TOXKOLOGY
AND
APPLIED
Persistent
PHARMACOLOGY
35,365-379 (1976)
Effects of Sarin and Dieldrin Primate Electroencephalogram
upon
the
JAMESL. BURCHFIEL,FRANK H. DUFFY, AND VAN M. SIM’ Seizure Unit Neurophysiology Laboratory, Children’s Hospital Medical Center, and Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115; and Biomedical Laboratory, Edgewood Arsenal, U.S. Army Aberdeen Proving Ground, Maryland 21010 Received August 4,1975; accepted October 26,197s
PersistentEffects of Sat-inand Dieldrin upon the Primate Electroencephalogram.BURCHHEL, J. L., DUFFY, F. H. AND SIM, V. M. (1976).Toxicol. Appl. Pharmacol. 35, 365-379. Rhesusmonkeys were injected with the organophosphatesarin or the chlorinatedhydrocarbon dieldrin according to oneof two schedules : (1) a single“large dose” (5 ,ug/kgof satin or 4 mg/ kg of die&in, iv) which produced overt signsof toxicity, or (2) a series of 10 “small doses”(1 pg/kg of sarin or 1 mg/kg of die&in, im, given 1 weekapart) which did not produceany major clinical signs.Electroencephalograms (EEG) were recorded from chronically implanted electrodes according to the following schedule:(1) three recording sessions prior to drug administration, (2) one at 24 hr post-drug, and (3) another three at 1 yr post-drug. Each recording sessionconsistedof runs in the following states: (1) awake and alert in a lighted environment, (2) awakeand alert in total darkness,and (3) drowsy. A seriesof control monkeys received equaltreatment but weregiven only drug diluent. Fast-Fourier transforms were performedon the EEGs with a PDP-12 computer to yield voltage vs frequency spectra.Resultsof statisticalanalysisof EEG spectrashowedthe following: For both sarinand dieldrin the singlelarge-doseadministration produced significantincreasesin the relative amount of beta voltage (1550 Hz) which persistedfor 1 yr. For sarin the predominanteffect wasin the EEG derivation from the temporalcortex, and for dieldrin from the frontal cortex. For both drugs,the beta increasewasmost prominentin the states of awake in darknessand drowsy. These results indicate that a single symptomatic exposure or a seriesof subclinical exposuresto satin or dieldrin can alter the frequency spectrumof the spontaneousEEG for up to 1 yr. Organophosphate (OP) and chlorinated hydrocarbon (CH) compounds are known to have potent effects upon the nervous systemsof a wide variety of organismsfrom simple invertebrates to man. This action has been used to considerable advantage in pest control where OP and CH compoundsconstitute two of the major classesof insecticides. The acute effects of OP and CH exposure upon the central nervous system(CNS) of man and other higher mammals include prominent electroencephalographic (EEG) changesand convulsions. In most instances,EEG abnormalities have been reported to disappear within 2 weeks of acute exposure or a termination of chronic exposure i BiomedicalLaboratory,EdgewoodArsenal,U.S. Army ProvingGround,Maryland. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
365
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BURCHFIEL,
DUFFY
AND
SIM
(Crescitelli and Gilman, 1946; Grob et al., 1947; Holmes and Gaon, 1956; Desi et al., 1966; Santolucito and Morrison, 1971). However, other studies indicate that the EEG effects of insecticide poisoning may persist for many weeks or months (Spiotta, 1951; Hoogendam et al., 1962; Dille and Smith, 1964; Kazantzis et al., 1964). Metcalf and Holmes (1969) suggested that OP exposure may lead to chronic EEG changes. They examined EEGs from a group of industrial workers with a past history of insecticide poisoning, but with no recent exposures, and reported a high incidence of abnormal low-to-medium voltage slow-wave activity in the theta range (4-6 Hz). The present study was undertaken to examine, under controlled conditions, the long-term effects of OP and CH exposure upon the primate brain as measured by changes in EEG activity. A representative OP compound (sarin) and a representative CH compound (dieldrin) were chosen because they were the two agents with the highest incidence of exposure in the Metcalf and Holmes study (personal communication). Dieldrin (85 % of the compound 1,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8aoctahydro-endo-exo-5,8-dimenthanonaphthalene and 15 y0 related compounds) is a widely used commercial insecticide. Sarin (GB; isopropyl methyl phosphonofluoridate), on the other hand, is not used commercially. It is ordinarily classified as a “nerve gas;” however, it shares a similar chemical structure and pharmacological action with more common OP insecticides such as malathion and parathion. Sarin differs from the commercial OP compounds in two major aspects. First, it is a fluorinated OP, and, second, it is considerably more potent. Rhesus monkeys were exposed to controlled doses of either dieldrin or sarin. These drug-treated animals, and appropriate controls, were then followed for 1 yr after acute exposure to assessthe presence of persistent changes in EEG activity. METHODS Experimental animal preparation. Twenty-two adolescent rhesus monkeys (Macaca mulatta) of both sexes, weighing 2.5-4.0 kg, were used in this study. Under general
anesthesia and aseptic conditions, cortical and depth electrodes were permanently implanted for chronic recording. The animals were initially anesthetized with a shortacting barbiturate (sodium methohexital) and maintained with methoxyfluorane and oxygen via an endotracheal tube. Cortical electrodes (O/80 stainless-steel screws) were placed into tapped holes in the skull so as to contact the intact dura. These were placed over the frontal, central, occipital, and temporal cortices bilaterally. Bipolar-depth electrodes (32-gauge insulated stainless-steel wire, l-mm tip separation, 0.5-mm tip exposure) were stereotaxically placed through small burr holes into the following sites : amygdala, hippocampus, mesencephalic reticular formation, and nucleus centrum medianum of the thalamus. Stereotaxic coordinates were based upon the atlas of Snider and Lee (1961). Electrode placements were histologically verified at postmortem. Insulated leads from the electrodes were soldered to two g-pin Winchester-type miniature connectors which were cemented to the skull with dental acrylic. Additional acrylic was used to form a durable, protective cap over the skull covering all leads and electrodes. Drug administration. Sarin for injection was prepared by dilution of an aliquot of purified drug (stored at -55°F) with normal saline. Biological effectiveness of this solution was determined routinely in rabbits and was never used more than 2 hr after
PERSISTENT
EEG EFFECTS
367
dilution. Chemical analysis of random samples indicated that sarin was always 90-95 % pure. Dieldrin was obtained in crystalline form (85 % technical grade) and dissolved in 100 % ethanol for injection. Sarin or dieldrin was administered according to one of two schedules. In one, animals received a single “large dose” of the drug: 5 ug/kg of sarin or 4 mg/kg of dieldrin, iv. In the other, animals received multiple “small doses” : a series of 10 injections of 1 pg/ kg of sarin or 1 mg/kg of dieldrin, im, given at intervals 1 week apart. Hereafter these will be referred to respectively as the “large-dose” and “small-dose” schedules. These dose levels were arrived at through pilot studies in which the attempt was made to find dose levels which would approximate, respectively, (1) a serious but sublethal exposure showing all the signs of poisoning and (2) a series of subclinical but near-threshold exposures showing few, if any, signs of overt poisoning. At the large-dose level of either sarin or dieldrin all animals had generalized seizures. During the pilot studies it was noted that prolonged periods of apnea often accompanied these seizures. Therefore, to prevent the possibility of secondary anoxic brain damage during large-dose administration, animals in the current study were paralyzed with gallamine triethiodide, and artificial respiration was achieved via an endotracheal tube. The drug was given by slow iv injection over 3 min. Animals were then allowed to recover from paralysis and breathe spontaneously when their EEG revealed a cessation of seizure activity. The average total length of paralysis for experimental animals was about 2.5 hr and never more than 4 hr. A group of control animals was similarly paralyzed and received an injection of an equal volume of drug diluent (saline for sarin or 100 % ethanol for dieldrin). The length of paralysis for controls was matched as nearly as possible to that of the experimentals. paralysis was not necessary for the animals receiving the multiple small doses. Injections were given im with the animal seated in a restraining chair. A group of control animals was handled in the same manner and received injections of drug diluent. Experimental design. Animals were randomly assigned to