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δ-aminolevulinic acid dehydratase enzyme activity in blood, brain, and liver of lead-dosed ducks
ENVlRONMENTAL
RESEARCH
19, 127-135
(1979)
SAminolevulinic Acid Dehydratase Enzyme Activity Blood, Brain, and Liver of Lead-Dosed Ducks M. P. DIETER*
in
AND M. T. FINLEY?
*U. S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, Maryland20811; tU. S. Fish and Wildlife Service, Fish Pesticide Research Laboratory. Columbia, Missouri 65201 Received
August
1, 1978
Mallard ducks were dosed with a single shotgun pellet (ca. 200 mg lead). After 1 month there was about 1 ppm lead in blood, 2.5 in liver, and 0.5 in brain. Lead-induced inhibition of Gaminolevulinic acid dehydratase enzyme in blood and cerebellum was much greater than in cerebral hemisphere or liver and was strongly correlated with the lead concentration in these tissues. The cerebellar portion of the brain was more sensitive to 8aminolevulinic acid dehydratase enzyme inhibition by lead than were the other tissues examined. There was also a greater increase in the glial cell marker enzyme, butyrylcholinesterase, in cerebellum than in cerebral hemisphere, suggesting that nonregenerating neuronal cells were destroyed by lead and replaced by glial cells in that portion of the brain. Even partial loss of cerebellar tissue is severely debilitating in waterfowl, because functions critical to survival such as visual, auditory, motor, and reflex responses are integrated at this brain center.
Inhibition of &aminolevulinic acid dehydratase (CALAD) has become a standard bioassay to detect acute and chronic lead exposure (Hemberg er al., 1970; Ohi ef al., 1974; Mouw et al., 1975; Hodson, 1976; Dieter et al., 1976). The enzyme is operative in the biosynthetic pathway of heme synthesis to maintain hemoglobin content in erythrocytes and to insure that adequate amounts of heme are available for incorporation into mitochondrial cytochromes (Sassaer al., 1975). The cytochtomes transport electrons through a redox potential and in the process generate high energy phosphate or ATP as a source of cellular energy. Inhibition of CALAD in cells of critical organs could inflict serious damage because the electron transport system, which is the energetic machinery of the cell, would be jeopardized. In addition, heme is a part of cytochrome P-450 that is necessary for some detoxification processes in the liver (Sassa et al., 1975). This study addresses the question of sublethal toxicity in migrating waterfowl that obtain balanced rations and do not immediately die from lead poisoning. We demonstrated that there was enough lead in one shotgun pellet to cause 60-90% inhibition of 6ALAD enzyme activity in erythroyctes, but not enough to kill waterfowl fed balanced diets (Finley ef al., 1976a,b). The inhibition persisted for 3 months (Dieter and Finley, 1978). Waterfowl ingesting one shotgun pellet may accumulate enough lead in their soft organs to disturb critical cellular functions but not enough to cause mortality. To initiate an examination of this hypothesis we have determined the extent of SALAD enzyme inhibition in blood, brain, and liver of lead-dosed ducks and have examined the relationships between lead accumulation and enzyme activity in these tissues. MATERIALS
AND METHODS
First-year-breeding mallard ducks (Anus plazyrhynchos) were paired and kept in vinylcoated wire pens on a long-day (16L:8D) light cycle. They were fed ad libitum diets of one-half yellow corn and one-half commercial breeder pellets ground to a mash. The 127 0013-9351/79/030127-09$02.00/O Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in any form reserved.
128
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FINLEY
pellets contained 3.1% calcium, 0.8% total phosphorous, and 850 IU vitamin D; the corn essentially diluted these concentrations in half. After 6 weeks half of the pairs of ducks were dosed with one No. 4 commercial lead shot. Average shot weight was 206 mg with a range between 195 and 215 mg. Shot was administered through a flexible plastic tube forced down to the proventriculus. Controls were untreated. At 1 month post-dosage blood samples were obtained from the alar vein, the ducks killed by cervical dislocation, and necropsies performed. Blood &ALAD (EC 4.2124) enzyme assays were carried out immediately (Burch and Siegel, 1971). The bone lead concentrations were compared and the results reported elsewhere (Finley and Dieter, 1978). Whole brain and liver were frozen at -80°C until enzyme and residue analyses. Liver samples were divided in half and used for enzyme and lead assays. One of the cerebral hemispheres and the cerebellum were separated from the whole brain and enzyme assays run on each part. Lead residues were determined with the remainder of brain sample. Results from brain lead residues of a subset of dosed ducks showed that the lead concentrations in one cerebral hemisphere, the cerebellum, and the remainder of the brain were not different. There was a highly significant correlation between the lead concentration in the remainder of the brain [0.61 + 0.19 (mean ppm lead + standard error)], the cerebellum (0.59 ? 0.19), and the cerebral hemisphere (0.51 f 0.16), withr = 0.95, P < 0.01, N = 15. This data permitted us to use a distinct portion of the brain for enzyme assays and the rest for lead assays. One cerebral hemisphere or the cerebellum was prepared in cold pH 7.4 Tris buffer with a glass homogenizer tube and a motorized Teflon pestle. Samples of whole homogenate were used for &ALAD enzyme assays (Burch and Siegel, 1971) or for acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) enzyme determinations using either acetylthiocholine or butyrylthiocholine as substrate (Bellman er al., 1961). Livers were prepared in a similar fashion for GALAD enzyme assays only. Total protein concentrations in the homogenates were determined by the Lowry (1951) method. Reactants were adjusted to insure there were no rate limiting conditions; activities of assays were proportional to homogenate added. Whole blood, brain, and liver were shipped frozen to Environmental Trace Substances Research Center, Columbia, Missouri, for lead determinations. A 50-~1 blood sample was diluted 1:9 with distilled water and the lead concentration determined by the method of standard additions followed with the analysis of a standard. Samples of brain and liver were homogenized prior to analysis, They were ashed and digested in an acid solution. All samples were assayed on a Perkin-Elmer model 303 atomic absorption spectrophotometer at 283.3 nm. [See also “Analytical Methods for Atomic Absorption Spectrophotometry,” Perkin-Elmer Corp., Norwalk, Conn. (1971).1 Limits of sensitivity were 0.02 ppm; residues are reported on a wet weight basis. RESULTS
There was no mortality or significant changes in body weight as a result of lead shot dosage. Fluoroscopy at 1 week insured that all ducks used for this study had retained the shotgun pellets given them. Most of the shot in the gizzard had been eroded by 1 month post-dosage. Egg production between dosed and undosed groups did not differ. Reproductive tracts and livers of laying hens were much larger than in non-layers. Almost 1 ppm lead remained in the circulation of lead-dosed ducks at 1 month post-
LEAD
AND
8-ALAD
IN
DUCK
TABLE
BRAIN
129
LIVER
1
TISSUE LEAD CONCENTRATIONS (ppm) IN MALLARD No. 4 COMMERCIAL LEAD SHOT
DUCKS 1 MONTH AVER DOSAGE WITH A (ca. 200 mg LEAD)~
Blood
BrainC
Liver
0.06 c_ 0.01 0.98 k 0.18
0.06 f 0.01 0.43 IT 0.08
0.16 f 0.07 2.42 2 0.89
TreatmenP None Dosedd
AND
0 Means 2 standard error; seven males and seven females per group. b There were no significant differences between sexes, and values were combined. c Lead residues in whole brain minus cerebellum and one cerebral hemisphere. tl There were significantly higher lead residues in all tissues of dosed ducks; P < 0.01, Student’s f test.
