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Tissue Residues and Ultrastructural Changes Induced by DDT in Chickens
Tissue Residues and Ultrastructural Changes Induced by DDT in Chickens MARIA DE LOS REYES 1 and EMILIO C. MORA Department of Poultry Science, Alabama Agricultural Experiment Station, Auburn University, Auburn, Alabama 36830 (Received for publication December 13, 1978)
1979 Poultry Science 58:1183-1191
INTRODUCTION T h e chlorinated h y d r o c a r b o n (organochlorine) insecticides have been used extensively in t h e c o n t r o l of insect pests for over 30 years. A m o n g these, l , l , l - t r i c h l o r o - 2 , 2 - b i s (p-chlorop h e n y l ) e t h a n e (DDT) is one of t h e best k n o w n and cheapest of t h e s y n t h e t i c insecticides. T h e use of t h e pesticide D D T has been b a n n e d , restricted, or phased o u t in developed countries like Canada, Scandinavian nations, and t h e United States because of alleged effects on non-target invertebrates and vertebrates, including beneficial insects and crustacae, birds and fishes, especially fish-eating raptorial birds, and its alleged effect on man and o t h e r m a m m a l s . T h e a m o u n t of research published since t h e discovery of D D T has been extensive. Toxicological effects have been studied in different species and storage and residue levels of D D T have been linked directly t o m e t a b o l i c studies. T h e pathological changes have been associated with exposure t o massive doses or t o m a n y relatively small doses of t h e pesticide, b u t t h e majority of t h e studies have been d o n e with light m i c r o s c o p y . T h e ultrastructural studies of
'Present address: Copiah-Lincoln Junior College, Wesson, MS 39191.
t h e histopathogenicity of D D T in chicken tissues were u n d e r t a k e n t o further u n d e r s t a n d t h e action of this pesticide in d o m e s t i c birds. Residue levels in different tissues were determ i n e d t o correlate t h e relationship b e t w e e n t h e a c c u m u l a t i o n of D D T in tissues and t h e p a t h o logical changes observed.
MATERIALS AND METHODS Twenty-four crossbred broiler chicks, rand o m sexed, 2 weeks of age, were used in this investigation. T h e chicks were caged in groups of six and watered and fed a commercial broiler ration ad libitum. T h e L D 5 0 dose value of 50% commercially available D D T for this e x p e r i m e n t was determ i n e d t o be 300 m g / k g of b o d y weight (St. Omer, 1 9 7 0 ; K o n s t and Plummer, 1 9 4 6 ; Draize et ah, 1944). Twelve chickens were used for controls and 12 chickens were treated with D D T . A daily dose of 100 m g / k g of b o d y weight was given orally t h r e e times p e r day for 10 days. Birds were weighed every 5 days, and t h e LD S 0 dose was adjusted accordingly. T h e tissues for t h e residue level analysis and for t h e histopathological studies were o b t a i n e d after signs of i n t o x i c a t i o n were manifested. Morbid animals were killed by cervical dislocation and specimens from liver, pancreas, heart,
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ABSTRACT Chickens were given DDT in an oral dose of 100 mg/kg of body weight 3 times a day for 10 days. Specimens of liver, pancreas, heart, skeletal muscle, brain, and sciatic nerve were obtained from morbid chicks for analysis of residue levels of DDT and metabolites, DDD and DDE, and for electron microscopic examination. Fat and sciatic nerve tissue contained the largest amount of DDT and metabolite residues, and skeletal muscle and brain tissue contained the smallest amount of residues. Only moderate amounts of detectable residues were present in the liver, pancreas, and heart muscle. The liver cells underwent more changes than any other organ and exhibited alterations in individual parenchymal cells as well as areas of tissue disorganization. Proliferation of bile duct epithelia and smooth endoplasmic reticulum was found. Disorganization of pancreatic endoplasmic reticulum and a decrease of zymogen granules were found. Displacement of sarcotubules and banding pattern in myocardium occurred. Alterations of the myelin sheath, increased amount of vesicles, and absence in some areas of the nucleated sheath of Schwann were the major changes observed in the sciatic nerve.
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RESULTS
Signs. Chickens in the control group had an initial weight range of 839.9 to 1,189.5 g with an average weight of 994.6 g. All birds gained weight and the final weight range was from 1,157.7 to 1,570.8 g with an average weight of 1,356.7 g. The DDT-treated group had an initial weight range of 776.3 to 1,080.5 g with an average weight of 935.9 g and a final average weight of 856.1 g. Initial signs of toxicity included barely perceptible tremors of the feathers and a curling of the toes. The toe curling increased in severity until the birds walked on the sides of their feet. At this stage the birds were frequently squatting but were able to rise voluntarily. The birds were also walking in an exaggerated and ataxic manner. Tremors increased to the point at which the birds could no longer rise. In the most severe cases, the muscles of the neck were under extreme tension and the neck was twisted into an S-shaped curve with the head in a fixed position over one wing. The birds became incapacitated to the point of being unable to eat or drink and the tremors became very severe and continuous, progressing into tonic convulsions. The convulsions were intermittent at first but later became continuous, generalized, and bilateral in nature. When convulsions were most severe, there were periods of post-seizure depression resembling exhaustion or loss of consciousness, but addi-
tional convulsions could be evoked by handling or touching a bird. The legs of the animals became cold and the body temperature was 1 to 2 below the normal average temperature of 42.6 C for controls. Death usually occurred within a few hours, but if the bird was handled or in any way excited, death occurred sooner, often within minutes. When the DDT treatment (10 days) was discontinued, all clinical signs in surviving birds gradually disappeared and after a period of 4 to 6 days the birds appeared normal when compared to the control group. The chickens with the most severe tremors did not have enough abdominal fat for the extraction procedure (1.0 g). Analysis of Residue Levels. The data obtained by quantitative analysis of the residues of DDT and metabolites, DDD and DDE, in the tissues of treated and control birds are summarized in Table 1. Histopathology. All electron micrographs are of specimens obtained from DDT-treated chicks. Analyses of control chick tissues determined that the changes were not due to fixation or processing artifacts. Liver. Large non-membrane bound vacuoles of variable sizes were found throughout the parenchymal cells and some had a flocculent material of low electron density (Fig. 1) and others appeared empty (Fig. 2). A mild to moderate proliferation of smooth endoplasmic reticulum was observed, with the greatest concentration being central or perinuclear (Fig. 3). Ribosomes could be identified around the cisternal membranes but there was no continuous ribosomal lining. The cisternae of smooth endoplasmic reticulum were not uniform in size or shape, and contained an electron dense material. An increase in lysosomes was found, mainly in areas close to bile ducts. Glycogen granules were not usually seen in treated animals. In the majority of parenchymal cells, the nucleoli contained a formation of granular and fibrillar components. In the treated animals, cells that appeared to be hepatocytes contained nuclei not characteristic of this cell type. The nuclei were pleomorphic with irregular outlines, more typical of connective tissue cells than of parenchymal cells. In some, the nuclear membranes appeared to be disrupted. In areas with more damage, it became increasingly difficult to differentiate between parenchymal and connective tissue cells, and positive identification was not always possible. A large number of
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skeletal muscle, brain, and sciatic nerve were taken. Samples of corresponding tissues from control animals were also obtained. Determination of Chlorinated Pesticides in Tissues. The determination of the levels of DDT residue and its metabolites, DDD and DDE, in the liver, pancreas, heart, skeletal muscle, brain, abdominal fat, and sciatic nerve of the treated chickens was carried out at the Alabama Pesticide Residue Laboratory, Division of Alabama Department of Agriculture and Industries, Auburn, Alabama, using analytical procedures recommended in Pesticide Analytical Manual (USDA, HEW, FDA, 1971). Electron Microscopy Techniques. For electron microscopy studies, specimens of liver, pancreas, heart, skeletal muscle, brain, and sciatic nerve were fixed in 3% glutaraldehyde in phosphate buffer, pH 7.3 and 1% osmium tetroxide (Millonig, 1961). The specimens were processed according to Luft (1961) and Reynolds (1963).