one of four drug-treatment groups: (1) single, large-dose sarin; (2) multiple, small-dose sarin; (3) single, large-dose dieldrin; or (4) multiple, small-dose dieldrin. There were three animals in each treatment group. These groups were matched with groups of control animals, three for each large-dose group and two for each small-dose group. Controls were subjected to the same injection schedules (including paralysis and artificial respiration for large-dose controls) but received only drug diluent. Each animal in the study was subjected to a series of EEG recording sessions. Prior to drug administration, three separate control sessions were performed on different days. For the single, large-dose animals, a recording was taken 24 hr after administration For the multiple, small-dose animals, a recording was taken 24 hr after administration ofthe last injection in the series. Subsequently, the EEGs of all animals were periodically monitored to check the integrity of the recording system and CNS activity. One year after exposure an additional three recordings were performed for all animals. During the l-yr interval the animals were maintained in separate cages in a temperaturecontrolled environment; they received no other drugs. EEG recording. The animal sat in a primate-restraining chair, with a mechanical head restraint, inside a closed, electrically shielded chamber. EEGs were recorded on seven-channel FM magnetic tape via revertor-coupled and calibrated outputs of a
368
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DUFFY
AND
SIM
Grass Model III polygraph with a bandpass of 0.5-80 Hz. An individual recording session consisted of the following runs: (1) Awake, alert in light, an epoch of at least 2-min duration in which the animal sat in the lighted chamber; alertness was maintained by intermittent auditory distraction; (2) Awake, alert in darkness, same as (l), but in darkness; (3) drowsy, an epoch of 15-30 min duration in which the animal sat in the darkened chamber and silence was maintained. For each run, bipolar recordings were obtained from the depth electrodes (amygdala, hippocampus, mesencephalic reticular formation, and nucleus centrum medianum of the thalamus) and from the cortical leads in the following bipolar pairings: frontalcentral, central-occipital, and occipital-temporal. Data analysis. Spectral analysis of the EEG data was accomplished on a PDP-12 computer using the comprehensive SIGSYS- 12 software system (Agrippa Data Systems). Signals were played back from FM tape through a Khrone-Hite filter set to bandpass OS-50 Hz (24 db per octave) and analog-to-digital converted by the computer at a rate of 256 Hz. Fast-Fourier transforms (FFTs) were performed, without smoothing, on 4-set epochs of EEG (computed on 1024 points over the frequency range O-128 Hz with a resolution of l/4 Hz). After FFT computation, the square root was taken of each frequency component to yield a voltage spectrum. Epochs containing excessive eye blinks, movement artifact, electrode artifact, or muscle activity were not analyzed. Consecutive epochs were averaged to yield a spectrum representing 1 min of EEG; i.e., the spectra of 15 4-set epochs were averaged. Two 1-min spectra were computed for each state in a recording session (i.e., awake in light, awake in darkness, and drowsy). The 2-min epoch of EEG most representative of drowsiness was selected by visual inspection of the polygraph tracing obtained in parallel with the analog tape recording. The selection of the drowsy epoch was made by an experienced electroencephalographer, based on the monkey EEG criteria of Reite et al. (1965). The electroencephalographer was unaware of a monkey’s exposure history at the time of selection. For each spectrum, a calculation was made of the percentage of total energy present in each of the classical EEG frequency bands defined as follows: delta = 0.5-3.75 Hz, theta = 3.75-7.75 Hz, alpha = 7.75-13.0 Hz, beta 1 = 13-22 Hz, and beta 2 = 22-50 Hz. The band from 50 to 128 Hz was not analyzed. For the final analysis, total spectra and frequency bands were averaged respectively for the three control sessions, the 24-hr session, and the three 1-yr sessions. This was done for each lead and each state. Thus, a final datum consisted of an average spectrum and frequency bands representing EEG activity from one electrode derivation, for one state, and for one set of sessions. Since two spectra were derived for each state and lead in a recording session, the values of N for the final averages were 6 for control, 2 for 24 hr, and 6 for 1 yr. In computing the final averages, sd were also calculated. Statistical analysis. Statistical analysis was performed in a two-step procedure. First, a longitudinal comparison was made for each animal using a two-tailed Student’s t test. The average spectrum and frequency bands derived from the control sessions (for a given lead and state) were compared first with that derived from the session at 24 hr post-drug and next with that from the sessions at 1 yr. For the average spectrum, the t test was applied at each point. As might be expected, this produced a large number of seemingly random “significant” differences. (Theoretically, one would expect an average of 5 % of the t tests to yield t values with a probability 60.05 merely by chance.)
PERSISTENT
EEG EFFECTS
369
Such differences at individual points throughout the spectrum proved very difficult to interpret. We felt that any criteria we might establish to determine when statistically significant points constituted a physiologically significant difference might suffer from subjective bias. For this reason we chose to base our results upon analysis of integrated frequency bands. Therefore, at the conclusion of the first step of statistical analysis, we had determined for each animal which frequency ranges were different by t test at 24 hr post-drug and at 1 yr post-drug as compared to the pre-drug control. In the longitudinal comparison, each animal served as his own control; data from animals within the same experimental group were not pooled for comparison. The next question, therefore, was which differences, if any, revealed by t test were consistently and exclusively seen among the drug-treated animals as opposed to control animals. This question was answered by the second step of the statistical analysis, Fisher’s test (Fisher, 1934). For this analysis the question was asked, how many drug-treated animals showed a significant change (by t test) in a particular frequency band and how many did not? The same question was asked of the control animals. From these answers a 2 x 2 contingency table was constructed and the exact probability of obtaining the resultant distribution in the table, or one more extreme, by chance alone was calculated. If this probability were ~0.05, then it was concluded that a significant change in frequency distribution of the EEG had occurred in a drug-treated group as compared to the controls. Statistical analysis among the various groups of control animals showed no consistent differences; therefore, for Fisher’s test all control animals were considered as a single group. RESULTS
Sarin
For either treatment schedule, no consistent, significant differences were found for any of the depth leads analyzed : amygdala, hippocampus, midbrain reticular formation, or nucleus centrum medianum of the thalamus. In the cortex, on the other hand, longterm alternations in EEG frequency spectra were observed for both the single, largedose and multiple, small-dose exposures. Large dose.The single large dose of sarin (5 pug/kg) produced a persistent increase in the relative amount of voltage in the beta frequency bands (13-22 and/or 22-50 Hz) of the occipital-temporal (O-T) EEG derivation. Figures 1 and 2 illustrate this for two of the sarin-treated animals during the state of awake in darkness. Examples of two control animals for the same lead and state are shown in Figs. 3 and 4. These examples illustrate some general findings which are applicable to all the drug treatment groups First of all, it can be seen in comparing the control records from these animals that a considerable degree of individual variability exists in the EEG frequency distributions. However, for a given animal, the spectra tended to be reasonably constant from day to day. The control animal shown in Fig. 3 is typical of the data from the 10 untreated animals. There are no significant differences among the spectra over the I-yr period of the study. Figure 4 shows an extreme case of variability for a control animal in which a number of apparently random alternations in frequency distribution occurred among the average spectra. It was because of the much greater degree of variability from animal to animal than from day to day for the same animal that the longitudinal method of statistical analysis described in the methods section was adopted.
