Von Deimling, O.; Grossarth, C. Esterase. IV. Postnatal development of the unspecific carboxylesterase in the kidney of the mouse. Histochimie 30:122-130; 1972. 47. Waddington, C. H. Induction by the endoderm in birds. Wilhelm Roux' Arch Entwicldungsmechanik Organismen 128:502; 1933. 48. Watanabe, M.; Takebe, S.; Kim, D. H.; Arakawa, R.; Kamimura, K.; Kobashi, K. Oxo-type organophosphate-resistant acetylcholinesterase from organophosphate-unsusceptible culex-tritaeniorhynchus. Chem. Pharm. Bull. 36:312-315; 1988. 49. Wehler, E.; Grossarth, C.; Von Deimling, O. Esterase. VI. Influence of testosterone on unspecific carboxylesterase in the mouse kidney during postnatal development. Histochimie 33:47-52; 1973.
0892-0362/91 $3.00 + .00
Development of Esterase Activities in the Chicken Before and After Hatching M. F A R A G E - E L A W A R 1
Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061 R e c e i v e d 14 A u g u s t 1989
FARAGE-ELAWAR, M. Developmentof esterase activities in the chicken before and after hatching. NEUROTOXICOL TERATOL 13(2) 147-152, 1991.--The embryonic chick has long been a model for developmental biology and has often been recommended as a model system in developmental toxicology. More recently, several investigators have shown that the chick embryo also provides a good model for identifying the neurotoxic effects of environmental pollutants, especially cholinesterase-inhibiting pesticides. Although numerous studies detail the structural development of chick embryos, few describe embryonic levels of enzyme synthesis and their changes during development. In this study, the development of esterase activity in chick embryos was measured from day 9 of incubation until 46 days after hatching. Brain acetylcholinesterase (AChE) activity was detected on day 9 of incubation at a concentration of 0.364 i~moles/min/g tissue. An increase between AChE activity and age of the embryos was observed. In the liver, the nonspecific cholinesterases (ChE) and carboxylesterase activities during incubation were not different from activities after the chicks had hatched. Plasma ChE and carboxylesterase activities did not change with age after hatching. Brain neuropathy target esterase (NTE) activity was not detected on day 9 of incubation and was extremely low (6.12 nmoles/15 min/mg protein) the next day, but increased rapidly with increasing age. This study demonstrates that chick embryos have developed esterase activities in the brain and liver by day 10 of incubation and again confh'ms that the insensitivity of chick embryos and young chicks to organophosphorus ester-induced delayed neurotoxicity is not due to absence of NTE. In addition, the results provide baseline data for evaluating the response of embryonic and immature chicks to neurotoxicants and teratogens. Chick embryos
Chickens
AChE
ChE
NTE
Carboxylesterase
Brain
Liver
Plasma
terase enzyme activities is limited. The enzyme acetylcholinesterase (ACHE) has been extensively studied and several studies have investigated the development of its activity in the developing chick nervous system (7, 17, 26, 30, 42, 45). The development of nonspecific cholinesterases (CHE) has been studied mostly in rodents and invertebrates (3, 9, 40, 43, 46, 48, 49). Levels of NTE during development have not been determined either for chick embryos or for young chicks. Young chicks, below the age of 55-70 days of age, are considered insensitive to OPIDN (24). However, many studies have shown that exposure of chicks in ovo to certain OP insecticides can lead to ataxia and paralysis at hatching (4, 16, 29, 33, 36). The relationship between the paralytic syndrome seen at hatching and delayed neurotoxicity or paralysis is not yet understood. The purpose of this paper is to investigate the developmental pattern of some esterase activities in the chicken during development both before and after hatching. The esterase activities to be measured were selected for their relevance to pesticide neurotoxicity.
