Contrasting
Actions Metabolite.
of Dieldrin and Aldrin-transdiol, on Cat CNS Function R. M.
Depurltnent
of Physiological
Sciences.
Receil)ed
Unirersify California October
JOY
of California, 956 16
21, I976:
Its
accepted
School
April
of Vererinacv
Medicine,
Du~~;s,
6, I977
Contrasting Actions of Dieldrin and Aldrin-transdiol, Its Metabolite,on Cat CNS Function. R. M. (1977). Toxicol. App/. Pharmacol. 42. 137-148. Dieldrin and aldrin-transdiol were administered to cats to test whether dieldrin is directly active or whether it requires conversion IO aldrin-transdiol for CNS effects. In cats 2-4 mg/kg dieldrin produced convulsive activity in 215 min. This was preceded by changes in the EEG and in responses evoked by sensory stimuli consistent with changes produced by pentylenetetrazol-type convulsants. Aldrin-transdiol in doses up to 20 mg/kg was without effect in cats. No convulsions were observed. No evidence 01 CNS action could be discerned from examination of the EEG or from responses to sensory stimulation. The results are consistent with the idea that dieldrin is directly active in the mammalian CNS. Its metabolite. aldrin-transdiol. when injected directly into the blood stream is either inactive or is incapable of entering the CNS from this route. The former alternative appears most likely. JOY.
The convulsant properties of dieldrin are well known. In mammals the acute toxicity of dieldrin is primarily of central nervous origin (Gowdey et al., 1954), and its actions at that level are similar to pentylenetetrazol (Joy, 1973, 1974, 1975). Following iv administration in cats of 2-4 mg/kg, convulsions develop in 2-15 min, suggesting that dieldrin is directly acting in the CNS (Joy, 1976). However, studies of the dieldrin metabolite, aldrin-transdiol, have caused others to conclude that the metabolite is largely responsible for the actions of dieldrin. Wang et a/. (197 1) proposed that dieldrin is converted to an active metabolite, basing this conclusion on the relative dose required and the temporal development of effects of dieldrin and metabolites in vitro. Van den Berken and Narahashi (1974) reported that aldrin-transdiol suppressespeak transient current in voltage-clamped squid axons while dieldrin is ineffective. Similarly, Akkermans et al. (1975a,b) have reported that aldrin-transdiol is both more potent and more rapid acting than dieldrin in a number of in vitro nerve and muscle preparations. However, Shroeder and Shankland (1975) have reported that, in cockroaches in vivo. internal concentrations of lo-) M aldrintransdiol maintained for 1 week were not toxic whereas dieldrin at similar concentrations gave 80% mortality in 10 hr. Korte and Arent (1965) have also reported that the oral LD50 for aldrin-transdiol in mice is at least 10 times higher than for dieldrin. In this report the CNS actions of dieldrin and aldrin-transdiol are compared to test the hypothesis that dieldrin must be converted to its metabolite before it can produce CNS activity in mammals. Dieldrin produces clearly recognizable changes in the CopyrIght 0 1911 hy Academic Press. Inc. All rights of reproducrmn in any form reserved. Pnnlcd I” Great Britam
137
ISSN 004 I -DOXX
138
K.M.JOY
electroencephalogram (EEG) and in evoked responses in sensory and motor pathways before precipitating tonic-clonic convulsions (Joy, 1973. 1974, 1976). If dieldrin must be converted to an active metabolite, then the metabolite would be expected to exert effects both at a lower dose and more rapidly. Aldrintransdiol was examined on these same systems to compare potency and temporal development against dieldrin. The results suggest that aldrin-transdiol is neither responsible for the convulsive properties ot dieldrin nor does this metabolite contribute significantly to it in mammals.