BURCHFIEL, DUFFY AND SIM
370 CONTROL 36
24
-BAND
PERCENT ~-ENERGY
S.E.
;
25.85 16.24
1.52 0.29
A
17.72 17.44 25.88
0.21 0.30 1.33
HOURS PERCEhC ENERGY
S.E.
t VALUE vs CONTROL
22.96 19.82 16.66 22.08 21.90
1.57 0.70 0.85 1.52 1.51
1.01 5.76" 1.89 5.13** 1.57
PERCENT ENERGY
S.E.
D T
22.09 14.61
0.90 0.35
2.13 3.58**
Iti B2
17.4s 19.37 29.27
0.32 0.50 0.52
0.48 4.43"" 2.38*
BAND A
1 YEAR w
"s
t VALUE CONTROL
FIG. 1. Example of average voltage spectra and frequency bands for one animal receiving a single large dose of sarin. Occipital-temporal EEG derivation in the state of awake in darkness. For the spectra, the ordinate scale is in microvolts and the abscissa scale is in hertz. Frequency bands give percentages of total voltage (O-50 Hz) over the following ranges : D = delta (0.5-3.75 Hz); T = theta (3.75-7.75 Hz); A = alpha (7.75-13.0 Hz); Bl = beta 1 (13-22 Hz); B2 = beta 2 (22-50 Hz). The t value is the result of Student’s I test between the indicated band and the corresponding band of the control spectrum. *,p < 0.5; **, p < 0.01.
CONTROL
D T
19.89 19.32
0.92 1.05
B"1 B2
32.50 18.72 13.25
0.94 0.?8 0.41
24 HOURS 30 uv
BAND :
PERCENT ENERGY S.E.
t VALUE YB CONTROL
26.80 19.05
0.65 2.12
0.14 3.53**
A
19.39
0.33
7.64*'
i:
19.65 18.26
0.22 1.44
6.68** 0.59
1 YEAR BAND
PERCENT ENERGY S.E.
t VALUE YS CONTROL
D T A
21.05 17.16 30.36
0.29 0.73 0.93
0.99 1.51 1.55
:t
18.64 16.29
1.23 0.58
2.77. 0.07
FIG.2. Example of average voltage spectra and frequency bands for another large-dose sarin animal. Same EEG derivation and recording state as animal in Fig. 1. For explanation of scales and legends see Fig. 1.
PERSISTENT
371
EEG EFFECTS
CONTROL
24 HOURS
1 YEAR
FIG. 3. Example of average voltage spectra and frequency bands for one control animal. Same EEG derivation and recording state as Figs. 1 and 2. For explanation of scales and legends see Fig. 1.
CONTROL
24 HOURS 25
-BAND
uv
:! 0 0
50
A Bl B2
PERCENT ENERGY S.E. 23.99 17.40 21.10 20.30 20.41
1.58 0.90 0.44 1.40 1.56
t VALUE YS CONTROL 0.55 0.53 5.43"" 0.71 3.86'"
1 YEAR
FIG. 4. Example of average voltage spectra and frequency bands for another control animal. Same EEG derivation and recording state as Figs. l-3. For explanation of scales and legends see Fig. 1.
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SIM
Similar degrees of variability were seen among the drug-treated animals. However, the key factor among the large-dose sarin animals was that, regardless of what other alternations in the spectra might occur, there was a consistent increase of relative voltage in the beta frequency bands. For instance, in Fig. 1 at 24 hr post-drug there was a significant increase in beta 1 which was still present 1 yr later. But, in addition, there was a significant increase in theta at 24 hr and an increase in beta 2 and a decrease in theta at 1 yr. Similarly, the animal shown in Fig. 2 displayed a persistent increase in relative beta voltage but also had other changes at 24 hr. A summary of the results oft test analysis for increased relative beta activity in the O-T frequency spectrum is given in Table 1. Each entry in the table is the proportion TABLE 1 SINGLE LARGE-DOSE ADMINISTRATION OF SARIN, OCCIPITALTEMPORAL EEG Recording
24 hr Sarin Control
state
Awake in light
Awake in
darkness
Drowsy
O/3 o/10
313 2110 (p = 0.035)b
213 l/10 (p = 0.105)b
213 2/10
213 o/10 (p = 0.038)b
313 2110 (p = 0.035)b
1 yr
Sarin Control
a Sarin, 5 pg/kg, iv. Proportion of animals showing a significant increase by t test in relative beta voltage. A percentage of total voltage of spectrum in the frequency bands 13-22 or 22-50 Hz of the EEG frequency spectrum at 24 hr and 1 yr post-drug. * Exact probability of distribution of drug-treated and control animals occurring by chance (2x2 Fisher Test).
of animals (control or treated) which showed a significant increase in beta activity by t test. For a given post-drug session and recording state, the proportion of treated animals showing an increase and the proportion of controls showing an increase form a 2 x 2 contingency table. Beneath each pair of proportions, in parentheses, is given the Fisher exact probability that such a distribution could arise by chance alone (where there is obviously no difference, the probability is omitted). It can be seen that the increase in relative beta voltage was most prominent in the state of awake in darkness. At 24 hr after administration, all three drug-treated animals showed a statistically significant increase in beta activity by I test, whereas only two of ten controls showed such an increase. The probability of this distribution arising by chance is 0.035. At 1 yr after sarin exposure two of three treated monkeys still showed a relative increase in beta activity; none of the controls showed any increase. This distribution has a chance probability of 0.0385.
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PERSISTENT EEG EFFECTS
For the drowsy state there was a significant increase in beta activity for treated animals at 1 yr (all sarin animals showing an increase as opposed to two of ten controls, p = 0.035). However, at 24 hr this change was not quite statistically valid (an increase in two of three treated animals and in one of ten controls, p = 0.105). In the state of awake in light, there was no significant increase of relative voltage in the beta bands. However, there would appear to be a trend toward increased beta at 1 yr after exposure (two of three treated animals showed a significant increase, while eight of ten controls did not, p = 0.188). Small dose.The animals receiving the multiple small doses of sarin (1 pg/kg once a week for 10 weeks) showed a persistent relative increase in O-T beta activity similar CONTROL
24
PERCENT ENERGY
S.E.