treatment; the brain lead concentration was about half, and the liver lead concentration about two times that in blood (Table 1). There were no significant differences between sexes. The extent .of &ALAD enzyme inhibition by lead was compared in the different tissues (Table 2). There were no significant differences in total protein concentrations in brain or liver homogenates. There was a 75% inhibition of &ALAD enzyme activity in the blood and a 42% inhibition in the liver, compared to controls. There was a 50% inhibition of 6ALAD enzyme activity in the cerebellum, and a 35% inhibition in the cerebral hemisphere of the brain. Figure 1 indicates that lead residues in the brain or Iiver accurately reflected those in the blood at 1 month post-dosage. There was also a highly predictable relationship in blood and in cerebellum between lead and inhibition of &ALAD enzyme activity in these tissues (Fig. 2). Table 3 lists the correlation coefficients for the various comparisons that were examined. Some measure of brain pathology was afforded by measurements of esterase enzyme activity in brain using acetylcholine or butyrylcholine as substrate. The latter substrate is specific for glial cells; enhanced butyrylcholinesterase activity is indicative of nerve cell damage and subsequent replacement by glial cells or supportive elements. There was a 28% increase in butytylcholinesterase activity in cerebellum and a 22% increase in the TABLE ACTIVITY
2
OF &AMINOLEVULINIC ACID DEHYDRATASE ENZYME IN TISSUES OF MALLARD DUCKS 1 MONTH ARER DOSAGE WITH A No. 4 COMMERCIAL LEAD SHOT (ca. 200 mg LEAD)”
Braind TreatmenP
BloodC
Liverd
None Dosed Changee
180.4 f 11.7 45.6 zk 10.8 -75%
138.2 2 11.9 80.7 2 8.7 -42%
Cerebellum
Cerebral hemisphere
144.9 2 10.3 72.7 ? 5.9 -50%
96.9 k 6.6 63.2 2 5.2 -35%
a Means f standard error; seven males and seven females per group. b There were no significant differences between sexes, and values were combined. c Enzyme activity expressed as increase in absorbance of 0.100 per hour per milliliter erythrocytes. d Enzyme activity expressed as micromoles substrate transformed per hour per 100 mg protein. e There were significantly lower enzyme activities in all tissues of dosed ducks; P <: 0.01, Student’s t test.
130 x n
DIETER
AND
FINLEY
2
.L fl
0.2
[I:
W _I w
.02
1 In 111 F 0
#02
0.2
.02
2
f3LIlllD
0.2
2
LEAD (PPM)
RG. 1. Correlation between lead concentration in blood and tissues of mallard ducks. The lines of fit are described by these power regressions: log brain lead = log 0.39 + 0.62 log blood lead; log liver lead = log 1.60 + 0.85 log blood lead. These relationships were statistically significant Qv = 28, P < 0.01).
cerebral hemisphere of the brain of lead-dosed ducks (Table 4). There were no significant changes in acetylcholinesterase activity. DISCUSSION Waterfowl are particularly vulnerable to lead poisoning because of their feeding habits and preference for grains. Ducks mistake spent pellets for grit and may ingest one or more lead shots. Ducks are particularly sensitive to lead if they are feeding on imbalanced diets such as corn or rice, and they die in about lo- 14 days after ingestion of a single lead shot (Finley and Dieter, 1978). Surveys over a 20-year period showed that 7% of the waterfowl annually ingested at least one spent lead shotgun pellet. It was conservatively estimated that 2% of these waterfowl die from lead poisoning (Bellrose, 1959). By subtraction, 5% of the population annually ingesting a lead shot survive if nutritionally adequate diets are available, but they cannot avoid the sublethal effects of lead poisoning. For example, given a waterfowl population of 2OO,OOO,or about the estimate for canvasback ducks in the Atlantic Flyway (U. S. Fish & Wildlife Service, 1972), 40 (2%) annually die from lead poisoning, and 10,OOO(5%) endure sublethal toxicity from a massive dose of lead. Other than waterfowl, only young children are routinely exposed to such extremes when they display pica and ingest paint chips containing high lead concentrations. We belabor an obvious point because a sublethal toxic challenge by lead imposes a
LEAD
AND
F-ALAD
IN
DUCK
BRAIN
AND
131
LIVER
2SB
SE
I0
10
CEREfKLLUM
f3LOilD
0 la
I
I
I
.I&!