METHODS General Procedures Twenty-four male cats. 2.5-4 kg, were subjects. Eight received dieldrin. eight received aldrin-transdiol, and eight served as controls. All procedures followed were in accord with the Guiding Principles in the Care and Use of Animals as approved by the Council of the American Physiological Society. Subjects were fasted overnight, anesthetized, tracheotomized, and placed in a stereotaxic frame. Halothane was the anesthetic for all surgical procedures. Both brachiocephalic veins and a femoral artery were cannulated for drug injection and to record blood pressure. The skin and muscles overlying the cranium were dissected laterally and holes were drilled through the bone to insert electrodes into subcortical structures. The bone overlying the sigmoid gyri was removed, the dura was dissected away, and the exposed cortex was covered with 38°C mineral oil to prevent drying. In 12 experiments the sciatic nerve was exposed and cut, and a Sherrington electrode was placed on the central end. After these procedures, a rectal probe was inserted, and 0.25% dibucaine was infiltrated into all wound margins and pressure points. All wound margins were wrapped in gauze kept moist with saline and mineral oil, and the corneas were coated with mineral oil to prevent drying. Then anesthesia was discontinued. Gallamine (-7 mg/kg/hr) was infused to produce paralysis, and respiration was supported by a Harvard 620 respirator. End-expiratory CO, was continually monitored with a Beckman LB1 analyzer, and respiration was adjusted to maintain end-expiratory CO, between 3.2 and 3.5%. Temperature was kept at 38OC with a heating pad and wraps. During all subsequent procedures, EEG, EKG, blood pressure, CO,, and temperature were monitored to assess physiological state and to assure freedom from pain. After recovery from anesthesia, all subjects displayed cycles of alert and relaxed or drowsy periods as monitored by EEG and autonomic activity. EEG Recording EEGs were obtained with chlorided silver ball electrodes, 0.5 mm in diameter, placed directly on the cortex or with stainless-steel electrodes tapped firmly into small burr holes made in the cranium overlying cortex. All records were monopolar, the stereotaxic frame serving as reference. EEGs were recorded on a Grass Model 78 polygraph, and selected samples before and after drug administration were chosen for illustrative purposes. In all figures an upward deflection in the EEG indicates negativity at the electrode with respect to the reference. Half-amplitude frequencies for all records are I and 90 Hz.
DIELDRIN
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Responses along Sensoty and Motor PathwaJts To provide stimuli for sensory pathway analysis, the sciatic nerve was stimulated in 12 experiments with supramaximal rectangular pulses (one per 2 set, 0.1 msec duration, 0.5-l mA). The stimuli were generated by a WPI Series 800 stimulator and were led to the electrode through a photon-coupled isolator. Stimuli were initiated at least 1 hr before data collection began to reduce the possibility of habituation during experii mental periods. Usually responses stabilized within 30-40 min (2.5 hr after termination of anesthesia). Responses were recorded at locations along the primary sensory pathway including the medial lemniscus (ML), coordinates from the stereotaxic atlas of Jasper and Ajmone-Marsan (1954), A 4.0, L 5.0. V -2.0: nucleus ventralis posterolateralis (VPL) of the thalamus (A 9.0, L 7.5, V 1.0); and from the posterior sigmoid gyrus (PSG) which, in the cat, represents primary somatosensory cortex. Bipolar stainless-steel electrodes were used to record responses in ML and VPL, and an Ag-AgCl ball electrode was used to record from the cortex. Subcortical electrodes were lowered to just above the desired recording site under stereotaxic control. Final placements were made while stimulating to produce a maximal amplitude evoked response. Cortical electrodes were positioned at the location yielding the largest response to stimulation. Responses during the experiment were recorded on tape (Hewlett-Packard 755C, O-2.5 kHz bandwidth) and analyzed at a later time. In 12 other experiments carried out in a similar manner, the VPL was stimulated at twice threshold intensity (one per 2 set, 0.1 msec, 0.6-1.5 mA). Responses were obtained from the posterior sigmoid gyrus (PSG). anterior sigmoid gyrus (ASG), and from corticofugal axons at the level of the cerebral peduncle (PED) (A 4.0, L 4.0, V -6.0). In every experiment 3&40 min of control data were collected which included 20 min of evoked-response data and l&20 min of spontaneous activity. Then specified amounts of solvent, dieldrin, or aldrin-transdiol were injected. Evoked responses were continuously elicited for 20 min; then additional compound was administered. This sequence was continued for the duration of the experiment. In four experiments with each compound, the time interval between injections was increased to 25 min to gather spontaneous activity. All doses indicated in the results are cumulative amounts and indicate the total amount given up to that point. At the end of the experiments the cats were sacrificed with pentobarbital, and the electrode locations were marked by passing current through them. The brains were perfused with formol-saline containing potassium ferricyanide and removed. Frozen sections, 50 ,um thick, were taken at the electrode locations and stained with Luxol fast blue. Actual locations were determined with the atlas of Jasper and Ajmone-Marsan (1954). In all cases, the locations of the electrodes were within the desired structure. Administration