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CHANGES INDUCED BY DDT TABLE l.Afean DDT and metabolic residues in tissues of morbid chickens Tissue
DDE
DDD
DDT
Total
Liver Control
52.54
137.45
81.96 No significant residues
271.95 a
Pancreas Control
52.09
75.89
189.49 No significant residues
317.47
Heart Control
41.48
40.84 148.34 No significant residues
230.66
Brain Control Sciatic nerve Control Fat Control
6.82
5.70
122.92 .19
135.44 .19
26.85
17.02
77.51 No significant residues
121.38
530.03
96.31
2,451.59 No significant residues
3,077.93
1,580.03 1.87
238.61
7,075.36 8.20
8,894.00 10.07
Results presented as parts per million, wet weight.
mitochondria of fairly constant size, shape, and intensity of staining was a main characteristic of the control group. In the treated animals, the number of mitochondria diminished. The majority of the mitochondria lacked the double layered membrane and the cristae were not visible. Only an amorphous matrix material was observed. The proliferation of bile duct canaliculi epithelium into partial or complete canaliculi was evident. In the control group, bile canaliculi were occasionally seen but in the treated birds, canaliculi were found frequently and often enlarged. Some canaliculi were composed of three cells joined to form a lumen. The apical portions of the cells terminated in microvilli, and the zonula occludens were seen close to canaliculi. Throughout these areas some degenerative changes of the cytoplasm were often seen. Pancreas. The nuclei of the acinar cells were often distorted and condensed (Fig. 4). In the DDT-treated specimens the endoplasmic reticulum was regularly organized around the nucleus (Fig. 5) except for damaged areas which had focal disorganization of the endoplasmic reticulum and few mitochondria (Fig. 6). Throughout the cytoplasm the cisternae of the rough endoplasmic reticulum were dilated to give a more circular than laminar appearance.
No other significant alterations of rough endoplasmic reticulum were noted. The number of zymogen granules present was much less in the treated animals than in the control group. The lumen formed by several acinar cells appeared to be wider (Fig. 6) and the free surfaces of the cells had few microvilli. Cytoplasmic degeneration was occasionally noted in areas where the rough endoplasmic reticulum was distorted. Heart. Mitochondria of normal cardiac tissue had excellent staining qualities with cristae sharply defined. In the treated chickens, some mitochondria were intact with welldefined membrane and cristae (Fig. 8) but most mitochondria had few cristae against a background of extremely low electron density. Nearby mitochondria contained only the amorphous matrix material. In many mitochondria the membranes were either damaged or entirely lost (Fig. 7 and 8). In the treated group, wide spaces occurred between organelles which appeared to be due to an increase in the diameters of the tubules of the T-system and to increased separation of organelles outside of the T-system. In areas in which the damage was more severe only the Z and I bands appeared clearly and in many areas only the Z bands were present. The sarcoplasmic reticulum appeared to be
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Breast muscle Control
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Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 3, 2015 All micrographs are of specimens-from DDT-treated chicks. FIG. 1. Typical proliferation of canaliculi (C) and perinuclear vacuolaction in liver from DDT-treated chicks. The cytoplasm has become hyaline and few organelles are visible. 6.840X. FIG. 2. Liver cells showing dilated cisternae (Ci) and mitochondria (M) with degenerated cristae. Canaliculus is dilated (arrow), 6.840X . FIG. 3. Perinuclear cytoplasmic degeneration of parenchymal cells of liver. Mitochondria have lost internal structures and are the only remaining organelles in hyalinized cytoplasm (arrow). 6,840X . FIG. 4. Cytoplasm of pancreatic cell swelling and irregular architecture of endoplasmic reticulum (ER). Zymogen granule (Z) still appears normal. 1O.620X.
CHANGES INDUCED BY DDT
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Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 3, 2015 FIG. 5. Cytoplasm of pancreatic cell with degeneration of endoplasmic reticulum. Few reticular strands remain (arrow). 15.960X. FIG. 6. Pancreatic acinar cells surrounding duct (D). The lumen of the duct has abnormally few microvilli (arrow). 8.000X. FIG. 7. DDT-induced myocardial degeneration. Progressive sarcomere degeneration is visible. Mitochondria have lost either internal architecture (M) or external membrane (arrow). 15.960X . FIG. 8. Myocardial degeneration as indicated by tubule displacement (arrows) toward interfibril separations. 12.540X.