34.46 19.79 10.40 15.83 15.09
1.96 0.87 1.39 0.83 1.17
HOURS PERCENT ENERGY
S.E.
t VALUE YS CONTROL
33.43 17.65 11.66 18.20 22.27
3.52 0.60 0.14 1.39 1.23
0.26 1.33 2.69* 1.44 3.25*'
-BAND
PERCENT ENERGY
S.E.
t VALUE YS CONTROL
D T A Bl 32
19.93 14.71 18.75 25.50 24.25
1.30 0.36 1.08 1.42 0.93
6.19" 4.93** 0.15 5.09" 6.15**
1 YEAR
FIG. 5. Example of average voltage spectra and frequency bands for one animal receiving multiple small-dose administration of sarin. Occipital-temporal EEG derivation in the state of awake in darkness. For explanation of scales and legends see Fig. 1.
to that seen with the large-dose animals. An example of spectra and frequency bands for one small-dose monkey is illustrated in Fig. 5. The results of t test analysis for increased beta activity in all small-dose animals is given in Table 2. At 24 hr after the last dose of sarin, all the treated animals showed a significant increase in beta for both the states of awake in darkness and drowsy. These increases were still present at 1 yr after exposure. In addition to the alternation in O-T EEG spectra, the small-dose animals also showed increases in relative beta activity in the frontal-central (F-C) EEG (Fig. 6 and Table 2). Such F-C alterations were not seen with the single large dose of sarin. The increase in F-C beta also differed from the increase in O-T beta in that it occurred only in the spectra at 1 yr post-drug; at 24 hr there was no significant increase (Table 2). In contrast, the spectra at 24 hr after termination of the small-dose schedule showed a
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consistent trend for all treated animals toward increases in relative voltage in the delta and theta bands and decreases in higher frequencies, particularly alpha. With time this frequency distribution reversed such that beta was increased and delta and theta decreased (see example in Fig. 6). The increase in relative beta at 1 yr was seen in all three recording states (Table 2). TABLE 2 MULTIPLE SMALL-DOSEADMINISTRATION OF SARIN, OccmTAL-TEMPORAL (O-T) AND FRONTAL-CENTRAL(F-C) EEG”. Recording state
A. O-T EEG 24 hr Sarin Control
1yr
Sarin Control
Awake in light
Awake in darkness
o/3 o/10
313 2/10 (p = 0.035y
213 2/10
213
o/10
(p=0.0385)*
B. F-C EEG 24 hr Sarin Control 1 Yr Sarin Control
Drowsy
313
l/10 (p = 0.014)b 313 2110 (p = 0.035)b
O/3
O/3
l/3
l/f3
218
118
313
313
313
118
l/8
l/8 (p = 0.024)*
(p = 0.024)*
(p=0.024)*
’ Sarin, 10 injections of 1 ,ug/kg, im, given at l-week intervals. Also seefootnote a, Table 1. b Seefootnote b, Table 1. Dieldrin
As with sarin, no consistent, significant differences were observed in the spectra from any of the subcortical structures. Also, like sarin, there were persistent increases in relative beta voltage in the cortical EEG spectra. Large dose.Increases in the relative amount of beta activity (13-22 and/or 22-50 Hz were observed in F-C EEG spectra. An example of this is illustrated for one animal in the state of awake in darkness in Fig. 7. The increase in F-C beta activity was seen at 24 hr post-drug in the states of awake in darkness and drowsy. At 1 yr post-drug it was still present during these states and also during the state of awake in light (Table 3). In general the alterations in frequency distribution tended to be more widespread at 1 yr after dieldrin exposure than at 24 hr. The example illustrated in Fig. 7 is typical
PERSISTENT EEG EFFECTS
375
CONTROL -BAND
PERCENT --ENERGY
S. E.
30.75 19.83 17.29 15.30 19.99
3.12 0.88 1.55 1.52 0.93
D i Bl BZ
24 HOURS PERCENT ENERGY
S. E.
t VALUE YS CONTROL
i:
34.93 20.97 15.30 14.57 17.53
0.59 0.02 0.98 1.02 0.85
0.73 0.71 0.69 0.26 1.40
BAND -
PERCENT ENERGY
S.E.
f VALUE YS CONTROL
19.59 13.90 17.40 26.03 26.07
1.10 0.37 1.34 1.41 0.74
3.37’ 6.21*’ 0.06 5.18’. 5.13**
BANo T” A
1 YEAR
T” A
FIG. 6. Example of average voltage spectra and frequency bands for the same animal as in Fig. 5 but for frontal+entral EEG derivation. Same recording state as in Fig. 5. For explanation of scales and legends see Fig. 1.
TABLE 3 SINGLELARGE-DOSEADMINISTRATIONOFDIELDRIN,FRONTAL -CENTRALEEG” Recording Awake in light 24 hr Dieldrin Control
state
Awake in darkness
u3
313
313
l/8
l/8
V3
(p = 0.024)b 1 yr Dieldrin Control
Drowsy
(p = 0.024)b
3/3
313
313
l/8
118
(p = 0.024)b
(p = 0.024)b
l/8
(p = 0.024)b
a Dieldrin, 4 mg/kg, iv. Also see footnote a, Table 1. b See footnote b, Table 1. of the treated animals in this respect. Note that at 24 hr after exposure, there was a significant change only in beta 1, whereas at 1 yr there were alterations in all frequency
bands except beta 2. At 1 yr, all the dieldrin-treated animals showed a significant decreasein the relative amount of theta voltage in the state of awake in darkness@ = 0.024,
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BURCHFIEL,
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AND
SIM
Fisher test) and a significant decrease in relative delta for the drowsy state (p = 0.024). At 24 hr, on the other hand, the only consistent change was the increase in relative beta. SmaZZ dose. The animals receiving the multiple, small-dose exposure to dieldrin exhibited EEG frequency changes in frontal cortex similar to those of the large-dose group. However, in the small-dose dieldrin group, one animal died before the 1-yr recording session. The two remaining animals both showed a statistically significant increase in beta activity at 1 yr for the states of awake in darkness and drowsy. Because of the small number of treated animals, this distribution (two of two treated animals showing an increase versus one of eight controls) did not quite reach the 0.05-level of significance by the Fisher test. The actual p value was 0.067. Had the other animal lived CONTROL 25 uv
BAND--
PERCENT ENERGY
S.E
;
17.54 28.95
1.10 0.57
A Bl B2
16.90 17.18 22.72
0.97 0.35 0.62
24 HOURS BAND-
PERCENT ENERGY
S.E.
T” A
28.37 15.97 17.48
0.16 3.67 2.54
0.22 1.51 0.27
;:
20.55 20.86
1.22 0.24
1.90 4.30**
S.E.
t VALUE vs CONTROL
“S
t VALUE CONTROL
1 YEAR BAND --
PERCENT ENERGY
D x
19.43 21.09 15.07
1.27 1.55 0.36
5.67** 3.66’ 2.30.