of Dieldrin and Aldrin-transdiol
Dieldrin (>99% HEOD) and aldrintransdiol (>95% pure) were provided by the Shell Chemical Company. Both compounds were verified for content and purity by gas chromatographic and mass spectral analyses. The chemicals were dissolved in ethanol at concentrations of 10 or 20 mg/kg/ml. Administration was accomplished by first injecting 0.05 ml of ethanol into the cannula, and then administering 0.1-0.5 ml of ethanol containing the cyclodiene derivative. The cannula was cleared with an additional 0.05 ml of ethanol followed by 2 ml of saline. Injection took l-2 min. In all
140
R. M. JOY
cases except the administration of aldrin-transdiol at 20 mg/kg, the total volume of ethanol administered was no greater than 1.1 ml, Ethanol in doses over 1 ml can produce transient signs of depression, but such effects are opposite to nature to and clearly distinguishable from the CNS actions of dieldrin.
Data Analysis In experiments where spontaneous EEGs were obtained, at least 5 min of data were gathered before and after each injection of chemical. EEGs were visually evaluated by two independent observers, and samples considered representative of a particular state were selected for illustration. Evoked responses in sensory and motor pathways were analyzed after the experiment. The taped responses were played back, and the average evoked response at each locus was determined at every minute interval with a computer of average transients. The peak-to-peak amplitude of the primary response was measured. These amplitudes provided a minute to minute measure of the evoked activity at various locations. For statistical analysis, the evoked-response amplitude during the last 10 min of data collection at each dose of solvent, dieldrin, or aldrin-transdiol were averaged for each animal. The results for four different subjects experiencing identical experimental conditions were used to generate means and SE for each condition. Finally the significance of the dieldrin and aldrin-transdiol groups as compared to the solvent control groups was determined by Student’s t test.
RESULTS
Electroencephalograms Intravenous administration of dieldrin induces a consistent sequence of EEG alterations in the cat (Fig. 1). Early signs of intoxication include the development of hypersynchronous activity distributed symmetrically across the cortical surface (Fig. 1B). This may occur with doses of 1 mg/kg or more. Later, epileptiform spikes or groups of spikes begin to appear (Figs. IC and 1D). These are distributed symmetrically between hemispheresand tend to be of larger voltage over anteriorly placed electrodes. They may occur spontaneously or may be evoked by sensory stimulation. The electrical correlates of convulsions (Fig. 1D) develop shortly thereafter. Seizures most often begin with the appearanceof high-amplitude synchronous waves. which are of largest amplitude from frontal cortical locations. These spread rapidly across the cortex and slow with time to generate the clonic phaseof the seizure. These events have beendescribed in detail in previous reports (Joy, 1973. 1976). The administration of aldrin-transdiol in dosesof l-20 mg/kg did not evoke any of the EEG actions characteristic of dieldrin (Fig. 2). No effects were observed at doses from 1 to 5 mg/kg. At 10 and 20 mg/kg there was an increase of slow waves and a disruption and slowing of spindles in the EEG. These changes are not typical of convulsive agents, but rather are more characteristic of sedative or CNS depressive agents. As similar changes occurred when equal volumes of solvent without aldrin transdiol were given, these changes are solvent related and can not be attributed to aldrin-transdiol. The important finding, however, is that aldrin-transdiol in doses 5-10
DIELDRIN
AND
ALDRIN-TRANSDIOL
141
times greater than the convulsant dose of dieldrin does not induce any evidence of hyperexcitability or preconvulsive changes in the EEG. In two subjects which had received 10 mg/kg aldrin-transdiol with no effect, dieldrin was administered. The EEG changes and convulsions reported for dieldrin occurred when 2 mg/kg were injected, indicating that the subjects were not unusually resistant to convulsive agents.