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In some areas of the sciatic nerve of DDTtreated chicks, the fibers appeared to be altered in the axoplasm and numerous vesicles were
seen. The most striking change observed in the treated group was the vacuolization within and around the myelin sheath (Fig. 11 and 12). DISCUSSION
Several investigators since the 1940's have pointed out that hyperexcitability, tremors, incoordination, and convulsions resulted from DDT poisoning (Hayes, 1959). Tremors, such as were observed in the chickens in the present study, are not limited to DDT poisoning; similar signs may be observed with avian encephalomyelitis, vitamin E deficiencies, and brain tumors (Biester and Schwarte, 1959). However, the signs observed in the treated birds indicated that the central nervous system was affected by DDT. Birds became cold to the touch as toxicity progressed. This finding is in agreement with that of others who reported that in DDT poisoning, rats, guinea pigs and rabbits became cold to the touch as the toxicity increased (Cameron and Burgess, 1945). Other investigators claimed that hyperthermia occurred shortly before death (Wooley and Barron, 1968). Gross pathological changes in the DDTtreated groups were not evident, except for depots of fat. When birds reached the point at which they were unable to eat or drink, mobilization of fat could have occurred and, consequently, fat deposits disappeared or diminished considerably. Residue analyses indicated that fat tissue had the greatest accumulation of DDT, followed by nervous tissue. The distribution of DDT and metabolites in different tissues depends upon the following factors: a) the lipid content of the tissue available for uptake and storage, b) the concentration of DDT in blood, c) the capillary density, d) ability of the species to excrete the compound, and e) general health of the animal (Ecobichon and Saschenbrecker, 1968). If these factors are taken into consideration in the evaluation of the amount of residue detected in the tissues studied, it can be reasoned that adipose tissue having a high lipid content and low metabolic rate is the primary tissue for storage. In this study, it was found that control chickens had detectable levels of residue from unknown sources. The tremors observed before death increased the amount of energy required by the bird, which could have caused increased utilization
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more abundant in treated than in the control tissue. Very few intercalated discs and glycogen granules were observed and no visible changes were found in the nuclei of the treated group. Skeletal Muscle. The nuclear membrane appeared to be complete, but the chromatin material was almost totally marginal and the nucleoli completely dispersed to the point that generally no nucleoli were seen. The mitochondria of the muscle tissue in the control group were small, mostly spherical shaped, and in the treated chicks mitochondria were elongate (Fig. 9). In some mitochondria, cristae were intact and the mitochondrial membranes were partially damaged. Wide spaces appeared between the organelles and some vacuolation was seen. The sarcotubules appeared to be disorganized and displaced throughout large areas of the muscle tissue. One of the most extensive changes occurring in the muscle tissue of the treated birds was the disarrangement of the muscle fibers observed in different areas. The banding pattern was absent in muscle segments with focal lesions. In some areas the Z bands were the only ones clearly seen. Brain Tissue — Cerebral Cortex. No gross lesions were found in the brain tissue of the DDT-treated chickens. The most consistent alteration in the treated group was a moderate increase in myelinated nerve fibers (Fig. 10). When neuroglia of the control and treated birds were compared, it was observed that interstitial cells were closer together in the control group than in the treated birds. The majority of mitochondria were intact and in some areas cristae were disrupted and amorphous material was present. Nissl substance was observed throughout the neuroplasm. Peripheral Nerve Tissue. The myelin sheath was present but the organized patttern observed in the control group was sometimes absent in treated birds. The distinct circular arrangement characteristic of this type of nerve fiber was present in only a few focal areas. The basement membrane of the nucleated sheath of Schwann was not as clear as in the control group and in some regions the membrane appeared to be absent. Mitochondria had disrupted cristae with matrix granules present (Fig. 11).
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Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 3, 2015 FIG. 9. Skeletal muscle with hyaline degeneration of fibrils (F) and disruption of mitochondrial structures (arrows). 20.52OX. FIG. 10. Cerebral cortex with numerous glial processes (G). Myelinated nerve has axoplasm retracted from myelin sheath (arrow). 10.260X. FIG. 11. Longitudinal section of sciatic nerve with marked vesiculation of axoplasm (Ax). Mitochondria are disrupted (arrow) and the axolema has separated from the myelin sheath (Al). 20,520X. FIG. 12. Cross section of sciatic nerve with degeneration of myelin sheath as shown by vacuolation (V) and confluence of myelin lines (arrow). 25.O80X .
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The concentrations of DDT and metabolites in the pancreas are relatively high when compared to that in muscle or brain, but they are similar when compared to liver residues. The pancreatic tissue used for the residue analysis was taken from the endocrine portion of the pancreas. The main residue found in this portion of the pancreas was DDT, probably because this portion of pancreas lacks the metabolic pathways found elsewhere; consequently DDT was metabolized very slowly. Cardiac muscle contained relatively high levels of DDT when compared with skeletal muscle. The transfer of DDT from storage sites into the blood and then into other organs was reported in sparrows and in starlings (Bernard, 1963; Harvey, 1967). It is known that cardiac tissue derives energy from lipopro-
teins (Gartner and Vahouny, 1966); thus, some of the lipoprotein that the heart utilizes can be DDT-bound lipoprotein and can cause an increase of DDT residues in cardiac tissue. The amounts of DDT and metabolite residue found in the breast muscle were low. It is not uncommon to find lipid droplets in skeletal muscles, but during periods of starvation the droplets diminished in size or completely disappeared. Because DDT and metabolites are lipid soluble the lack of lipid material in the muscle tissue can be the cause of relatively low levels of residue in the skeletal muscle. Proliferation of smooth endoplasmic reticulum was observed in all liver specimens studied and probably proliferated because of an increase in microsomal enzyme activity of the liver cells. At the present time, it is widely accepted that lipid-soluble insecticides like DDT enhance the development of smooth endoplasmic reticulum. The fact that the cisternae were dilated suggested hypertrophy of liver cells. Although pancreatic involvement has not been a primary feature of DDT poisoning, the pancreatic acinar cells were examined to determine possible changes because of the high amount of rough endoplasmic reticulum. The amount of DDT and metabolite residues found in the pancreas was 317.47 ppm, higher than the values for liver and heart. The most consistent changes in pancreatic tissues were alterations of mitochondria and focal disorganization of endoplasmic reticulum. Pancreatic acinar cells produce large amounts of digestive enzymes, which are secreted in zymogen granules. A decrease in zymogen granules would represent visual evidence of a decrease in the formation of digestive enzymes. The DDTtreated pancreatic tissue revealed a marked decrease in zymogen granules, thus indicating that production of digestive enzymes was partially inhibited. Myocardial mitochondria appeared to be affected by the treatment and could have produced a decrease in energy production, which could in turn have led to a breakdown in the selective permeability of the mitochondrial membranes. To overcome the decrease in energy supply, more DDT-bound lipoprotein could enter the myocardium and in turn could cause the relatively higher amounts of DDT found in this tissue. DDT and metabolite residues found in the breast muscle were low, and the most notice-
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of stored fat. Consequently, in the later stages of poisoning, fat could be mobilized and DDT shifted from fat to other sites, increasing the amount of DDT in other tissues. Peripheral nerve tissue had the second largest amount of DDT residue. Myelin appeared to retain DDT longer than other lipids found in the central nervous system; thus, myelin can store DDT that may diffuse to other places during the later stages of poisoning. The residue levels of DDT and metabolites found in the liver indicated that DDD was the most prominent residue. These results are in disagreement with results of others who reported DDE the major metabolite found in bird livers (Ecobinchon and Saschenbrecker, 1968; Gartner and Vahouny, 1966; Moore, 1966). Liver is chiefly a metabolic organ and, therefore, DDT can be metabolized further to DDD or DDE. There are five principal routes of DDT metabolism in various animals: a) oxidation to DDA bis (p-chlorophenyl) acetic acid, b) oxidation to kelthane, c) oxidation to dichlorobenzyphenone, d) dehydrochlorination to DDE l,l-dichloro-2,2-bis (p-chlorophenyl) ethane. The metabolism of DDT was not a parameter studied in this investigation, but the fact that both metabolites, DDD and DDE, were found in liver tissue indicated that birds could use both routes for the metabolism of DDT. The presence of large amounts of DDD in the tissues is of interest in the DDT metabolism. The metabolite could have been formed by intestinal flora of the birds and absorbed in this form has been observed in mice (Barker et al, 1965), or the metabolite could have been formed by enzymatic reaction.