Bl B2
25.34 22.68
0.74 0.69
9.86.. 0.03
FIG. 7. Example of average voltage spectra and frequency bands for large-dose dieldrin animal. Frontal
AND
APPLIED
Persistent
PHARMACOLOGY
35,365-379 (1976)
Effects of Sarin and Dieldrin Primate Electroencephalogram
upon
the
JAMESL. BURCHFIEL,FRANK H. DUFFY, AND VAN M. SIM’ Seizure Unit Neurophysiology Laboratory, Children’s Hospital Medical Center, and Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115; and Biomedical Laboratory, Edgewood Arsenal, U.S. Army Aberdeen Proving Ground, Maryland 21010 Received August 4,1975; accepted October 26,197s
PersistentEffects of Sat-inand Dieldrin upon the Primate Electroencephalogram.BURCHHEL, J. L., DUFFY, F. H. AND SIM, V. M. (1976).Toxicol. Appl. Pharmacol. 35, 365-379. Rhesusmonkeys were injected with the organophosphatesarin or the chlorinatedhydrocarbon dieldrin according to oneof two schedules : (1) a single“large dose” (5 ,ug/kgof satin or 4 mg/ kg of die&in, iv) which produced overt signsof toxicity, or (2) a series of 10 “small doses”(1 pg/kg of sarin or 1 mg/kg of die&in, im, given 1 weekapart) which did not produceany major clinical signs.Electroencephalograms (EEG) were recorded from chronically implanted electrodes according to the following schedule:(1) three recording sessions prior to drug administration, (2) one at 24 hr post-drug, and (3) another three at 1 yr post-drug. Each recording sessionconsistedof runs in the following states: (1) awake and alert in a lighted environment, (2) awakeand alert in total darkness,and (3) drowsy. A seriesof control monkeys received equaltreatment but weregiven only drug diluent. Fast-Fourier transforms were performedon the EEGs with a PDP-12 computer to yield voltage vs frequency spectra.Resultsof statisticalanalysisof EEG spectrashowedthe following: For both sarinand dieldrin the singlelarge-doseadministration produced significantincreasesin the relative amount of beta voltage (1550 Hz) which persistedfor 1 yr. For sarin the predominanteffect wasin the EEG derivation from the temporalcortex, and for dieldrin from the frontal cortex. For both drugs,the beta increasewasmost prominentin the states of awake in darknessand drowsy. These results indicate that a single symptomatic exposure or a seriesof subclinical exposuresto satin or dieldrin can alter the frequency spectrumof the spontaneousEEG for up to 1 yr. Organophosphate (OP) and chlorinated hydrocarbon (CH) compounds are known to have potent effects upon the nervous systemsof a wide variety of organismsfrom simple invertebrates to man. This action has been used to considerable advantage in pest control where OP and CH compoundsconstitute two of the major classesof insecticides. The acute effects of OP and CH exposure upon the central nervous system(CNS) of man and other higher mammals include prominent electroencephalographic (EEG) changesand convulsions. In most instances,EEG abnormalities have been reported to disappear within 2 weeks of acute exposure or a termination of chronic exposure i BiomedicalLaboratory,EdgewoodArsenal,U.S. Army ProvingGround,Maryland. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
365
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BURCHFIEL,
DUFFY
AND
SIM
(Crescitelli and Gilman, 1946; Grob et al., 1947; Holmes and Gaon, 1956; Desi et al., 1966; Santolucito and Morrison, 1971). However, other studies indicate that the EEG effects of insecticide poisoning may persist for many weeks or months (Spiotta, 1951; Hoogendam et al., 1962; Dille and Smith, 1964; Kazantzis et al., 1964). Metcalf and Holmes (1969) suggested that OP exposure may lead to chronic EEG changes. They examined EEGs from a group of industrial workers with a past history of insecticide poisoning, but with no recent exposures, and reported a high incidence of abnormal low-to-medium voltage slow-wave activity in the theta range (4-6 Hz). The present study was undertaken to examine, under controlled conditions, the long-term effects of OP and CH exposure upon the primate brain as measured by changes in EEG activity. A representative OP compound (sarin) and a representative CH compound (dieldrin) were chosen because they were the two agents with the highest incidence of exposure in the Metcalf and Holmes study (personal communication). Dieldrin (85 % of the compound 1,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8aoctahydro-endo-exo-5,8-dimenthanonaphthalene and 15 y0 related compounds) is a widely used commercial insecticide. Sarin (GB; isopropyl methyl phosphonofluoridate), on the other hand, is not used commercially. It is ordinarily classified as a “nerve gas;” however, it shares a similar chemical structure and pharmacological action with more common OP insecticides such as malathion and parathion. Sarin differs from the commercial OP compounds in two major aspects. First, it is a fluorinated OP, and, second, it is considerably more potent. Rhesus monkeys were exposed to controlled doses of either dieldrin or sarin. These drug-treated animals, and appropriate controls, were then followed for 1 yr after acute exposure to assessthe presence of persistent changes in EEG activity. METHODS Experimental animal preparation. Twenty-two adolescent rhesus monkeys (Macaca mulatta) of both sexes, weighing 2.5-4.0 kg, were used in this study. Under general
anesthesia and aseptic conditions, cortical and depth electrodes were permanently implanted for chronic recording. The animals were initially anesthetized with a shortacting barbiturate (sodium methohexital) and maintained with methoxyfluorane and oxygen via an endotracheal tube. Cortical electrodes (O/80 stainless-steel screws) were placed into tapped holes in the skull so as to contact the intact dura. These were placed over the frontal, central, occipital, and temporal cortices bilaterally. Bipolar-depth electrodes (32-gauge insulated stainless-steel wire, l-mm tip separation, 0.5-mm tip exposure) were stereotaxically placed through small burr holes into the following sites : amygdala, hippocampus, mesencephalic reticular formation, and nucleus centrum medianum of the thalamus. Stereotaxic coordinates were based upon the atlas of Snider and Lee (1961). Electrode placements were histologically verified at postmortem. Insulated leads from the electrodes were soldered to two g-pin Winchester-type miniature connectors which were cemented to the skull with dental acrylic. Additional acrylic was used to form a durable, protective cap over the skull covering all leads and electrodes. Drug administration. Sarin for injection was prepared by dilution of an aliquot of purified drug (stored at -55°F) with normal saline. Biological effectiveness of this solution was determined routinely in rabbits and was never used more than 2 hr after
PERSISTENT
EEG EFFECTS
367
dilution. Chemical analysis of random samples indicated that sarin was always 90-95 % pure. Dieldrin was obtained in crystalline form (85 % technical grade) and dissolved in 100 % ethanol for injection. Sarin or dieldrin was administered according to one of two schedules. In one, animals received a single “large dose” of the drug: 5 ug/kg of sarin or 4 mg/kg of dieldrin, iv. In the other, animals received multiple “small doses” : a series of 10 injections of 1 pg/ kg of sarin or 1 mg/kg of dieldrin, im, given at intervals 1 week apart. Hereafter these will be referred to respectively as the “large-dose” and “small-dose” schedules. These dose levels were arrived at through pilot studies in which the attempt was made to find dose levels which would approximate, respectively, (1) a serious but sublethal exposure showing all the signs of poisoning and (2) a series of subclinical but near-threshold exposures showing few, if any, signs of overt poisoning. At the large-dose level of either sarin or dieldrin all animals had generalized seizures. During the pilot studies it was noted that prolonged periods of apnea often accompanied these seizures. Therefore, to prevent the possibility of secondary anoxic brain damage during large-dose administration, animals in the current study were paralyzed with gallamine triethiodide, and artificial respiration was achieved via an endotracheal tube. The drug was given by slow iv injection over 3 min. Animals were then allowed to recover from paralysis and breathe spontaneously when their EEG revealed a cessation of seizure activity. The average total length of paralysis for experimental animals was about 2.5 hr and never more than 4 hr. A group of control animals was similarly paralyzed and received an injection of an equal volume of drug diluent (saline for sarin or 100 % ethanol for dieldrin). The length of paralysis for controls was matched as nearly as possible to that of the experimentals. paralysis was not necessary for the animals receiving the multiple small doses. Injections were given im with the animal seated in a restraining chair. A group of control animals was handled in the same manner and received injections