0.2
2
TI!ZilJE
0
0
l
0.2
2
LERD (PPM)
lead concentration and kminolevulinic acid dehydratase enzyme (ALAD) activity in blood and brain of mallard ducks. The line of fit is described by these power regressions: log blood ALAD activity = log 27.6 - 0.59 log blood lead; log cerebellum ALAD activity = log 47.7 - 0.37 log brain lead. These relationships were statistically significant @’ = 28, P < 0.01). FIG. 2. Correlation
between
TABLE 3 CORRELATIONSBETWEEN LEAD AND SALAD ENZYME ACTIVITY IN TISSUES OF MALLARD DUCKS 1 MONTH ARER DOSAGE WITH A No. 4 COMMERCIAL LEAD SHOT”.* Comparisons’ (independent variable vs dependent variable) Blood lead vs brain leadd Blood lead vs liver lead Blood lead vs SALAD in blood SALAD in blood vs SALAD in liver SALAD in blood vs SALAD in cerebral hemisphere SALAD in blood vs &ALAD in cerebellum Brain lead vs GALAD in cerebral hemisphere Brain lead vs SALAD in cerebellum Liver lead vs GALAD in liver
Correlation coefftcient (r*) + + + + + -
0.82 0.72 0.77 0.41 0.43 0.59 0.47 0.75
- 0.34
(1Values are product-moment correlation coefftcients following log transformation values for both treatment groups (n = 28). b N = 28 (seven pairs dosed and seven pairs undosed ducks). e P < 0.01 for all comparisons, linear regression analysis. d Lead residues in whole brain minus cerebellum and one cerebral hemisphere
of combined
132
DIETER
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TABLE ACTIVITY
OF ESTERASE WITH
A
ENZYMES
No. 4
IN BRAIN
COMMERCIAL
FINLEY
4
OF MALLARD LEAD
SHOT
Acetylcholinesterase’
DUCKS 1 MONTH (ca. 200 mg LEAD)”
AFTER
DOSAGE
ButyrylcholinesteraseC
TreatmenP
Cerebellum
Cerebral hemisphere
Cerebellum
Cerebral hemisphere
None Dosed Change
63.7 k 4.1 78.7 k 6.4 N.S.
48.3 k 4.2 57.1 2 4.0 N.S.
12.01 + 0.71 15.40 2 0.74 +28%d
8.58 -+ 0.57 10.43 2 0.59 +22%’
Q Means t standard error; seven males and seven females per group. b There were no significant differences between sexes and values were combined. c Enzyme activity expressed as micromoles of substrate transformed per minute per 100 mg protein. d.e Significant increase above controls at dP < 0.01 or eP < 0.05, Student’s t test; no significant difference in acetylcholinesterase.
serious physiological threat whether or not animals die. Lead is truly a systemic poison that adversely affects virtually all of the body systems to which it is distributed, especially the nervous system and the digestive system. Lead is such an efficient poison because it operates at the molecular level by inhibiting activities of enzymes that are required by all cells. Recent reviews of lead toxicity contain long lists of these sublethal effects (cf. National Academy of Sciences, 1972; Vallee and Ulmer, 1972; Coyer and Rhyne, 1973; Clemenser al., 1975; Kehoe, 1976). The common theme running through all these articles is that only parts-per-billion concentrations of lead exert sublethal toxic effects, and that development of more sensitive physiological measurements has resulted in a continual decline of the “no effect” level for lead. We have demonstrated in mallard ducks that 1 month after a single lead shot was ingested the blood lead concentration was about 1 ppm, and that the ratio between circulating lead and residues in two critical organs, the brain and liver, was 0.5 and 2.0, uncomfortably close to or above unity. This confirmed earlier field-collected data from canvasback ducks, where the ratio of lead residues between blood and organs was unity or greater (Dieter, 1978). We know then that blood lead levels can closely reflect brain and liver levels of lead, and that thousands of waterfowl are actually carrying these lead levels after ingesting spent lead shot. We have also demonstrated that this amount of lead inhibited blood SALAD enzyme activity by 75% and brain and liver SALAD enzyme activities by 35-50%. We can conclude that blood, brain, and liver lead levels, and the degree of SALAD enzyme inhibition in brain and liver, are reflected by blood SALAD enzyme inhibition with a reasonable degree of accuracy. Similar conclusions were reached by Mouw et al. (1975) in mammalian studies. They compared lead in blood, liver, brain, and other organs of urban and rural rats, and in those with elevated levels found up to six times as much lead in liver, and two times as much in brain, as in blood. They reported an inverse correlation between SALAD enzyme activity and lead in blood and kidney (r* = -0.60, P < O.OOl), showing that 50% inhibition corresponded to about 0.3 ppm blood lead and 5 ppm kidney lead. Hammond (1973) measured SALAD inhibition in rat liver after a single dose or up to 27 days of treatment with lead in the drinking water. He found a significant inverse correlation between lead and SALAD enzyme activity in blood, and also in liver (r2 = -0.69, P < 0.01). In another study lead and SALAD in rat tissues
LEAD
AND
8-ALAD
IN
DUCK
BRAIN
AND
LIVER
133
were examined after 3 and 6 weeks of chronic treatment with lead (Buchet et al., 1976). They observed that lead concentrations in blood and brain, liver, or kidney were highly correlated (r 2 = 0.9, P < 0.01). There were significant inverse correlations between lead and ij-ALAD enzyme activities in the kidney of both sexes and the brain of females exposed to lead for 6 weeks (r2 = -0.7, P < 0.01). It is evident then that lead concentrations that result in 8 -ALAD enzyme inhibition in blood also inhibit the enzyme in brain, liver, and kidney. It is not certain whether this magnitude of inhibition in tissues affects heme production to the extent that cellular processes dependent on heme, such as electron transport and ATP generation or cytochrome P-450 activity and hepatic detoxification, are seriously affected. It has been shown, for instance, that low lead concentrations, such as those we are reporting in tissues, inhibited adenyl cyclase (Nathanson and Bloom, 1975) and cytochrome P-450 activity (Scoppa et al., 1973) in ratbrain and liver and energy metabolism in rat brain (Bull et al., 1975). We noted that SALAD enzyme in the cerebellum of ducks was more sensitive to lead (50% inhibition) than the enzyme in the cerebral hemisphere (35% inhibition) and far more sensitive than that in the liver, where there was six times as mpch lead but only 42% inhibition of SALAD enzyme activity. This was also evident in the studies of Hammond (1973) and Buchet et al. (1976), who reported only slight SALAD enzyme inhibition in rat liver. Perhaps the lead in liver is bound in metallothionein complexes and is biologically inactive. We also demonstrated a significant increase in butyrylcholinesterase enzyme activity in mallard ducks that