CHANGES INDUCED BY DDT
T h e p r o m i n e n t damages caused b y D D T a t t h e ultrastructural level in chicken organs and tissues were identified in this study. Most of t h e gross signs of D D T intoxication could be explained on t h e basis of t h e ultrastructural involvement. It is obvious t h a t m o r e studies are needed. This was indicated b y t h e fact t h a t chickens recovered rapidly after removal of t h e
D D T from t h e ration as n o t e d b y their recovery from t r e m o r s , ataxia, and o t h e r gross signs. REFERENCES Barker, P. S., F. O. Morrison, and R. S. Whitaker, 1965. Conversion of DDT to DDD by Proteus vulgaris, a bacterium isolated from the intestinal flora of a mouse. Nature 205:621. Bernard, R. F., 1963. Studies on the effects of DDT on birds. Publications of the Museum, Michigan State University 2:159. Biester, H. E., and L. H. Schwarte, 1959. Diseases of poultry. Iowa State University Press, Ames, IA. Cameron, G. R., and F. Burgess, 1945. The toxicity of 2,2-bis (p-chlorophenyl) 1,1,1-trichloethane (D.D.T.). Brit. Med. J. 1:865-871. Draize, J. H., G. Woodard, O. G. Fitzhugh, A. A. Nelson, R. B. Smith, Jr., and H. O. Calvery, 1944. Summary of toxicological studies of the insecticide DDT. Chem. Eng. News 22:1503. Ecobichon, D. J., and P. W. Saschenbrecker, 1968. Pharmacodynamic study of DDT in cockerels. Can. J. Physiol. Pharmacol. 46:785-794. Gartner, S. L., and G. V. Vahouny, 1966. Activation of soluble heart lipoproteins. Amer. J. Physiol. 211:1063. Harvey, J. M., 1967. Excretion of DDT by migratory birds. Can. J. Zool. 45:629. Hayes, W. J., 1959. DDT: The insecticide dichlorodiphenyltrichloroethane and its significance. Page 11—247 in Human and veterinary medicine. Vol. 11. P. Muller, ed. Birkhauser-Verlag, Basel. Konst, H., and P. J. G. Plummer, 1946. Studies on the toxicity of DDT for domestic and laboratory animals. Can. J. Comp. Med. 10:128. Luft, J. H., 1961. Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414. Millonig, G., 1961. A modified procedure for lead staining of thin section. J. Biophys. Biochem. Cytol. 11:739-746. Moore, N. W., 1966. Pesticides in the environment and their effect on wildlife. J. Appl. Ecol. Suppl. 3. Narahashi, T., and T. Yamosaki, 1960. Mechanisms of increase in negative afterpotential by cicopharum (DDT) in the giant axon of the cockroach. J. Physiol. 152:122-140. Pesticide Analytical Manual, 1971. Vol. 1 USDA, HEW, FDA. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208. St. Omer, V. V., 1970. Chronic and acute toxicity of chlorinated hydrocarbon insecticides in mammals and birds. Can. Vet. J. 11:215-216. Wooley, D. E., and B. A. Barron, 1968. Effects of DDT on brain electrical activity in awake unrestrained rats. Toxicol. Appl. Pharmacol. 12:440— 454.
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able change in skeletal muscle was displacem e n t of sarcotubules and banding. Similar effects caused by D D T were observed in cardiac muscle, b u t t h e histological changes in skeletal muscle were less p r o b a b l y because of t h e smaller a m o u n t of D D T a c c u m u l a t e d in this tissue. T h e a m o u n t s of D D T and m e t a b o l i t e residues found in t h e brain of t h e treated chickens were t h e lowest of all t h e organs studied. T h e results o b t a i n e d were higher ( 1 2 1 . 3 8 p p m ) than values r e p o r t e d in t h e literature. Although D D T appeared t o be relatively less soluble in myelin lipids t h a n in o t h e r lipids, t h e myelin appeared t o retain D D T longer t h a n o t h e r lipids of t h e brain. In this s t u d y , an increased a m o u n t of m y e l i n a t e d cross sections of nerve fibers were seen, suggesting t h e possibility t h a t lipid myelin could be s o m e w h a t m o r e a b u n d a n t in treated animals t h a n in control birds. Of t h e tissues studied, t h e sciatic nerve had t h e greatest a m o u n t of residues of D D T a n d m e t a b o l i t e s . A striking change observed in t h e treated group was t h e lack of t h e orderly circular arrangement of t h e myelin sheath and vacuolization within and a r o u n d t h e myelin sheath. Myelin is n o t essential for t h e action of D D T because similar residue levels were r e p o r t e d in b o t h m y e l i n a t e d and u n m y e l i n a t e d nerves (Narahashi and Yamosaki, 1960). R e t e n t i o n of D D T within myelin could p r o d u c e deterioration and breakd o w n of t h e nerve tissue. In s o m e areas, t h e nucleated sheath of S c h w a n n appeared t o be absent. It appeared t h a t D D T held t h e sodium channels in nerve m e m b r a n e s open for an abnormal period of t i m e allowing t h e sodium current flow for a longer time which could elicit a train of responses in nerves and muscle. A b n o r m a l periods of stimuli could p r o d u c e a proliferation of synaptic vesicles n o t only at t h e nerve endings b u t t o s o m e e x t e n t t h r o u g h o u t t h e axoplasm.