TOXICOLOGICAL studies often depend on animal models, particularly when, as with neurotoxic or teratogenic agents, consequences of exposure may be irreversible. The use of the chick embryo has been popular because the eggs are inexpensive and the embryos easy to handle. Moreover, the chick embryo has been used extensively in developmental biology (32, 38, 47) so its embryology is well defined. Capitalizing on this extensive data base, toxicologists have suggested the chick embryo be used in development toxicity screens (20, 21, 41) and have used both chick embryos and young chicks to test a wide range of chemicals (6, 15, 18, 27, 29). In toxicity studies, chemicals have been injected into eggs at various times during incubation, ranging from day 0 to day 15 of incubation (16, 27, 29, 33). Adult chickens are also used in toxicity testing. The hen is the accepted animal model to determine if insecticidal organophosphorus esters (OPs) are capable of causing organophosphate-induced delayed neuropathy (OPIDN) (1,31). The clinical signs of poisoning in the hen most closely resemble those seen in humans, whereas the diverse effects of OPs on the central as well as the peripheral nervous system are not all duplicated in rodent models (2,44). Although the chick embryo and the adult chicken are widely used in testing, information on the sequential development of es-
METHOD
Animals Fertile chicken eggs, hybrids between Peterson strain roosters and Hubbard hens, were obtained from Rocco Chicken Farms,
~Present address: The Procter and Gamble Co., Ivorydale Technical Center, 5299 Spring Grove Ave., Cincinnati, OH 45217.
147
148
FARAGE-ELAWAR
40-
----D---
Body welght
2010, 1680 •
30.
~ |
1350' ~ .
+
:
~ +I
.
.
.
7
.
.
.
.
13 15 Days of Incubation
.
.
.
21
.
.
0
10
20 30 Days after Hatching
40
50
FIG. 1. Body weight of chickens versus days of incubation and days after hatching. Results are expressed in g ___SE, n = 6 for unhatched and 12 for hatched chicks.
for the determination of ACHE. Plasma and liver carboxylesterase were determined as described by Levine and Murphy (28). For the NTE assay, 1% brain homogenate was prepared in ice-cold Tris-EDTA buffer and measured as described by Sprague et al. (39). The protein content of tissue samples was determined by the dye-binding method of Bradford (5). In addition, the body, brain and liver weights were recorded on each day of the biochemical assays.
Harrisonburg, VA. They were incubated at 38°C and candled on days 4 and 14 of incubation to discard infertile and dead eggs. After hatching, chicks were fed a corn-soybean diet, without antibiotics or other additives, designed for chicks at Virginia Tech. Both food and water were provided ad lib throughout the study. Chicks were kept in thermostatically controlled starter batteries for 2.5 weeks, then moved to cages containing 4-6 birds each.
Esterase Activity Measurements Statistics
Activities of brain and liver enzymes were determined at different times during incubation and after hatching; plasma activities were only determined after hatching. Brains and livers were quickly removed and put on ice and all esterase activities were determined on the day of sacrifice. Acetylthiocholine (purchased from Sigma, St. Louis, MO) was used as the substrate for both AChE and nonspecific ChE. Acetylthiocholine can be used as the substrate to measure chicken's ChE (34). et-Naphthyl acetate (purchased from Sigma, St. Louis, MO) was used as the substrate to measure carboxylesterase. Activities of plasma and liver cholinesterases (ChE) and brain acetylcholinesterase (ACHE) were measured using the method of Ellman et al. (12). The tissues were homogenized in cold phosphate buffer (pH 8.0, 0.1 M). A 1% brain homogenate was used
Analysis of variance was used to compare activities during incubation and after hatching, using the statistical package "Epistar" (written by T. L. Gustafson, 1984). A p-value of less than 0.05 (two-sided) was considered statistically significant. RESULTS As expected, body, liver and brain weights increased steadily with increase in age (Fig. 1). The body weight on day 9 of incubation was 1.84---0.007 g ( m e a n - S E , N = 6 ) and reached 1567---252 g ( m e a n _ S E , N = 12) on day 46 after hatching (Fig. 1). The liver was extremely small on day 10 of incubation (0.035±0.004 g, m e a n - S E , n = 6 ) ; its size increased to
brain weight liver weight
• 1.0 -
~ ~
40 "
0.8
30
0.6
~ ~
~° 0.4 10
0.2
0.0
,
7
9
11
13
15
17
Days of Incubation
19
21
•
0
!