of drug diluent. Experimental design. Animals were randomly assigned to one of four drug-treatment groups: (1) single, large-dose sarin; (2) multiple, small-dose sarin; (3) single, large-dose dieldrin; or (4) multiple, small-dose dieldrin. There were three animals in each treatment group. These groups were matched with groups of control animals, three for each large-dose group and two for each small-dose group. Controls were subjected to the same injection schedules (including paralysis and artificial respiration for large-dose controls) but received only drug diluent. Each animal in the study was subjected to a series of EEG recording sessions. Prior to drug administration, three separate control sessions were performed on different days. For the single, large-dose animals, a recording was taken 24 hr after administration For the multiple, small-dose animals, a recording was taken 24 hr after administration ofthe last injection in the series. Subsequently, the EEGs of all animals were periodically monitored to check the integrity of the recording system and CNS activity. One year after exposure an additional three recordings were performed for all animals. During the l-yr interval the animals were maintained in separate cages in a temperaturecontrolled environment; they received no other drugs. EEG recording. The animal sat in a primate-restraining chair, with a mechanical head restraint, inside a closed, electrically shielded chamber. EEGs were recorded on seven-channel FM magnetic tape via revertor-coupled and calibrated outputs of a
368
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Grass Model III polygraph with a bandpass of 0.5-80 Hz. An individual recording session consisted of the following runs: (1) Awake, alert in light, an epoch of at least 2-min duration in which the animal sat in the lighted chamber; alertness was maintained by intermittent auditory distraction; (2) Awake, alert in darkness, same as (l), but in darkness; (3) drowsy, an epoch of 15-30 min duration in which the animal sat in the darkened chamber and silence was maintained. For each run, bipolar recordings were obtained from the depth electrodes (amygdala, hippocampus, mesencephalic reticular formation, and nucleus centrum medianum of the thalamus) and from the cortical leads in the following bipolar pairings: frontalcentral, central-occipital, and occipital-temporal. Data analysis. Spectral analysis of the EEG data was accomplished on a PDP-12 computer using the comprehensive SIGSYS- 12 software system (Agrippa Data Systems). Signals were played back from FM tape through a Khrone-Hite filter set to bandpass OS-50 Hz (24 db per octave) and analog-to-digital converted by the computer at a rate of 256 Hz. Fast-Fourier transforms (FFTs) were performed, without smoothing, on 4-set epochs of EEG (computed on 1024 points over the frequency range O-128 Hz with a resolution of l/4 Hz). After FFT computation, the square root was taken of each frequency component to yield a voltage spectrum. Epochs containing excessive eye blinks, movement artifact, electrode artifact, or muscle activity were not analyzed. Consecutive epochs were averaged to yield a spectrum representing 1 min of EEG; i.e., the spectra of 15 4-set epochs were averaged. Two 1-min spectra were computed for each state in a recording session (i.e., awake in light, awake in darkness, and drowsy). The 2-min epoch of EEG most representative of drowsiness was selected by visual inspection of the polygraph tracing obtained in parallel with the analog tape recording. The selection of the drowsy epoch was made by an experienced electroencephalographer, based on the monkey EEG criteria of Reite et al. (1965). The electroencephalographer was unaware of a monkey’s exposure history at the time of selection. For each spectrum, a calculation was made of the percentage of total energy present in each of the classical EEG frequency bands defined as follows: delta = 0.5-3.75 Hz, theta = 3.75-7.75 Hz, alpha = 7.75-13.0 Hz, beta 1 = 13-22 Hz, and beta 2 = 22-50 Hz. The band from 50 to 128 Hz was not analyzed. For the final analysis, total spectra and frequency bands were averaged respectively for the three control sessions, the 24-hr session, and the three 1-yr sessions. This was done for each lead and each state. Thus, a final datum consisted of an average spectrum and frequency bands representing EEG activity from one electrode derivation, for one state, and for one set of sessions. Since two spectra were derived for each state and lead in a recording session, the values of N for the final averages were 6 for control, 2 for 24 hr, and 6 for 1 yr. In computing the final averages, sd were also calculated. Statistical analysis. Statistical analysis was performed in a two-step procedure. First, a longitudinal comparison was made for each animal using a two-tailed Student’s t test. The average spectrum and frequency bands derived from the control sessions (for a given lead and state) were compared first with that derived from the session at 24 hr post-drug and next with that from the sessions at 1 yr. For the average spectrum, the t test was applied at each point. As might be expected, this produced a large number of seemingly random “significant” differences. (Theoretically, one would expect an average of 5 % of the t tests to yield t values with a probability 60.05 merely by chance.)
PERSISTENT
EEG EFFECTS
369
Such differences at individual points throughout the spectrum proved very difficult to interpret. We felt that any criteria we might establish to determine when statistically significant points constituted a physiologically significant difference might suffer from subjective bias. For this reason we chose to base our results upon analysis of integrated frequency bands. Therefore, at the conclusion of the first step of statistical analysis, we had determined for each animal which frequency ranges were different by t test at 24 hr post-drug and at 1 yr post-drug as compared to the pre-drug control. In the longitudinal comparison, each animal served as his own control; data from animals within the same experimental group were not pooled for comparison. The next question, therefore, was which differences, if any, revealed by t test were consistently and exclusively seen among the drug-treated animals as opposed to control animals. This question was answered by the second step of the statistical analysis, Fisher’s test (Fisher, 1934). For this analysis the question was asked, how many drug-treated animals showed a significant change (by t test) in a particular frequency band and how many did not? The same question was asked of the control animals. From these answers a 2 x 2 contingency table was constructed and the exact probability of obtaining the resultant distribution in the table, or one more extreme, by chance alone was calculated. If this probability were ~0.05, then it was concluded that a significant change in frequency distribution of the EEG had occurred in a drug-treated group as compared to the controls. Statistical analysis among the various groups of control animals showed no consistent differences; therefore, for Fisher’s test all control animals were considered as a single group. RESULTS
Sarin
For either treatment schedule, no consistent, significant differences were found for any of the depth leads analyzed : amygdala, hippocampus, midbrain reticular formation, or nucleus centrum medianum of the thalamus. In the cortex, on the other hand, longterm alternations in EEG frequency spectra were observed for both the single, largedose and multiple, small-dose exposures. Large dose.The single large dose of sarin (5 pug/kg) produced a persistent increase in the relative amount of voltage in the beta frequency bands (13-22 and/or 22-50 Hz) of the occipital-temporal (O-T) EEG derivation. Figures 1 and 2 illustrate this for two of the sarin-treated animals during the state of awake in darkness. Examples of two control animals for the same lead and state are shown in Figs. 3 and 4. These examples illustrate some general findings which are applicable to all the drug treatment groups First of all, it can be seen in comparing the control records from these animals that a considerable degree of individual variability exists in the EEG frequency distributions. However, for a given animal, the spectra tended to be reasonably constant from day to day. The control animal shown in Fig. 3 is typical of the data from the 10 untreated animals. There are no significant differences among the spectra over the I-yr period of the study. Figure 4 shows an extreme case of variability for a control animal in which a number of apparently random alternations in frequency distribution occurred among the average spectra. It was because of the much greater degree of variability from animal to animal than from day to day for the same animal that the longitudinal method of statistical analysis described in the methods section was adopted.