was greater in cerebellum (28% increase) than in cerebral hemisphere (22% increase). Butyrylcholinesterase is regarded as a marker enzyme for glial or supportive cells and other non-neuronal elements (Atherton and Zimmerman, 1974; Giacobini, 1964; Valcana and Timiras, 1974). An increase in butyrylcholinesterase activity suggested evidence of brain pathology, e.g., the biochemical change may be a reflection of a decrease in the number of neurons with a subsequent replacement by supportive cells and other elements. The biochemical lesions we have demonstrated in the brain, particularly in the cerebellum, precede the symptoms evident in the latter stages of lead poisoning. In waterfowl these include wing and head “droop,” flaccidity, and inability to fly and walk, which combined with impaction and starvation soon lead to death. Many of these symptoms are a result of cerebellar damage. The varied role of the cerebellum includes integrating visual and auditory input with motor function and organizing phasic reflex responses and skilled voluntary motion. This occurs when sensory input received by the cerebral cortex is passed to and from the cerebellum for modulation of response. Another important cerebellar function in waterfowl is modulation of the extensive reflex adjustments necessary for diving. The extent of brain pathology that can occur without permanent damage will await further studies correlating loss of function with brain lead accumulation. Present data indicate that lead levels found in brain and liver of ducks after ingestion of a single lead shot causes severe biochemical lesions. Lead-induced enzyme inhibition in critical organs may indirectly contribute to the unspecified mortality that occurs in waterfowl populations. REFERENCES Atherton,R. w., andZimmerman,G. D. (1974). Comparisonsbetweenacetyland butyrylcholinesterase activity
134
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in hibernating and non-hibernating ground squirrels, Spermophilus tridecemlineatus. Environ. Physiol. Biochem. 4, 97-103. Bellrose, F. C. (1959). Lead poisoning as a mortality factor in waterfowl populations. Ill. Nat. Hist. Surv. Bull. 27(3), 235-288. Buchet, J. P., Roels, H., Hubermont, G., and Lauwerys, R. (1976). Effect of lead on some parameters of the heme biosynthetic pathway in rat tissues in vivo. Toxicology 6, 21-34. Bull, R. J., Stanaszek, P. M., O’Neill, J. J., and Lutkenhoff, S. D. (1975). Specificity of the effects of lead on brain energy metabolism for substrates donating a cytoplasmic reducing equivalent. Environ. Health Perspecl. 12, 89-95. Burch, H. B., and Siegel, A. L. (1971). Improved method for measurement of delta-aminolevulinic acid dehydratase activity of human erythrocytes. Clin. Chem. 17, 1038. Clemens, E. T., Krook, L., Amnson, A. L., and Stevens, C. E. (1975). Pathogenesis of lead shot poisoning in the mallard duck. Cornell Vet. 65, 248-285. Dieter, M. P., Perry, M. C., and Mulhem, B. M. (1976). Lead and PCB’s in canvasback ducks: Relationship between enzyme levels and residues in blood. Arch. Environ. Contam. Toxicol. 5, l-13. Dieter, M. P. (In press). Use of the ALAD blood enzyme bioassay to monitor lead contamination in the canvasback duck population. In “Proceedings of International Symposium on Pathobiology of Environmental Pollutants.” Dieter, M. P., and Finley, M. T. (1978). Erythrocyte Gaminolevulinic acid dehydmtase activity in mallard ducks: Dmation of inhibition after lead shot dosage. J. wild. Manage. 42(3), 621-625. Ellman, G. L., Courtney, K. D., Andres V., Jr., and Featherstone, R. M. (1961). A new and rapid calorimetric determination of acetvlcholinesterase activity. Biochem. Pharmacol. 7, 88. Finley, M. T., Dieter, M. P., and Locke, L. N. (1976a). GAminolevulinic acid dehydratase: Inhibition in ducks dosed with lead shot. Environ. Res. 12, 243-249. Finley, M. T., Dieter, M. P., and Locke, L. N. (1976b). Lead in tissues of mallard ducks dosed with two types of lead shot. Bull. Environ. Contam. Toxicol. 16(3), 261-269. Finley, M. T., and Dieter, M. P. (1978). Toxicity of an experimental lead-iron shot versus commercial lead shot in mallards. J. Wildl. Manage. 42, 32-39. Giacobini,. E. (1964). Metabolic relations between glia and neurons studied in single cells. In “Morphological and Biochemical Correlates of Neural Activity” (M. M. Cohen and R. S. Snider, Eds.), pp. 15-38. Harper, New York. Goyer, R. A., and Rhyne, B. C. (1973). Pathological effects of lead. Int. Rev. Exp. Parhol. l2, l-77. Hammond, P. B. (1973). The relationship between inhibition of &aminolevulinic acid dehydratase by lead and lead mobilization by ethylenediamine tetraacetate (EDTA). Toxicol. Appl. Pharmacol. 26, 466-475. Hemberg, S., Nikkanen, J., Mellin, G., and Lilius, H. (1970). GAminolevulinic acid dehydrase as a measure of lead exposure. Arch. Environ. Health 21, 140-145. Hodson, P. V. (1976). SAminolevulinic acid dehydratase activity of fish blood as an indicator of a harmful exposure to lead. J. Fish. Res. Board Canad. 33, 268-271. Kehoe, R. A. (1976). Pharmacology and toxicology of heavy metals: Lead. Pharmac. Ther. 1, 161-188. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265. Mouw, D., Kalitis, K., Anver, M., Schwartz, J., Constan, A., Hartung, R., Cohen, B., and Ringler, D. (1975). Lead: Possible toxicity in urban vs rural rats. Arch. Environ. Health 30, 276-280. Nathanson, J. A., and Bloom, F. E. (1975). Lead-induced inhibition of brain adenyl cyclase. Nature (London) 255, 419-420. National Academy of Sciences. (1972). Committee on Biologic Effects of Atmospheric Pollutants. In “Lead Airborne Lead in Perspective,” pp. 205-212. Washington, D.C. Ohi, G., Seki, H., Akiyama, K., and Yagyu, H. (1974). The pigeon, a sensor of lead pollution. Bull. Environ. Contam. Toxicof. U(l), 92-98. Sassa, S., Granick, S., and Kappas, A. (1975). Effect of lead and genetic factors on heme biosynthesis in the human red cell. Ann. N.Y. Acad. Sci. 244, 419-440. Scoppa, P., Roumengous, M., and Penning, W. (1973). Hepatic drug metabolizing activity in lead-poisoned rats. Experientia 29, 970-972. U. S. Fish and Wildlife Service. (1972). Waterfowl Status Report. Spec. Sci. Rep. Wild]. No. 166.
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Valcana, T., and Timiras, P. S. (1974). Effects of X-radiation on the development of the cholinergic systemof the rat brain. I. Study of alterations in choline acetyltransferase and acetylcholmesterase activity and acetylcholinesterase synthesis. Environ. Physiol. Biochem. 4, 41-57. Vallee, B. L.. and Ulmer, D. D. (1972). Biochemical effects of mercury, cadmium, and lead. Annu. Rev. Biochem. 41, 91-128.