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1979 Poultry Science 58:1183-1191
INTRODUCTION T h e chlorinated h y d r o c a r b o n (organochlorine) insecticides have been used extensively in t h e c o n t r o l of insect pests for over 30 years. A m o n g these, l , l , l - t r i c h l o r o - 2 , 2 - b i s (p-chlorop h e n y l ) e t h a n e (DDT) is one of t h e best k n o w n and cheapest of t h e s y n t h e t i c insecticides. T h e use of t h e pesticide D D T has been b a n n e d , restricted, or phased o u t in developed countries like Canada, Scandinavian nations, and t h e United States because of alleged effects on non-target invertebrates and vertebrates, including beneficial insects and crustacae, birds and fishes, especially fish-eating raptorial birds, and its alleged effect on man and o t h e r m a m m a l s . T h e a m o u n t of research published since t h e discovery of D D T has been extensive. Toxicological effects have been studied in different species and storage and residue levels of D D T have been linked directly t o m e t a b o l i c studies. T h e pathological changes have been associated with exposure t o massive doses or t o m a n y relatively small doses of t h e pesticide, b u t t h e majority of t h e studies have been d o n e with light m i c r o s c o p y . T h e ultrastructural studies of
'Present address: Copiah-Lincoln Junior College, Wesson, MS 39191.
t h e histopathogenicity of D D T in chicken tissues were u n d e r t a k e n t o further u n d e r s t a n d t h e action of this pesticide in d o m e s t i c birds. Residue levels in different tissues were determ i n e d t o correlate t h e relationship b e t w e e n t h e a c c u m u l a t i o n of D D T in tissues and t h e p a t h o logical changes observed.
MATERIALS AND METHODS Twenty-four crossbred broiler chicks, rand o m sexed, 2 weeks of age, were used in this investigation. T h e chicks were caged in groups of six and watered and fed a commercial broiler ration ad libitum. T h e L D 5 0 dose value of 50% commercially available D D T for this e x p e r i m e n t was determ i n e d t o be 300 m g / k g of b o d y weight (St. Omer, 1 9 7 0 ; K o n s t and Plummer, 1 9 4 6 ; Draize et ah, 1944). Twelve chickens were used for controls and 12 chickens were treated with D D T . A daily dose of 100 m g / k g of b o d y weight was given orally t h r e e times p e r day for 10 days. Birds were weighed every 5 days, and t h e LD S 0 dose was adjusted accordingly. T h e tissues for t h e residue level analysis and for t h e histopathological studies were o b t a i n e d after signs of i n t o x i c a t i o n were manifested. Morbid animals were killed by cervical dislocation and specimens from liver, pancreas, heart,
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ABSTRACT Chickens were given DDT in an oral dose of 100 mg/kg of body weight 3 times a day for 10 days. Specimens of liver, pancreas, heart, skeletal muscle, brain, and sciatic nerve were obtained from morbid chicks for analysis of residue levels of DDT and metabolites, DDD and DDE, and for electron microscopic examination. Fat and sciatic nerve tissue contained the largest amount of DDT and metabolite residues, and skeletal muscle and brain tissue contained the smallest amount of residues. Only moderate amounts of detectable residues were present in the liver, pancreas, and heart muscle. The liver cells underwent more changes than any other organ and exhibited alterations in individual parenchymal cells as well as areas of tissue disorganization. Proliferation of bile duct epithelia and smooth endoplasmic reticulum was found. Disorganization of pancreatic endoplasmic reticulum and a decrease of zymogen granules were found. Displacement of sarcotubules and banding pattern in myocardium occurred. Alterations of the myelin sheath, increased amount of vesicles, and absence in some areas of the nucleated sheath of Schwann were the major changes observed in the sciatic nerve.
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RESULTS
Signs. Chickens in the control group had an initial weight range of 839.9 to 1,189.5 g with an average weight of 994.6 g. All birds gained weight and the final weight range was from 1,157.7 to 1,570.8 g with an average weight of 1,356.7 g. The DDT-treated group had an initial weight range of 776.3 to 1,080.5 g with an average weight of 935.9 g and a final average weight of 856.1 g. Initial signs of toxicity included barely perceptible tremors of the feathers and a curling of the toes. The toe curling increased in severity until the birds walked on the sides of their feet. At this stage the birds were frequently squatting but were able to rise voluntarily. The birds were also walking in an exaggerated and ataxic manner. Tremors increased to the point at which the birds could no longer rise. In the most severe cases, the muscles of the neck were under extreme tension and the neck was twisted into an S-shaped curve with the head in a fixed position over one wing. The birds became incapacitated to the point of being unable to eat or drink and the tremors became very severe and continuous, progressing into tonic convulsions. The convulsions were intermittent at first but later became continuous, generalized, and bilateral in nature. When convulsions were most severe, there were periods of post-seizure depression resembling exhaustion or loss of consciousness, but addi-
tional convulsions could be evoked by handling or touching a bird. The legs of the animals became cold and the body temperature was 1 to 2 below the normal average temperature of 42.6 C for controls. Death usually occurred within a few hours, but if the bird was handled or in any way excited, death occurred sooner, often within minutes. When the DDT treatment (10 days) was discontinued, all clinical signs in surviving birds gradually disappeared and after a period of 4 to 6 days the birds appeared normal when compared to the control group. The chickens with the most severe tremors did not have enough abdominal fat for the extraction procedure (1.0 g). Analysis of Residue Levels. The data obtained by quantitative analysis of the residues of DDT and metabolites, DDD and DDE, in the tissues of treated and control birds are summarized in Table 1. Histopathology. All electron micrographs are of specimens obtained from DDT-treated chicks. Analyses of control chick tissues determined that the changes were not due to fixation or processing artifacts. Liver. Large non-membrane bound vacuoles of variable sizes were found throughout the parenchymal cells and some had a flocculent material of low electron density (Fig. 1) and others appeared empty (Fig. 2). A mild to moderate proliferation of smooth endoplasmic reticulum was observed, with the greatest concentration being central or perinuclear (Fig. 3). Ribosomes could be identified around the cisternal membranes but there was no continuous ribosomal lining. The cisternae of smooth endoplasmic reticulum were not uniform in size or shape, and contained an electron dense material. An increase in lysosomes was found, mainly in areas close to bile ducts. Glycogen granules were not usually seen in treated animals. In the majority of parenchymal cells, the nucleoli contained a formation of granular and fibrillar components. In the treated animals, cells that appeared to be hepatocytes contained nuclei not characteristic of this cell type. The nuclei were pleomorphic with irregular outlines, more typical of connective tissue cells than of parenchymal cells. In some, the nuclear membranes appeared to be disrupted. In areas with more damage, it became increasingly difficult to differentiate between parenchymal and connective tissue cells, and positive identification was not always possible. A large number of
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skeletal muscle, brain, and sciatic nerve were taken. Samples of corresponding tissues from control animals were also obtained. Determination of Chlorinated Pesticides in Tissues. The determination of the levels of DDT residue and its metabolites, DDD and DDE, in the liver, pancreas, heart, skeletal muscle, brain, abdominal fat, and sciatic nerve of the treated chickens was carried out at the Alabama Pesticide Residue Laboratory, Division of Alabama Department of Agriculture and Industries, Auburn, Alabama, using analytical procedures recommended in Pesticide Analytical Manual (USDA, HEW, FDA, 1971). Electron Microscopy Techniques. For electron microscopy studies, specimens of liver, pancreas, heart, skeletal muscle, brain, and sciatic nerve were fixed in 3% glutaraldehyde in phosphate buffer, pH 7.3 and 1% osmium tetroxide (Millonig, 1961). The specimens were processed according to Luft (1961) and Reynolds (1963).