FIG. I. EEG changes developing after dieldrin administration. (A) Before injecting 2 mg/kg dieldrin. (H) Five minutes after dieldrin injection. This record demonstrates the high-amplitude hypersynchrony. which is the first indication of developing hyperexcitahility. (C) Ten minutes later the EEG shows intermittent bursts of hypersynchrony with single or multiple superimposed spikes. It is from this background that convulsions most often develop. (D) Single or grouped spikes occurring occasionally before convulsions. (E) Twenty-five minutes after dieldrin administration a convulsion develops spontaneously on a background of intermittent hypersynchrony. Seizure is followed by a period of post-ictal depression. Lower right: Amplitude calibration. A and B. 100 ,uV: C-E. 200 pV. Time marks indicate I and 5 sec.
Changes in cardiovascular function were seen with 10 and 20 mg/kg aldrintransdiol. All subjects developed transient hypotension accompanied by transient or sustained arrythmias. One subject died of ventricular fibrillation after 20 mg/kg. Evoked Responses to Sciatic Nerzle Stimulatiorl The effects of the various treatments upon the amplitudes of responses evoked by sciatic stimulation are given in Table 1. Administration of ethanol solvent in doses up to 0.8 ml/kg did not alter these responses. With dieldrin. responses along the somatosensory pathway in ML and VPL were reduced. The cortical responses. however, were increased in amplitude. The degree of enhancement was dose dependent. A more complete analysis of these effects (Joy, 1974) has demonstrated that they are due to an increased response of cortical neurons to afferent stimuli. In contrast to dieidrin, aldrin-transdiol has no effect upon these responses in doses up to IO mg/kg.
42
R. M. JOY
FIG. 2. EEG activity during aldrintransdiol administration. EEG activity was unaffected at doses of 220 mg/kg aldrintransdiol. The slowing of the EEG and the disruption of EEG spindle waves at IO and 20 mg/kg are due to the ethanol employed as a solvent. The straight line segment in the bottom trace at 20 mg/kg is an artifact. Lower right: Amplitude calibrations. all records 50 pV. Time marks represent I and 5 sec.
DIELDRIN
AND
ALDRIN-TRANSDIOL
143
FIG. 3. Effect of dieldrin on evoked-response amplitudes in sensory and motor pathways, Graphs indicate the changes in amplitudes. as percentage of control amplitudes, during dieldrin administration. Amplitudes were averaged for every 2-min period and are plotted continuously over time from IO min before dieldrin injection until appearance of seizures. (A) Evoked-response amplitudes along the somatosensory pathway to sciatic nerve stimulation. (B) Evoked-response amplitudes in sensorimotor pathway to VPL stimulation.
144
R.M..lOY TABLE
AMPLITUDES
OF
EVOKED
RESPONSES
1
IN SOMATOSENSORY STIMULATION Amplitude
PATHWAY
of evoked
TO SCIATIC
response”
NERVI:
(X -t SE)
Dose Compound Ethanol
Dieldrin
administered 0.1 ml/kg 0.2 ml/kg 0.4 ml/kg 0.8 ml/kg 1 mg/kg
2 mg/kg 3 mg/kg Aldrin-transdiol
1 mg/kg 2.5 mg/kg 5 mg/kg
10 mg/kg
N 4 4 4 4 4 3’ 4 4 4 4
ML 98i 3 96+ 5 95+ 3 901 9 86 * 4h 16 + lh 69& 13 952 5 952 4 862 I 80F 5
VPL 935 4 92k 7 901 IO 91+ 9 82 k 4h 7li 7fi 74+ 10 92+ 3 855 4 872 2 81+ 4
PSG 98+ 995 97& 99t 147 + 177 + 210 & 108 & 101 * 103 * 98+
3 9 IO 9 12” IS” 36:’ I I 5 16 3
” Expressed as percentage of preinjection control amplitude. “p < 0.05 as compared to solvent group. V One subject had a seizure before sufficient data could be collected.