BURCHFIEL, DUFFY AND SIM
370 CONTROL 36
24
-BAND
PERCENT ~-ENERGY
S.E.
;
25.85 16.24
1.52 0.29
A
17.72 17.44 25.88
0.21 0.30 1.33
HOURS PERCEhC ENERGY
S.E.
t VALUE vs CONTROL
22.96 19.82 16.66 22.08 21.90
1.57 0.70 0.85 1.52 1.51
1.01 5.76" 1.89 5.13** 1.57
PERCENT ENERGY
S.E.
D T
22.09 14.61
0.90 0.35
2.13 3.58**
Iti B2
17.4s 19.37 29.27
0.32 0.50 0.52
0.48 4.43"" 2.38*
BAND A
1 YEAR w
"s
t VALUE CONTROL
FIG. 1. Example of average voltage spectra and frequency bands for one animal receiving a single large dose of sarin. Occipital-temporal EEG derivation in the state of awake in darkness. For the spectra, the ordinate scale is in microvolts and the abscissa scale is in hertz. Frequency bands give percentages of total voltage (O-50 Hz) over the following ranges : D = delta (0.5-3.75 Hz); T = theta (3.75-7.75 Hz); A = alpha (7.75-13.0 Hz); Bl = beta 1 (13-22 Hz); B2 = beta 2 (22-50 Hz). The t value is the result of Student’s I test between the indicated band and the corresponding band of the control spectrum. *,p < 0.5; **, p < 0.01.
CONTROL
D T
19.89 19.32
0.92 1.05
B"1 B2
32.50 18.72 13.25
0.94 0.?8 0.41
24 HOURS 30 uv
BAND :
PERCENT ENERGY S.E.
t VALUE YB CONTROL
26.80 19.05
0.65 2.12
0.14 3.53**
A
19.39
0.33
7.64*'
i:
19.65 18.26
0.22 1.44
6.68** 0.59
1 YEAR BAND
PERCENT ENERGY S.E.
t VALUE YS CONTROL
D T A
21.05 17.16 30.36
0.29 0.73 0.93
0.99 1.51 1.55
:t
18.64 16.29
1.23 0.58
2.77. 0.07
FIG.2. Example of average voltage spectra and frequency bands for another large-dose sarin animal. Same EEG derivation and recording state as animal in Fig. 1. For explanation of scales and legends see Fig. 1.
PERSISTENT
371
EEG EFFECTS
CONTROL
24 HOURS
1 YEAR
FIG. 3. Example of average voltage spectra and frequency bands for one control animal. Same EEG derivation and recording state as Figs. 1 and 2. For explanation of scales and legends see Fig. 1.
CONTROL
24 HOURS 25
-BAND
uv
:! 0 0
50
A Bl B2
PERCENT ENERGY S.E. 23.99 17.40 21.10 20.30 20.41
1.58 0.90 0.44 1.40 1.56
t VALUE YS CONTROL 0.55 0.53 5.43"" 0.71 3.86'"
1 YEAR
FIG. 4. Example of average voltage spectra and frequency bands for another control animal. Same EEG derivation and recording state as Figs. l-3. For explanation of scales and legends see Fig. 1.
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Similar degrees of variability were seen among the drug-treated animals. However, the key factor among the large-dose sarin animals was that, regardless of what other alternations in the spectra might occur, there was a consistent increase of relative voltage in the beta frequency bands. For instance, in Fig. 1 at 24 hr post-drug there was a significant increase in beta 1 which was still present 1 yr later. But, in addition, there was a significant increase in theta at 24 hr and an increase in beta 2 and a decrease in theta at 1 yr. Similarly, the animal shown in Fig. 2 displayed a persistent increase in relative beta voltage but also had other changes at 24 hr. A summary of the results oft test analysis for increased relative beta activity in the O-T frequency spectrum is given in Table 1. Each entry in the table is the proportion TABLE 1 SINGLE LARGE-DOSE ADMINISTRATION OF SARIN, OCCIPITALTEMPORAL EEG Recording
24 hr Sarin Control
state
Awake in light
Awake in
darkness
Drowsy
O/3 o/10
313 2110 (p = 0.035)b
213 l/10 (p = 0.105)b
213 2/10
213 o/10 (p = 0.038)b
313 2110 (p = 0.035)b
1 yr
Sarin Control
a Sarin, 5 pg/kg, iv. Proportion of animals showing a significant increase by t test in relative beta voltage. A percentage of total voltage of spectrum in the frequency bands 13-22 or 22-50 Hz of the EEG frequency spectrum at 24 hr and 1 yr post-drug. * Exact probability of distribution of drug-treated and control animals occurring by chance (2x2 Fisher Test).
of animals (control or treated) which showed a significant increase in beta activity by t test. For a given post-drug session and recording state, the proportion of treated animals showing an increase and the proportion of controls showing an increase form a 2 x 2 contingency table. Beneath each pair of proportions, in parentheses, is given the Fisher exact probability that such a distribution could arise by chance alone (where there is obviously no difference, the probability is omitted). It can be seen that the increase in relative beta voltage was most prominent in the state of awake in darkness. At 24 hr after administration, all three drug-treated animals showed a statistically significant increase in beta activity by I test, whereas only two of ten controls showed such an increase. The probability of this distribution arising by chance is 0.035. At 1 yr after sarin exposure two of three treated monkeys still showed a relative increase in beta activity; none of the controls showed any increase. This distribution has a chance probability of 0.0385.
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PERSISTENT EEG EFFECTS
For the drowsy state there was a significant increase in beta activity for treated animals at 1 yr (all sarin animals showing an increase as opposed to two of ten controls, p = 0.035). However, at 24 hr this change was not quite statistically valid (an increase in two of three treated animals and in one of ten controls, p = 0.105). In the state of awake in light, there was no significant increase of relative voltage in the beta bands. However, there would appear to be a trend toward increased beta at 1 yr after exposure (two of three treated animals showed a significant increase, while eight of ten controls did not, p = 0.188). Small dose.The animals receiving the multiple small doses of sarin (1 pg/kg once a week for 10 weeks) showed a persistent relative increase in O-T beta activity similar CONTROL
24
PERCENT ENERGY
S.E.