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CHANGES INDUCED BY DDT TABLE l.Afean DDT and metabolic residues in tissues of morbid chickens Tissue
DDE
DDD
DDT
Total
Liver Control
52.54
137.45
81.96 No significant residues
271.95 a
Pancreas Control
52.09
75.89
189.49 No significant residues
317.47
Heart Control
41.48
40.84 148.34 No significant residues
230.66
Brain Control Sciatic nerve Control Fat Control
6.82
5.70
122.92 .19
135.44 .19
26.85
17.02
77.51 No significant residues
121.38
530.03
96.31
2,451.59 No significant residues
3,077.93
1,580.03 1.87
238.61
7,075.36 8.20
8,894.00 10.07
Results presented as parts per million, wet weight.
mitochondria of fairly constant size, shape, and intensity of staining was a main characteristic of the control group. In the treated animals, the number of mitochondria diminished. The majority of the mitochondria lacked the double layered membrane and the cristae were not visible. Only an amorphous matrix material was observed. The proliferation of bile duct canaliculi epithelium into partial or complete canaliculi was evident. In the control group, bile canaliculi were occasionally seen but in the treated birds, canaliculi were found frequently and often enlarged. Some canaliculi were composed of three cells joined to form a lumen. The apical portions of the cells terminated in microvilli, and the zonula occludens were seen close to canaliculi. Throughout these areas some degenerative changes of the cytoplasm were often seen. Pancreas. The nuclei of the acinar cells were often distorted and condensed (Fig. 4). In the DDT-treated specimens the endoplasmic reticulum was regularly organized around the nucleus (Fig. 5) except for damaged areas which had focal disorganization of the endoplasmic reticulum and few mitochondria (Fig. 6). Throughout the cytoplasm the cisternae of the rough endoplasmic reticulum were dilated to give a more circular than laminar appearance.
No other significant alterations of rough endoplasmic reticulum were noted. The number of zymogen granules present was much less in the treated animals than in the control group. The lumen formed by several acinar cells appeared to be wider (Fig. 6) and the free surfaces of the cells had few microvilli. Cytoplasmic degeneration was occasionally noted in areas where the rough endoplasmic reticulum was distorted. Heart. Mitochondria of normal cardiac tissue had excellent staining qualities with cristae sharply defined. In the treated chickens, some mitochondria were intact with welldefined membrane and cristae (Fig. 8) but most mitochondria had few cristae against a background of extremely low electron density. Nearby mitochondria contained only the amorphous matrix material. In many mitochondria the membranes were either damaged or entirely lost (Fig. 7 and 8). In the treated group, wide spaces occurred between organelles which appeared to be due to an increase in the diameters of the tubules of the T-system and to increased separation of organelles outside of the T-system. In areas in which the damage was more severe only the Z and I bands appeared clearly and in many areas only the Z bands were present. The sarcoplasmic reticulum appeared to be
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Breast muscle Control
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Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 3, 2015 All micrographs are of specimens-from DDT-treated chicks. FIG. 1. Typical proliferation of canaliculi (C) and perinuclear vacuolaction in liver from DDT-treated chicks. The cytoplasm has become hyaline and few organelles are visible. 6.840X. FIG. 2. Liver cells showing dilated cisternae (Ci) and mitochondria (M) with degenerated cristae. Canaliculus is dilated (arrow), 6.840X . FIG. 3. Perinuclear cytoplasmic degeneration of parenchymal cells of liver. Mitochondria have lost internal structures and are the only remaining organelles in hyalinized cytoplasm (arrow). 6,840X . FIG. 4. Cytoplasm of pancreatic cell swelling and irregular architecture of endoplasmic reticulum (ER). Zymogen granule (Z) still appears normal. 1O.620X.
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Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 3, 2015 FIG. 5. Cytoplasm of pancreatic cell with degeneration of endoplasmic reticulum. Few reticular strands remain (arrow). 15.960X. FIG. 6. Pancreatic acinar cells surrounding duct (D). The lumen of the duct has abnormally few microvilli (arrow). 8.000X. FIG. 7. DDT-induced myocardial degeneration. Progressive sarcomere degeneration is visible. Mitochondria have lost either internal architecture (M) or external membrane (arrow). 15.960X . FIG. 8. Myocardial degeneration as indicated by tubule displacement (arrows) toward interfibril separations. 12.540X.