In Fig. 3A the temporal onset of the changes dieldrin produces in these evoked responses are shown. Response amplitudes were determined every 2 set throughout the experiment, and the average amplitude for each successive 2-min period was calculated. The graphs indicate the changes occurring in amplitude over a 5%min period starting 10 min before injecting dieldrin and ending at the onset of the first convulsive seizure. The actions of dieldrin develop rapidly upon administration. The growth in amplitude of the cortical response was steady throughout the experiment. No sudden or abrupt transitions in the responses occurred, suggesting that cortical excitability increases progressively with time to finally reach a level at which spontaneous seizures can occur. A similar experiment with aldrin-transdiol is shown in Fig. 4A. Data were obtained as described above and plotted over time. The figure indicates evoked-response amplitudes over a 2-hr period. These results demonstrate that even over this long interval of time. there is no indication of alteration in evoked responses by aidrin-transdioi.
Evoked Responses in Sensory and Motor Cortex to Stimulation Thalarnic Somatosensory RelaJT Nucleus, VPL
of the Spec$c
The action of dieidrin on cortical excitability can be more clearly demonstrated by stimulating the thalamic nuclei conveying somatosensory activity to the cortex. Such stimuli reach the PSG, which contains the primary somatosensory receiving area of the cortex, and the ASG, which serves as a multimodality sensory integration area for the cat. Both of these areas provide the bulk of corticofugal fibers that project from the cortex to activate subcortical and spinal motor systems. A comparison of the effects of dieldrin and aldrin-transdiol administration on evoked responses at these loci are given in Table 2. Ethanol in doses up to 0.8 ml/kg was without effect. Dieldrin increased the amplitude of evoked responses at all levels. At each locus the extent of the increase was
DIELDRIN
AND
ALDRIN
145
TRANSDIOL
dependent upon the amount of dieldrin administered. Dieldrin consistently increased the responses evoked in ASG and PED to a greater extent than responses in PSG. This effect is predictable from the fact that the pathway from VPL to ASG is largely multisynaptic, whereas that from VPLto PSG is monosynaptic. These results are in agreement
200
CONTROL ALORIN
TRANSDIOL
- mg/hg
OIELDRIN
FIG. 4. Effect of aldrintransdiol on evoked-response amplitudes in sensory and motor pathways. Graphs indicate the changes in amplitudes, as percentage of control, during aldrin-transdiol administration. Amplitudes were averaged for every 2-min period and are plotted continuously over time beginning 20 min before aldrin-transdiol administration. (A) Evoked-response amplitudes along the somatosensory pathway to sciatic nerve stimulation. (B) Evoked-response amplitudes in sensorimotor pathway to VPL stimulation,
with the concept that dieldrin produces a generalized increase in cortical responsiveness. In contrast, aldrin-transdiol had no effect on these responses in doses up to 10 mg/kg. Figure 3B shows the temporal onset of changes in evoked-response amplitudes to VPL stimulation after injecting dieldrin. The graph shows changes developing over a 60-min period. In this particular subject, changes were not evident until 2 mg/kg had been administered. Subsequently, evoked-response amplitudes at all loci increased progressively until the hyperexcitability reached a level where convulsions developed.
146
K.M.JOY TABLE
AMPLITUDES
2
OF EVOKED RESPONSES IN SENWRIMOTOR PATHWAY -1'0 SUMULAUON THALAMICSOMATOSENSORYRELAYNUCLEUS, VPL Amplitude
Compound Ethanol
Dieldrin
Aldrin-transdiol
Dose administered 0.1 0.2 0.4 0.8
ml/kg ml/kg ml/kg ml/kg
I mglkg
2 mg/kg 3 mg/kg I w/kg 2.5 mg/kg 5 mg/kg
IO mg/kg
N
PSG
4 4 4 4 4
4 2’ 4 4 4 4
IOOi 4 972 4 102-t 4 95 L IO 132-t 7h
l96k 15h 212 97i 112-t lO4i lO5i
7 3h 3 6
ofevoked (A’ f SE)
OI
response”
ASG
PED
107-t II 91-t 9 102 * 12 89i I3 I55 * l2h 293 rfr 25” 286 -95i 4 99+ 6 103+ 5 1035 8
93-t 8 8x+ 6 84 i IO 79* II 216 f 12” 430 t 48/’ 391 86& 6 x5, 5 882 4 83+ 6
‘I Expressed as percentage of preinjection control amplitude. h p < 0.05 as compared to solvent group. c Two subjects had a seizure before sufficient data could be collected
Figure 4B shows the temporal development of evoked-response amplitude after aldrin-transdiol administration. The graph covers 160 min. Aldrin-transdiol did not significantly alter the responsesduring an 80.min period in which a total of 10 mg/kg were injected. When dieldrin was given, however, the amplitude of the evoked responses did increase as previously described. This indicates that subjects not responding to aldrin-transdiol were responsive to dieldrin.