34.46 19.79 10.40 15.83 15.09
1.96 0.87 1.39 0.83 1.17
HOURS PERCENT ENERGY
S.E.
t VALUE YS CONTROL
33.43 17.65 11.66 18.20 22.27
3.52 0.60 0.14 1.39 1.23
0.26 1.33 2.69* 1.44 3.25*'
-BAND
PERCENT ENERGY
S.E.
t VALUE YS CONTROL
D T A Bl 32
19.93 14.71 18.75 25.50 24.25
1.30 0.36 1.08 1.42 0.93
6.19" 4.93** 0.15 5.09" 6.15**
1 YEAR
FIG. 5. Example of average voltage spectra and frequency bands for one animal receiving multiple small-dose administration of sarin. Occipital-temporal EEG derivation in the state of awake in darkness. For explanation of scales and legends see Fig. 1.
to that seen with the large-dose animals. An example of spectra and frequency bands for one small-dose monkey is illustrated in Fig. 5. The results of t test analysis for increased beta activity in all small-dose animals is given in Table 2. At 24 hr after the last dose of sarin, all the treated animals showed a significant increase in beta for both the states of awake in darkness and drowsy. These increases were still present at 1 yr after exposure. In addition to the alternation in O-T EEG spectra, the small-dose animals also showed increases in relative beta activity in the frontal-central (F-C) EEG (Fig. 6 and Table 2). Such F-C alterations were not seen with the single large dose of sarin. The increase in F-C beta also differed from the increase in O-T beta in that it occurred only in the spectra at 1 yr post-drug; at 24 hr there was no significant increase (Table 2). In contrast, the spectra at 24 hr after termination of the small-dose schedule showed a
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consistent trend for all treated animals toward increases in relative voltage in the delta and theta bands and decreases in higher frequencies, particularly alpha. With time this frequency distribution reversed such that beta was increased and delta and theta decreased (see example in Fig. 6). The increase in relative beta at 1 yr was seen in all three recording states (Table 2). TABLE 2 MULTIPLE SMALL-DOSEADMINISTRATION OF SARIN, OccmTAL-TEMPORAL (O-T) AND FRONTAL-CENTRAL(F-C) EEG”. Recording state
A. O-T EEG 24 hr Sarin Control
1yr
Sarin Control
Awake in light
Awake in darkness
o/3 o/10
313 2/10 (p = 0.035y
213 2/10
213
o/10
(p=0.0385)*
B. F-C EEG 24 hr Sarin Control 1 Yr Sarin Control
Drowsy
313
l/10 (p = 0.014)b 313 2110 (p = 0.035)b
O/3
O/3
l/3
l/f3
218
118
313
313
313
118
l/8
l/8 (p = 0.024)*
(p = 0.024)*
(p=0.024)*
’ Sarin, 10 injections of 1 ,ug/kg, im, given at l-week intervals. Also seefootnote a, Table 1. b Seefootnote b, Table 1. Dieldrin
As with sarin, no consistent, significant differences were observed in the spectra from any of the subcortical structures. Also, like sarin, there were persistent increases in relative beta voltage in the cortical EEG spectra. Large dose.Increases in the relative amount of beta activity (13-22 and/or 22-50 Hz were observed in F-C EEG spectra. An example of this is illustrated for one animal in the state of awake in darkness in Fig. 7. The increase in F-C beta activity was seen at 24 hr post-drug in the states of awake in darkness and drowsy. At 1 yr post-drug it was still present during these states and also during the state of awake in light (Table 3). In general the alterations in frequency distribution tended to be more widespread at 1 yr after dieldrin exposure than at 24 hr. The example illustrated in Fig. 7 is typical
PERSISTENT EEG EFFECTS
375
CONTROL -BAND
PERCENT --ENERGY
S. E.
30.75 19.83 17.29 15.30 19.99
3.12 0.88 1.55 1.52 0.93
D i Bl BZ
24 HOURS PERCENT ENERGY
S. E.
t VALUE YS CONTROL
i:
34.93 20.97 15.30 14.57 17.53
0.59 0.02 0.98 1.02 0.85
0.73 0.71 0.69 0.26 1.40
BAND -
PERCENT ENERGY
S.E.
f VALUE YS CONTROL
19.59 13.90 17.40 26.03 26.07
1.10 0.37 1.34 1.41 0.74
3.37’ 6.21*’ 0.06 5.18’. 5.13**
BANo T” A
1 YEAR
T” A
FIG. 6. Example of average voltage spectra and frequency bands for the same animal as in Fig. 5 but for frontal+entral EEG derivation. Same recording state as in Fig. 5. For explanation of scales and legends see Fig. 1.
TABLE 3 SINGLELARGE-DOSEADMINISTRATIONOFDIELDRIN,FRONTAL -CENTRALEEG” Recording Awake in light 24 hr Dieldrin Control
state
Awake in darkness
u3
313
313
l/8
l/8
V3
(p = 0.024)b 1 yr Dieldrin Control
Drowsy
(p = 0.024)b
3/3
313
313
l/8
118
(p = 0.024)b
(p = 0.024)b
l/8
(p = 0.024)b
a Dieldrin, 4 mg/kg, iv. Also see footnote a, Table 1. b See footnote b, Table 1. of the treated animals in this respect. Note that at 24 hr after exposure, there was a significant change only in beta 1, whereas at 1 yr there were alterations in all frequency
bands except beta 2. At 1 yr, all the dieldrin-treated animals showed a significant decreasein the relative amount of theta voltage in the state of awake in darkness@ = 0.024,
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SIM
Fisher test) and a significant decrease in relative delta for the drowsy state (p = 0.024). At 24 hr, on the other hand, the only consistent change was the increase in relative beta. SmaZZ dose. The animals receiving the multiple, small-dose exposure to dieldrin exhibited EEG frequency changes in frontal cortex similar to those of the large-dose group. However, in the small-dose dieldrin group, one animal died before the 1-yr recording session. The two remaining animals both showed a statistically significant increase in beta activity at 1 yr for the states of awake in darkness and drowsy. Because of the small number of treated animals, this distribution (two of two treated animals showing an increase versus one of eight controls) did not quite reach the 0.05-level of significance by the Fisher test. The actual p value was 0.067. Had the other animal lived CONTROL 25 uv
BAND--
PERCENT ENERGY
S.E
;
17.54 28.95
1.10 0.57
A Bl B2
16.90 17.18 22.72
0.97 0.35 0.62
24 HOURS BAND-
PERCENT ENERGY
S.E.
T” A
28.37 15.97 17.48
0.16 3.67 2.54
0.22 1.51 0.27
;:
20.55 20.86
1.22 0.24
1.90 4.30**
S.E.
t VALUE vs CONTROL
“S
t VALUE CONTROL
1 YEAR BAND --
PERCENT ENERGY
D x
19.43 21.09 15.07
1.27 1.55 0.36
5.67** 3.66’ 2.30.
Bl B2
25.34 22.68
0.74 0.69
9.86.. 0.03
FIG. 7. Example of average voltage spectra and frequency bands for large-dose dieldrin animal. Frontal