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In some areas of the sciatic nerve of DDTtreated chicks, the fibers appeared to be altered in the axoplasm and numerous vesicles were
seen. The most striking change observed in the treated group was the vacuolization within and around the myelin sheath (Fig. 11 and 12). DISCUSSION
Several investigators since the 1940's have pointed out that hyperexcitability, tremors, incoordination, and convulsions resulted from DDT poisoning (Hayes, 1959). Tremors, such as were observed in the chickens in the present study, are not limited to DDT poisoning; similar signs may be observed with avian encephalomyelitis, vitamin E deficiencies, and brain tumors (Biester and Schwarte, 1959). However, the signs observed in the treated birds indicated that the central nervous system was affected by DDT. Birds became cold to the touch as toxicity progressed. This finding is in agreement with that of others who reported that in DDT poisoning, rats, guinea pigs and rabbits became cold to the touch as the toxicity increased (Cameron and Burgess, 1945). Other investigators claimed that hyperthermia occurred shortly before death (Wooley and Barron, 1968). Gross pathological changes in the DDTtreated groups were not evident, except for depots of fat. When birds reached the point at which they were unable to eat or drink, mobilization of fat could have occurred and, consequently, fat deposits disappeared or diminished considerably. Residue analyses indicated that fat tissue had the greatest accumulation of DDT, followed by nervous tissue. The distribution of DDT and metabolites in different tissues depends upon the following factors: a) the lipid content of the tissue available for uptake and storage, b) the concentration of DDT in blood, c) the capillary density, d) ability of the species to excrete the compound, and e) general health of the animal (Ecobichon and Saschenbrecker, 1968). If these factors are taken into consideration in the evaluation of the amount of residue detected in the tissues studied, it can be reasoned that adipose tissue having a high lipid content and low metabolic rate is the primary tissue for storage. In this study, it was found that control chickens had detectable levels of residue from unknown sources. The tremors observed before death increased the amount of energy required by the bird, which could have caused increased utilization
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more abundant in treated than in the control tissue. Very few intercalated discs and glycogen granules were observed and no visible changes were found in the nuclei of the treated group. Skeletal Muscle. The nuclear membrane appeared to be complete, but the chromatin material was almost totally marginal and the nucleoli completely dispersed to the point that generally no nucleoli were seen. The mitochondria of the muscle tissue in the control group were small, mostly spherical shaped, and in the treated chicks mitochondria were elongate (Fig. 9). In some mitochondria, cristae were intact and the mitochondrial membranes were partially damaged. Wide spaces appeared between the organelles and some vacuolation was seen. The sarcotubules appeared to be disorganized and displaced throughout large areas of the muscle tissue. One of the most extensive changes occurring in the muscle tissue of the treated birds was the disarrangement of the muscle fibers observed in different areas. The banding pattern was absent in muscle segments with focal lesions. In some areas the Z bands were the only ones clearly seen. Brain Tissue — Cerebral Cortex. No gross lesions were found in the brain tissue of the DDT-treated chickens. The most consistent alteration in the treated group was a moderate increase in myelinated nerve fibers (Fig. 10). When neuroglia of the control and treated birds were compared, it was observed that interstitial cells were closer together in the control group than in the treated birds. The majority of mitochondria were intact and in some areas cristae were disrupted and amorphous material was present. Nissl substance was observed throughout the neuroplasm. Peripheral Nerve Tissue. The myelin sheath was present but the organized patttern observed in the control group was sometimes absent in treated birds. The distinct circular arrangement characteristic of this type of nerve fiber was present in only a few focal areas. The basement membrane of the nucleated sheath of Schwann was not as clear as in the control group and in some regions the membrane appeared to be absent. Mitochondria had disrupted cristae with matrix granules present (Fig. 11).
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Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 3, 2015 FIG. 9. Skeletal muscle with hyaline degeneration of fibrils (F) and disruption of mitochondrial structures (arrows). 20.52OX. FIG. 10. Cerebral cortex with numerous glial processes (G). Myelinated nerve has axoplasm retracted from myelin sheath (arrow). 10.260X. FIG. 11. Longitudinal section of sciatic nerve with marked vesiculation of axoplasm (Ax). Mitochondria are disrupted (arrow) and the axolema has separated from the myelin sheath (Al). 20,520X. FIG. 12. Cross section of sciatic nerve with degeneration of myelin sheath as shown by vacuolation (V) and confluence of myelin lines (arrow). 25.O80X .
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The concentrations of DDT and metabolites in the pancreas are relatively high when compared to that in muscle or brain, but they are similar when compared to liver residues. The pancreatic tissue used for the residue analysis was taken from the endocrine portion of the pancreas. The main residue found in this portion of the pancreas was DDT, probably because this portion of pancreas lacks the metabolic pathways found elsewhere; consequently DDT was metabolized very slowly. Cardiac muscle contained relatively high levels of DDT when compared with skeletal muscle. The transfer of DDT from storage sites into the blood and then into other organs was reported in sparrows and in starlings (Bernard, 1963; Harvey, 1967). It is known that cardiac tissue derives energy from lipopro-
teins (Gartner and Vahouny, 1966); thus, some of the lipoprotein that the heart utilizes can be DDT-bound lipoprotein and can cause an increase of DDT residues in cardiac tissue. The amounts of DDT and metabolite residue found in the breast muscle were low. It is not uncommon to find lipid droplets in skeletal muscles, but during periods of starvation the droplets diminished in size or completely disappeared. Because DDT and metabolites are lipid soluble the lack of lipid material in the muscle tissue can be the cause of relatively low levels of residue in the skeletal muscle. Proliferation of smooth endoplasmic reticulum was observed in all liver specimens studied and probably proliferated because of an increase in microsomal enzyme activity of the liver cells. At the present time, it is widely accepted that lipid-soluble insecticides like DDT enhance the development of smooth endoplasmic reticulum. The fact that the cisternae were dilated suggested hypertrophy of liver cells. Although pancreatic involvement has not been a primary feature of DDT poisoning, the pancreatic acinar cells were examined to determine possible changes because of the high amount of rough endoplasmic reticulum. The amount of DDT and metabolite residues found in the pancreas was 317.47 ppm, higher than the values for liver and heart. The most consistent changes in pancreatic tissues were alterations of mitochondria and focal disorganization of endoplasmic reticulum. Pancreatic acinar cells produce large amounts of digestive enzymes, which are secreted in zymogen granules. A decrease in zymogen granules would represent visual evidence of a decrease in the formation of digestive enzymes. The DDTtreated pancreatic tissue revealed a marked decrease in zymogen granules, thus indicating that production of digestive enzymes was partially inhibited. Myocardial mitochondria appeared to be affected by the treatment and could have produced a decrease in energy production, which could in turn have led to a breakdown in the selective permeability of the mitochondrial membranes. To overcome the decrease in energy supply, more DDT-bound lipoprotein could enter the myocardium and in turn could cause the relatively higher amounts of DDT found in this tissue. DDT and metabolite residues found in the breast muscle were low, and the most notice-
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of stored fat. Consequently, in the later stages of poisoning, fat could be mobilized and DDT shifted from fat to other sites, increasing the amount of DDT in other tissues. Peripheral nerve tissue had the second largest amount of DDT residue. Myelin appeared to retain DDT longer than other lipids found in the central nervous system; thus, myelin can store DDT that may diffuse to other places during the later stages of poisoning. The residue levels of DDT and metabolites found in the liver indicated that DDD was the most prominent residue. These results are in disagreement with results of others who reported DDE the major metabolite found in bird livers (Ecobinchon and Saschenbrecker, 1968; Gartner and Vahouny, 1966; Moore, 1966). Liver is chiefly a metabolic organ and, therefore, DDT can be metabolized further to DDD or DDE. There are five principal routes of DDT metabolism in various animals: a) oxidation to DDA bis (p-chlorophenyl) acetic acid, b) oxidation to kelthane, c) oxidation to dichlorobenzyphenone, d) dehydrochlorination to DDE l,l-dichloro-2,2-bis (p-chlorophenyl) ethane. The metabolism of DDT was not a parameter studied in this investigation, but the fact that both metabolites, DDD and DDE, were found in liver tissue indicated that birds could use both routes for the metabolism of DDT. The presence of large amounts of DDD in the tissues is of interest in the DDT metabolism. The metabolite could have been formed by intestinal flora of the birds and absorbed in this form has been observed in mice (Barker et al, 1965), or the metabolite could have been formed by enzymatic reaction.