DISCUSSION
There is no evidence from these studies that aldrin-transdiol contributes to the toxicity seen following dieldrin exposure. Intravenous administration of it produced no indication of CNS actions, whereas dieldrin in much lower doseswas effective. Cardiovascular effects, somewhat like those reported for aldrin (Gowdey and Stavraky, 1955), were seen with aldrin-transdiol but only at dose levels well above the convulsive dose 100 for dieldrin in cats. In this respect, the data are consistent with the findings of Shroeder and Shankland (1975) in insects where continuous maintenance of internal concentrations of 1O-3 M aldrin-transdiol was without effect. They are also in accord with previous data that the LDSO for orally administered aldrin-transdiol in mice is l215 times higher than for dieldrin (Korte and Arent, 1965). The data indicate that aldrin-transdiol either has little or no CNS activity or that it cannot gain accessto the CNS upon iv administration. Although specific data about the penetration of aldrin-transdiol is unavailable, other evidence suggeststhat it should enter the CNS readily. Aldrin-transdiol is soluble in N-hexane (Ludwig and Korte,
DIELDRIN
AND
ALDRIN-TRANSDIOL
147
1965) readily extracted from aqueous solutions by ether (Korte and Arent, 1965; Matthews and Matsumura, 1969), and chromatographs in systems employing various mobile phases in a manner consistent with a compound expected to cross the bloodbrain barrier (Korte and Arent, 1965: Matthews and Matsumura, 1969). These data are very difficult to reconcile with the theory proposed by Wang et al. (1971) and supported by others (van den Berken and Narahashi, 1974; Akkermans et a/.. 1975a,b) that dieldrin is first converted to aldrin-transdiol before it can exert its neurotoxic action. If aldrin-transdiol does penetrate into the CNS, then the theory is incompatible with the findings reported herein. Even if aldrin-transdiol does not gain entry into the CNS, it would be necessary to assume that dieldrin reaches the CNS and then is rapidly converted to aldrin-transdiol. Such conversion would have to be rapid, as convulsions can occur within minutes of dieldrin administration. Although specific data are lacking, the brain is not considered as a significant metabolic site of dieldrin (Matsumura, 1975). Certainly, the bulk of evidence, particularly in intact mammals, is much more consistent with the belief that dieldrin is a direct acting neurotoxicant. A satisfactory explanation for the activity of aldrin-transdiol and dieldrin in in vitro systems is not presently available. However, certain possibilities merit consideration. Typically, dieldrin and aldrin-transdiol are applied in vitro as an alcoholic solution. When added to aqueous bathing solution, they form opalescent suspensions. The extremely low water solubility of dieldrin of 0.18 ppm (Park and Bruce, 1968) makes solutions in physiological media of > lo-’ M unattainable. In most reports in the literature “solutions” of 10mm4to 10ms M are described indicating they are at best dispersions. Schneider (1975) has demonstrated that for DDT, the amount of DDT bound to membranes is limited by the amount of DDT present, not by its concentration. The membranes behave more as a separate phase containing a solvent for DDT than as a protein with a limited number of binding sites. As a similar phenomenon can be expected to occur for dieldrin and aldrin-transdiol, concentrations used in in vitro studies become meaningless and cannot be correlated with effective in vioo concentrations. The effective membrane concentrations of organochlorine insecticides are likely to be greatly underestimated in in uitro studies, and it is questionable whether changes observed in such systems can be considered relevant to toxicity developing in intact organisms. A second distinction between in uivo and in vitro experiments relates to the quality and quantity of nervous tissue present. It is known for pentylenetetrazol, a water-soluble convulsant. that the brain is more sensitive to its actions than is the spinal cord (Lewin and Esplin, 196 1). Peripheral nerves are even less responsive (Eyzaguirre and Lilienthal, 1949). A similar progression of sensitivity has been found by Gowdey et al. (1954) for dieldrin. Dieldrin is comparatively inactive in vivo on peripheral nerves and striated muscle. For aldrin-transdiol, no activity comparable to dieldrin in mammals has yet been reported. In conclusion, it appears unlikely that aldrin-transdiol contributes significantly to the toxic CNS properties of dieldrin in mammals. For these species. dieldrin seems directly active and does not require metabolism for effect. On the basis of the evidence presented by Shroeder and Shankland (1975), it also seems unlikely that the metabolic conversion of dieldrin to aldrin-transdiol contributes significantly to the insecticidal actions of dieldrin.