RESEARCH
19, 127-135
(1979)
SAminolevulinic Acid Dehydratase Enzyme Activity Blood, Brain, and Liver of Lead-Dosed Ducks M. P. DIETER*
in
AND M. T. FINLEY?
*U. S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, Maryland20811; tU. S. Fish and Wildlife Service, Fish Pesticide Research Laboratory. Columbia, Missouri 65201 Received
August
1, 1978
Mallard ducks were dosed with a single shotgun pellet (ca. 200 mg lead). After 1 month there was about 1 ppm lead in blood, 2.5 in liver, and 0.5 in brain. Lead-induced inhibition of Gaminolevulinic acid dehydratase enzyme in blood and cerebellum was much greater than in cerebral hemisphere or liver and was strongly correlated with the lead concentration in these tissues. The cerebellar portion of the brain was more sensitive to 8aminolevulinic acid dehydratase enzyme inhibition by lead than were the other tissues examined. There was also a greater increase in the glial cell marker enzyme, butyrylcholinesterase, in cerebellum than in cerebral hemisphere, suggesting that nonregenerating neuronal cells were destroyed by lead and replaced by glial cells in that portion of the brain. Even partial loss of cerebellar tissue is severely debilitating in waterfowl, because functions critical to survival such as visual, auditory, motor, and reflex responses are integrated at this brain center.
Inhibition of &aminolevulinic acid dehydratase (CALAD) has become a standard bioassay to detect acute and chronic lead exposure (Hemberg er al., 1970; Ohi ef al., 1974; Mouw et al., 1975; Hodson, 1976; Dieter et al., 1976). The enzyme is operative in the biosynthetic pathway of heme synthesis to maintain hemoglobin content in erythrocytes and to insure that adequate amounts of heme are available for incorporation into mitochondrial cytochromes (Sassaer al., 1975). The cytochtomes transport electrons through a redox potential and in the process generate high energy phosphate or ATP as a source of cellular energy. Inhibition of CALAD in cells of critical organs could inflict serious damage because the electron transport system, which is the energetic machinery of the cell, would be jeopardized. In addition, heme is a part of cytochrome P-450 that is necessary for some detoxification processes in the liver (Sassa et al., 1975). This study addresses the question of sublethal toxicity in migrating waterfowl that obtain balanced rations and do not immediately die from lead poisoning. We demonstrated that there was enough lead in one shotgun pellet to cause 60-90% inhibition of 6ALAD enzyme activity in erythroyctes, but not enough to kill waterfowl fed balanced diets (Finley ef al., 1976a,b). The inhibition persisted for 3 months (Dieter and Finley, 1978). Waterfowl ingesting one shotgun pellet may accumulate enough lead in their soft organs to disturb critical cellular functions but not enough to cause mortality. To initiate an examination of this hypothesis we have determined the extent of SALAD enzyme inhibition in blood, brain, and liver of lead-dosed ducks and have examined the relationships between lead accumulation and enzyme activity in these tissues. MATERIALS
AND METHODS
First-year-breeding mallard ducks (Anus plazyrhynchos) were paired and kept in vinylcoated wire pens on a long-day (16L:8D) light cycle. They were fed ad libitum diets of one-half yellow corn and one-half commercial breeder pellets ground to a mash. The 127 0013-9351/79/030127-09$02.00/O Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in any form reserved.
128
DIETER
AND
FINLEY
pellets contained 3.1% calcium, 0.8% total phosphorous, and 850 IU vitamin D; the corn essentially diluted these concentrations in half. After 6 weeks half of the pairs of ducks were dosed with one No. 4 commercial lead shot. Average shot weight was 206 mg with a range between 195 and 215 mg. Shot was administered through a flexible plastic tube forced down to the proventriculus. Controls were untreated. At 1 month post-dosage blood samples were obtained from the alar vein, the ducks killed by cervical dislocation, and necropsies performed. Blood &ALAD (EC 4.2124) enzyme assays were carried out immediately (Burch and Siegel, 1971). The bone lead concentrations were compared and the results reported elsewhere (Finley and Dieter, 1978). Whole brain and liver were frozen at -80°C until enzyme and residue analyses. Liver samples were divided in half and used for enzyme and lead assays. One of the cerebral hemispheres and the cerebellum were separated from the whole brain and enzyme assays run on each part. Lead residues were determined with the remainder of brain sample. Results from brain lead residues of a subset of dosed ducks showed that the lead concentrations in one cerebral hemisphere, the cerebellum, and the remainder of the brain were not different. There was a highly significant correlation between the lead concentration in the remainder of the brain [0.61 + 0.19 (mean ppm lead + standard error)], the cerebellum (0.59 ? 0.19), and the cerebral hemisphere (0.51 f 0.16), withr = 0.95, P < 0.01, N = 15. This data permitted us to use a distinct portion of the brain for enzyme assays and the rest for lead assays. One cerebral hemisphere or the cerebellum was prepared in cold pH 7.4 Tris buffer with a glass homogenizer tube and a motorized Teflon pestle. Samples of whole homogenate were used for &ALAD enzyme assays (Burch and Siegel, 1971) or for acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) enzyme determinations using either acetylthiocholine or butyrylthiocholine as substrate (Bellman er al., 1961). Livers were prepared in a similar fashion for GALAD enzyme assays only. Total protein concentrations in the homogenates were determined by the Lowry (1951) method. Reactants were adjusted to insure there were no rate limiting conditions; activities of assays were proportional to homogenate added. Whole blood, brain, and liver were shipped frozen to Environmental Trace Substances Research Center, Columbia, Missouri, for lead determinations. A 50-~1 blood sample was diluted 1:9 with distilled water and the lead concentration determined by the method of standard additions followed with the analysis of a standard. Samples of brain and liver were homogenized prior to analysis, They were ashed and digested in an acid solution. All samples were assayed on a Perkin-Elmer model 303 atomic absorption spectrophotometer at 283.3 nm. [See also “Analytical Methods for Atomic Absorption Spectrophotometry,” Perkin-Elmer Corp., Norwalk, Conn. (1971).1 Limits of sensitivity were 0.02 ppm; residues are reported on a wet weight basis. RESULTS
There was no mortality or significant changes in body weight as a result of lead shot dosage. Fluoroscopy at 1 week insured that all ducks used for this study had retained the shotgun pellets given them. Most of the shot in the gizzard had been eroded by 1 month post-dosage. Egg production between dosed and undosed groups did not differ. Reproductive tracts and livers of laying hens were much larger than in non-layers. Almost 1 ppm lead remained in the circulation of lead-dosed ducks at 1 month post-
LEAD
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LIVER
1
TISSUE LEAD CONCENTRATIONS (ppm) IN MALLARD No. 4 COMMERCIAL LEAD SHOT
DUCKS 1 MONTH AVER DOSAGE WITH A (ca. 200 mg LEAD)~
Blood
BrainC
Liver
0.06 c_ 0.01 0.98 k 0.18
0.06 f 0.01 0.43 IT 0.08
0.16 f 0.07 2.42 2 0.89
TreatmenP None Dosedd
AND
0 Means 2 standard error; seven males and seven females per group. b There were no significant differences between sexes, and values were combined. c Lead residues in whole brain minus cerebellum and one cerebral hemisphere. tl There were significantly higher lead residues in all tissues of dosed ducks; P < 0.01, Student’s f test.