CHANGES INDUCED BY DDT
T h e p r o m i n e n t damages caused b y D D T a t t h e ultrastructural level in chicken organs and tissues were identified in this study. Most of t h e gross signs of D D T intoxication could be explained on t h e basis of t h e ultrastructural involvement. It is obvious t h a t m o r e studies are needed. This was indicated b y t h e fact t h a t chickens recovered rapidly after removal of t h e
D D T from t h e ration as n o t e d b y their recovery from t r e m o r s , ataxia, and o t h e r gross signs. REFERENCES Barker, P. S., F. O. Morrison, and R. S. Whitaker, 1965. Conversion of DDT to DDD by Proteus vulgaris, a bacterium isolated from the intestinal flora of a mouse. Nature 205:621. Bernard, R. F., 1963. Studies on the effects of DDT on birds. Publications of the Museum, Michigan State University 2:159. Biester, H. E., and L. H. Schwarte, 1959. Diseases of poultry. Iowa State University Press, Ames, IA. Cameron, G. R., and F. Burgess, 1945. The toxicity of 2,2-bis (p-chlorophenyl) 1,1,1-trichloethane (D.D.T.). Brit. Med. J. 1:865-871. Draize, J. H., G. Woodard, O. G. Fitzhugh, A. A. Nelson, R. B. Smith, Jr., and H. O. Calvery, 1944. Summary of toxicological studies of the insecticide DDT. Chem. Eng. News 22:1503. Ecobichon, D. J., and P. W. Saschenbrecker, 1968. Pharmacodynamic study of DDT in cockerels. Can. J. Physiol. Pharmacol. 46:785-794. Gartner, S. L., and G. V. Vahouny, 1966. Activation of soluble heart lipoproteins. Amer. J. Physiol. 211:1063. Harvey, J. M., 1967. Excretion of DDT by migratory birds. Can. J. Zool. 45:629. Hayes, W. J., 1959. DDT: The insecticide dichlorodiphenyltrichloroethane and its significance. Page 11—247 in Human and veterinary medicine. Vol. 11. P. Muller, ed. Birkhauser-Verlag, Basel. Konst, H., and P. J. G. Plummer, 1946. Studies on the toxicity of DDT for domestic and laboratory animals. Can. J. Comp. Med. 10:128. Luft, J. H., 1961. Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414. Millonig, G., 1961. A modified procedure for lead staining of thin section. J. Biophys. Biochem. Cytol. 11:739-746. Moore, N. W., 1966. Pesticides in the environment and their effect on wildlife. J. Appl. Ecol. Suppl. 3. Narahashi, T., and T. Yamosaki, 1960. Mechanisms of increase in negative afterpotential by cicopharum (DDT) in the giant axon of the cockroach. J. Physiol. 152:122-140. Pesticide Analytical Manual, 1971. Vol. 1 USDA, HEW, FDA. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208. St. Omer, V. V., 1970. Chronic and acute toxicity of chlorinated hydrocarbon insecticides in mammals and birds. Can. Vet. J. 11:215-216. Wooley, D. E., and B. A. Barron, 1968. Effects of DDT on brain electrical activity in awake unrestrained rats. Toxicol. Appl. Pharmacol. 12:440— 454.
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able change in skeletal muscle was displacem e n t of sarcotubules and banding. Similar effects caused by D D T were observed in cardiac muscle, b u t t h e histological changes in skeletal muscle were less p r o b a b l y because of t h e smaller a m o u n t of D D T a c c u m u l a t e d in this tissue. T h e a m o u n t s of D D T and m e t a b o l i t e residues found in t h e brain of t h e treated chickens were t h e lowest of all t h e organs studied. T h e results o b t a i n e d were higher ( 1 2 1 . 3 8 p p m ) than values r e p o r t e d in t h e literature. Although D D T appeared t o be relatively less soluble in myelin lipids t h a n in o t h e r lipids, t h e myelin appeared t o retain D D T longer t h a n o t h e r lipids of t h e brain. In this s t u d y , an increased a m o u n t of m y e l i n a t e d cross sections of nerve fibers were seen, suggesting t h e possibility t h a t lipid myelin could be s o m e w h a t m o r e a b u n d a n t in treated animals t h a n in control birds. Of t h e tissues studied, t h e sciatic nerve had t h e greatest a m o u n t of residues of D D T a n d m e t a b o l i t e s . A striking change observed in t h e treated group was t h e lack of t h e orderly circular arrangement of t h e myelin sheath and vacuolization within and a r o u n d t h e myelin sheath. Myelin is n o t essential for t h e action of D D T because similar residue levels were r e p o r t e d in b o t h m y e l i n a t e d and u n m y e l i n a t e d nerves (Narahashi and Yamosaki, 1960). R e t e n t i o n of D D T within myelin could p r o d u c e deterioration and breakd o w n of t h e nerve tissue. In s o m e areas, t h e nucleated sheath of S c h w a n n appeared t o be absent. It appeared t h a t D D T held t h e sodium channels in nerve m e m b r a n e s open for an abnormal period of t i m e allowing t h e sodium current flow for a longer time which could elicit a train of responses in nerves and muscle. A b n o r m a l periods of stimuli could p r o d u c e a proliferation of synaptic vesicles n o t only at t h e nerve endings b u t t o s o m e e x t e n t t h r o u g h o u t t h e axoplasm.
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