0
I0
!
2'0 310 Days alter Halchlng
40
50
FIG. 2. Brain and liver weights versus days of incubation and days after hatching. Results are expressed in g __.SE, n = 6 for unhatched and 12 for hatched chicks.
DEVELOPMENT OF ESTERASES IN CHICKENS
149
14.
Brain AChE 12t
Liver ChE
10.
u ~
1.4
1.2 ¢ l.U
1.0
0.8 0.6' 0.4' 0.2' 0.0
Days after Hatching
FIG. 6. Plasma carboxylesterase activity in chickens versus days after hatching. Results are expressed as ~moles ct-naphthyl acetate hydrolyzed/ min/ml _+SE, n = 6 for unhatched and 12 for hatched chicks.
DEVELOPMENT OF ESTERASES IN CHICKENS
151
800 -
700
600
o
~.
500
w
o
E
300
200 ,
100
O
11
13
15
17
19
2
0
10
2°
30
40
Hatch
Days o! Incubation
Days after Hatching
FIG. 7. Brain neuropathy target esterase activity in chickens versus days of incubation and days after hatching. Results are expressed as nmoles phenylvalerate hydrolyzed/15 mirgmg protein- SE, n = 6 for unhatched and 12 for hatched chicks.
viously (10, 11, 14, 16, 25). One interesting observation was that the activity of liver carboxylesterase in the 10-day-old embryo was similar to that of the adult chicken. Liver carboxylesterase is a very important enzyme that detoxifies many xenobiotics and usually serves as a nonspecific binding site for organophosphate and carbamate insecticides (8,10). Therefore, the high activity of the enzyme early during embryogenesis may provide protection to the embryo from early exposure to foreign materials. The physiological and biochemical functions of the brain enzyme known as neuropathy target esterase (NTE) are still unknown. However, this enzyme is of great importance in the delayed neurotoxicity associated with organophosphorus esters (OPs). Johnson (22,23) considers the phosphorylation and aging of >75% of this enzyme in adult hens to be a prerequisite for the delayed neuropathy. The activity of NTE was not detectable in the chick embryo before day 10 of incubation. Like the brain ACHE, NTE activities increased with the age of the embryo, mainly from day 10 of incubation until hatching. The most significant increase (5-fold) was noticeable between day 16 of incubation and hatching. A similar pattern of increase in the brain NTE was seen in mallard embryos (19). Many studies using pesticides have demonstrated the inhibition of esterases in chick embryos (16, 19, 33). Young chicks are nonetheless relatively
insensitive to OPIDN, and the age for the development of the paralytic symptoms lies between 55-70 days of age (24). However, chick embryos and very young chicks have been reported to show paralysis and alterations in muscle morphology after in ovo exposure to certain OPs (4, 16, 29, 33, 36). Even though these studies showed that in ovo exposure of chicks to OPs caused an ataxic syndrome that bears some resemblance to OPIDN, the relationship between the paralytic syndrome seen in chicks at hatching to OPIDN is still not well understood. It is accepted that the period of organogenesis of the chick embryo ends on day 10 of incubation when all the major structures except feathers have differentiated. This study is important because it demonstrates the presence of enzyme activities in the chick embryos before day 10 of incubation. In addition, it demonstrates that the chick embryo could be sensitive to chemicals that inhibit esterases in the young or adult chickens. Therefore, the chick embryo could be as or more susceptible to some pesticide neurotoxicity than originally thought. ACKNOWLEDGEMENTS The author would like to express her appreciation to Drs. M. Ehrich and B. Magnus Francis for the critical review of this manuscript and their valuable advice.
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