treatment; the brain lead concentration was about half, and the liver lead concentration about two times that in blood (Table 1). There were no significant differences between sexes. The extent .of &ALAD enzyme inhibition by lead was compared in the different tissues (Table 2). There were no significant differences in total protein concentrations in brain or liver homogenates. There was a 75% inhibition of &ALAD enzyme activity in the blood and a 42% inhibition in the liver, compared to controls. There was a 50% inhibition of 6ALAD enzyme activity in the cerebellum, and a 35% inhibition in the cerebral hemisphere of the brain. Figure 1 indicates that lead residues in the brain or Iiver accurately reflected those in the blood at 1 month post-dosage. There was also a highly predictable relationship in blood and in cerebellum between lead and inhibition of &ALAD enzyme activity in these tissues (Fig. 2). Table 3 lists the correlation coefficients for the various comparisons that were examined. Some measure of brain pathology was afforded by measurements of esterase enzyme activity in brain using acetylcholine or butyrylcholine as substrate. The latter substrate is specific for glial cells; enhanced butyrylcholinesterase activity is indicative of nerve cell damage and subsequent replacement by glial cells or supportive elements. There was a 28% increase in butytylcholinesterase activity in cerebellum and a 22% increase in the TABLE ACTIVITY
2
OF &AMINOLEVULINIC ACID DEHYDRATASE ENZYME IN TISSUES OF MALLARD DUCKS 1 MONTH ARER DOSAGE WITH A No. 4 COMMERCIAL LEAD SHOT (ca. 200 mg LEAD)”
Braind TreatmenP
BloodC
Liverd
None Dosed Changee
180.4 f 11.7 45.6 zk 10.8 -75%
138.2 2 11.9 80.7 2 8.7 -42%
Cerebellum
Cerebral hemisphere
144.9 2 10.3 72.7 ? 5.9 -50%
96.9 k 6.6 63.2 2 5.2 -35%
a Means f standard error; seven males and seven females per group. b There were no significant differences between sexes, and values were combined. c Enzyme activity expressed as increase in absorbance of 0.100 per hour per milliliter erythrocytes. d Enzyme activity expressed as micromoles substrate transformed per hour per 100 mg protein. e There were significantly lower enzyme activities in all tissues of dosed ducks; P <: 0.01, Student’s t test.
130 x n
DIETER
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2
.L fl
0.2
[I:
W _I w
.02
1 In 111 F 0
#02
0.2
.02
2
f3LIlllD
0.2
2
LEAD (PPM)
RG. 1. Correlation between lead concentration in blood and tissues of mallard ducks. The lines of fit are described by these power regressions: log brain lead = log 0.39 + 0.62 log blood lead; log liver lead = log 1.60 + 0.85 log blood lead. These relationships were statistically significant Qv = 28, P < 0.01).
cerebral hemisphere of the brain of lead-dosed ducks (Table 4). There were no significant changes in acetylcholinesterase activity. DISCUSSION Waterfowl are particularly vulnerable to lead poisoning because of their feeding habits and preference for grains. Ducks mistake spent pellets for grit and may ingest one or more lead shots. Ducks are particularly sensitive to lead if they are feeding on imbalanced diets such as corn or rice, and they die in about lo- 14 days after ingestion of a single lead shot (Finley and Dieter, 1978). Surveys over a 20-year period showed that 7% of the waterfowl annually ingested at least one spent lead shotgun pellet. It was conservatively estimated that 2% of these waterfowl die from lead poisoning (Bellrose, 1959). By subtraction, 5% of the population annually ingesting a lead shot survive if nutritionally adequate diets are available, but they cannot avoid the sublethal effects of lead poisoning. For example, given a waterfowl population of 2OO,OOO,or about the estimate for canvasback ducks in the Atlantic Flyway (U. S. Fish & Wildlife Service, 1972), 40 (2%) annually die from lead poisoning, and 10,OOO(5%) endure sublethal toxicity from a massive dose of lead. Other than waterfowl, only young children are routinely exposed to such extremes when they display pica and ingest paint chips containing high lead concentrations. We belabor an obvious point because a sublethal toxic challenge by lead imposes a
LEAD
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BRAIN
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LIVER
2SB
SE
I0
10
CEREfKLLUM
f3LOilD
0 la
I
I
I
.I&!
0.2
2
TI!ZilJE
0
0
l
0.2
2
LERD (PPM)
lead concentration and kminolevulinic acid dehydratase enzyme (ALAD) activity in blood and brain of mallard ducks. The line of fit is described by these power regressions: log blood ALAD activity = log 27.6 - 0.59 log blood lead; log cerebellum ALAD activity = log 47.7 - 0.37 log brain lead. These relationships were statistically significant @’ = 28, P < 0.01). FIG. 2. Correlation
between
TABLE 3 CORRELATIONSBETWEEN LEAD AND SALAD ENZYME ACTIVITY IN TISSUES OF MALLARD DUCKS 1 MONTH ARER DOSAGE WITH A No. 4 COMMERCIAL LEAD SHOT”.* Comparisons’ (independent variable vs dependent variable) Blood lead vs brain leadd Blood lead vs liver lead Blood lead vs SALAD in blood SALAD in blood vs SALAD in liver SALAD in blood vs SALAD in cerebral hemisphere SALAD in blood vs &ALAD in cerebellum Brain lead vs GALAD in cerebral hemisphere Brain lead vs SALAD in cerebellum Liver lead vs GALAD in liver
Correlation coefftcient (r*) + + + + + -
0.82 0.72 0.77 0.41 0.43 0.59 0.47 0.75
- 0.34
(1Values are product-moment correlation coefftcients following log transformation values for both treatment groups (n = 28). b N = 28 (seven pairs dosed and seven pairs undosed ducks). e P < 0.01 for all comparisons, linear regression analysis. d Lead residues in whole brain minus cerebellum and one cerebral hemisphere
of combined
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OF ESTERASE WITH
A
ENZYMES
No. 4
IN BRAIN
COMMERCIAL
FINLEY
4
OF MALLARD LEAD
SHOT
Acetylcholinesterase’
DUCKS 1 MONTH (ca. 200 mg LEAD)”
AFTER
DOSAGE
ButyrylcholinesteraseC
TreatmenP
Cerebellum
Cerebral hemisphere
Cerebellum
Cerebral hemisphere
None Dosed Change
63.7 k 4.1 78.7 k 6.4 N.S.
48.3 k 4.2 57.1 2 4.0 N.S.
12.01 + 0.71 15.40 2 0.74 +28%d
8.58 -+ 0.57 10.43 2 0.59 +22%’
Q Means t standard error; seven males and seven females per group. b There were no significant differences between sexes and values were combined. c Enzyme activity expressed as micromoles of substrate transformed per minute per 100 mg protein. d.e Significant increase above controls at dP < 0.01 or eP < 0.05, Student’s t test; no significant difference in acetylcholinesterase.
serious physiological threat whether or not animals die. Lead is truly a systemic poison that adversely affects virtually all of the body systems to which it is distributed, especially the nervous system and the digestive system. Lead is such an efficient poison because it operates at the molecular level by inhibiting activities of enzymes that are required by all cells. Recent reviews of lead toxicity contain long lists of these sublethal effects (cf. National Academy of Sciences, 1972; Vallee and Ulmer, 1972; Coyer and Rhyne, 1973; Clemenser al., 1975; Kehoe, 1976). The common theme running through all these articles is that only parts-per-billion concentrations of lead exert sublethal toxic effects, and that development of more sensitive physiological measurements has resulted in a continual decline of the “no effect” level for lead. We have demonstrated in mallard ducks that 1 month after a single lead shot was ingested the blood lead concentration was about 1 ppm, and that the ratio between circulating lead and residues in two critical organs, the brain and liver, was 0.5 and 2.0, uncomfortably close to or above unity. This confirmed earlier field-collected data from canvasback ducks, where the ratio of lead residues between blood and organs was unity or greater (Dieter, 1978). We know then that blood lead levels can closely reflect brain and liver levels of lead, and that thousands of waterfowl are actually carrying these lead levels after ingesting spent lead shot. We have also demonstrated that this amount of lead inhibited blood SALAD enzyme activity by 75% and brain and liver SALAD enzyme activities by 35-50%. We can conclude that blood, brain, and liver lead levels, and the degree of SALAD enzyme inhibition in brain and liver, are reflected by blood SALAD enzyme inhibition with a reasonable degree of accuracy. Similar conclusions were reached by Mouw et al. (1975) in mammalian studies. They compared lead in blood, liver, brain, and other organs of urban and rural rats, and in those with elevated levels found up to six times as much lead in liver, and two times as much in brain, as in blood. They reported an inverse correlation between SALAD enzyme activity and lead in blood and kidney (r* = -0.60, P < O.OOl), showing that 50% inhibition corresponded to about 0.3 ppm blood lead and 5 ppm kidney lead. Hammond (1973) measured SALAD inhibition in rat liver after a single dose or up to 27 days of treatment with lead in the drinking water. He found a significant inverse correlation between lead and SALAD enzyme activity in blood, and also in liver (r2 = -0.69, P < 0.01). In another study lead and SALAD in rat tissues
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133
were examined after 3 and 6 weeks of chronic treatment with lead (Buchet et al., 1976). They observed that lead concentrations in blood and brain, liver, or kidney were highly correlated (r 2 = 0.9, P < 0.01). There were significant inverse correlations between lead and ij-ALAD enzyme activities in the kidney of both sexes and the brain of females exposed to lead for 6 weeks (r2 = -0.7, P < 0.01). It is evident then that lead concentrations that result in 8 -ALAD enzyme inhibition in blood also inhibit the enzyme in brain, liver, and kidney. It is not certain whether this magnitude of inhibition in tissues affects heme production to the extent that cellular processes dependent on heme, such as electron transport and ATP generation or cytochrome P-450 activity and hepatic detoxification, are seriously affected. It has been shown, for instance, that low lead concentrations, such as those we are reporting in tissues, inhibited adenyl cyclase (Nathanson and Bloom, 1975) and cytochrome P-450 activity (Scoppa et al., 1973) in ratbrain and liver and energy metabolism in rat brain (Bull et al., 1975). We noted that SALAD enzyme in the cerebellum of ducks was more sensitive to lead (50% inhibition) than the enzyme in the cerebral hemisphere (35% inhibition) and far more sensitive than that in the liver, where there was six times as mpch lead but only 42% inhibition of SALAD enzyme activity. This was also evident in the studies of Hammond (1973) and Buchet et al. (1976), who reported only slight SALAD enzyme inhibition in rat liver. Perhaps the lead in liver is bound in metallothionein complexes and is biologically inactive. We also demonstrated a significant increase in butyrylcholinesterase enzyme activity in mallard ducks that was greater in cerebellum (28% increase) than in cerebral hemisphere (22% increase). Butyrylcholinesterase is regarded as a marker enzyme for glial or supportive cells and other non-neuronal elements (Atherton and Zimmerman, 1974; Giacobini, 1964; Valcana and Timiras, 1974). An increase in butyrylcholinesterase activity suggested evidence of brain pathology, e.g., the biochemical change may be a reflection of a decrease in the number of neurons with a subsequent replacement by supportive cells and other elements. The biochemical lesions we have demonstrated in the brain, particularly in the cerebellum, precede the symptoms evident in the latter stages of lead poisoning. In waterfowl these include wing and head “droop,” flaccidity, and inability to fly and walk, which combined with impaction and starvation soon lead to death. Many of these symptoms are a result of cerebellar damage. The varied role of the cerebellum includes integrating visual and auditory input with motor function and organizing phasic reflex responses and skilled voluntary motion. This occurs when sensory input received by the cerebral cortex is passed to and from the cerebellum for modulation of response. Another important cerebellar function in waterfowl is modulation of the extensive reflex adjustments necessary for diving. The extent of brain pathology that can occur without permanent damage will await further studies correlating loss of function with brain lead accumulation. Present data indicate that lead levels found in brain and liver of ducks after ingestion of a single lead shot causes severe biochemical lesions. Lead-induced enzyme inhibition in critical organs may indirectly contribute to the unspecified mortality that occurs in waterfowl populations. REFERENCES Atherton,R. w., andZimmerman,G. D. (1974). Comparisonsbetweenacetyland butyrylcholinesterase activity
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in hibernating and non-hibernating ground squirrels, Spermophilus tridecemlineatus. Environ. Physiol. Biochem. 4, 97-103. Bellrose, F. C. (1959). Lead poisoning as a mortality factor in waterfowl populations. Ill. Nat. Hist. Surv. Bull. 27(3), 235-288. Buchet, J. P., Roels, H., Hubermont, G., and Lauwerys, R. (1976). Effect of lead on some parameters of the heme biosynthetic pathway in rat tissues in vivo. Toxicology 6, 21-34. Bull, R. J., Stanaszek, P. M., O’Neill, J. J., and Lutkenhoff, S. D. (1975). Specificity of the effects of lead on brain energy metabolism for substrates donating a cytoplasmic reducing equivalent. Environ. Health Perspecl. 12, 89-95. Burch, H. B., and Siegel, A. L. (1971). Improved method for measurement of delta-aminolevulinic acid dehydratase activity of human erythrocytes. Clin. Chem. 17, 1038. Clemens, E. T., Krook, L., Amnson, A. L., and Stevens, C. E. (1975). Pathogenesis of lead shot poisoning in the mallard duck. Cornell Vet. 65, 248-285. Dieter, M. P., Perry, M. C., and Mulhem, B. M. (1976). Lead and PCB’s in canvasback ducks: Relationship between enzyme levels and residues in blood. Arch. Environ. Contam. Toxicol. 5, l-13. Dieter, M. P. (In press). Use of the ALAD blood enzyme bioassay to monitor lead contamination in the canvasback duck population. In “Proceedings of International Symposium on Pathobiology of Environmental Pollutants.” Dieter, M. P., and Finley, M. T. (1978). Erythrocyte Gaminolevulinic acid dehydmtase activity in mallard ducks: Dmation of inhibition after lead shot dosage. J. wild. Manage. 42(3), 621-625. Ellman, G. L., Courtney, K. D., Andres V., Jr., and Featherstone, R. M. (1961). A new and rapid calorimetric determination of acetvlcholinesterase activity. Biochem. Pharmacol. 7, 88. Finley, M. T., Dieter, M. P., and Locke, L. N. (1976a). GAminolevulinic acid dehydratase: Inhibition in ducks dosed with lead shot. Environ. Res. 12, 243-249. Finley, M. T., Dieter, M. P., and Locke, L. N. (1976b). Lead in tissues of mallard ducks dosed with two types of lead shot. Bull. Environ. Contam. Toxicol. 16(3), 261-269. Finley, M. T., and Dieter, M. P. (1978). Toxicity of an experimental lead-iron shot versus commercial lead shot in mallards. J. Wildl. Manage. 42, 32-39. Giacobini,. E. (1964). Metabolic relations between glia and neurons studied in single cells. In “Morphological and Biochemical Correlates of Neural Activity” (M. M. Cohen and R. S. Snider, Eds.), pp. 15-38. Harper, New York. Goyer, R. A., and Rhyne, B. C. (1973). Pathological effects of lead. Int. Rev. Exp. Parhol. l2, l-77. Hammond, P. B. (1973). The relationship between inhibition of &aminolevulinic acid dehydratase by lead and lead mobilization by ethylenediamine tetraacetate (EDTA). Toxicol. Appl. Pharmacol. 26, 466-475. Hemberg, S., Nikkanen, J., Mellin, G., and Lilius, H. (1970). GAminolevulinic acid dehydrase as a measure of lead exposure. Arch. Environ. Health 21, 140-145. Hodson, P. V. (1976). SAminolevulinic acid dehydratase activity of fish blood as an indicator of a harmful exposure to lead. J. Fish. Res. Board Canad. 33, 268-271. Kehoe, R. A. (1976). Pharmacology and toxicology of heavy metals: Lead. Pharmac. Ther. 1, 161-188. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265. Mouw, D., Kalitis, K., Anver, M., Schwartz, J., Constan, A., Hartung, R., Cohen, B., and Ringler, D. (1975). Lead: Possible toxicity in urban vs rural rats. Arch. Environ. Health 30, 276-280. Nathanson, J. A., and Bloom, F. E. (1975). Lead-induced inhibition of brain adenyl cyclase. Nature (London) 255, 419-420. National Academy of Sciences. (1972). Committee on Biologic Effects of Atmospheric Pollutants. In “Lead Airborne Lead in Perspective,” pp. 205-212. Washington, D.C. Ohi, G., Seki, H., Akiyama, K., and Yagyu, H. (1974). The pigeon, a sensor of lead pollution. Bull. Environ. Contam. Toxicof. U(l), 92-98. Sassa, S., Granick, S., and Kappas, A. (1975). Effect of lead and genetic factors on heme biosynthesis in the human red cell. Ann. N.Y. Acad. Sci. 244, 419-440. Scoppa, P., Roumengous, M., and Penning, W. (1973). Hepatic drug metabolizing activity in lead-poisoned rats. Experientia 29, 970-972. U. S. Fish and Wildlife Service. (1972). Waterfowl Status Report. Spec. Sci. Rep. Wild]. No. 166.
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Valcana, T., and Timiras, P. S. (1974). Effects of X-radiation on the development of the cholinergic systemof the rat brain. I. Study of alterations in choline acetyltransferase and acetylcholmesterase activity and acetylcholinesterase synthesis. Environ. Physiol. Biochem. 4, 41-57. Vallee, B. L.. and Ulmer, D. D. (1972). Biochemical effects of mercury, cadmium, and lead. Annu. Rev. Biochem. 41, 91-128.