148
R. M. JOY ACKNOWLEDGMENTS
The author thanks Mr. R. Zimmerman and Mr. D. Wong for their technical assistance. 1 thank the Shell Chemical Company for their generous gift of dieldrin and aldrin-transdiol. This work was supported by a UCD Faculty Research Grant. REFERENCES AKKERMANS. L. M. A., VAN DEN BERKEN, J., AND VAN DER ZALM, J. M. (1975a). Effects of aldrin-transdiol on neuromuscular facilitation and depression. Eur. J. Pharmacol. 3 I. I66175. AKKERMANS, L. M. A., VAN DEN BERKEN. J., AND VEKSLUIJS-HELDER, M. (1975b). Excitatory and depressant effects of dieldrin and aldrin-transdiol in the spinal cord of the toad (Xer7opus laevis). Eur. J. Pharmacol. 34, 133- 142. EYZAGUIRRE. C.. AND LILIENTHAL, J. C., JR. (1949). Veratrinic effects of pentamethylenetetrazol (Metrazol) and 2.2.bis(p-chlorophenyl)1.1 ,l -trichloroethane (DDT) on mammalian neuromuscular function. Proc. Sot. Exp. Biol. Med. 70, 272-275. GOWDEY. C. W.. GRAHAM, A. R., SEGUIN, J. J.. AND STAVRAKY, G. W. (1954). The pharmacological properties of the insecticide dieldrin. Canad. J. Biochem. Phwiol. 32. 498503. GOWDEY. C. W.. AND STAVRAKY. G. W. (1955). A study of the autonomic manifestations seen in acute aldrin and dieldrin poisoning. Canad. J. Biochem. Physiol. 33, 272-282. JASPER, H. H.. AND AJMONE-MARSAN, C. (1954). A Stereotaxic Atlas of the Diencephalon oJ‘ the Cat. National Research Council of Canada, Ottawa, Canada. JOY. R. M. (1973). Electrical correlates of preconvulsive and convulsive doses of chlorinated hydrocarbon insecticides in the CNS. Neuropharmacologj~ 12,63-76. JOY. R. M. (1974). Alteration of sensory and motor evoked responses by dieldrin. Neuropharmacology 13.93- 110. JOY. R. M. (1975). Comparative effects of convulsants on the antidromic cortical response to pyramidal tract stimulation. Neuropharmacology 14. 869-88 1. JOY, R. M. (1976). Convulsive properties of chlorinated hydrocarbon insecticides in the cat central nervous system. Toxicol. Appl. Pharmacol. 35, 95- 106. KORTE. F.. AND ARENT, H. (1965). Metabolism of insecticides, IX(l). Isolation and identification of dieldrin metabolites from urine of rabbits after oral administration of dieldrinmiJC. Lij2 Sri. 4. 2017-2026. LEWIN, J.. AND ESPLIN, D. W. (1961). Analysis of the spinal excitatory action of pentylenetetrazol. J. Pharrnacol. Esp. Therap. 132. 245-250. LIIDWIG. G.. AND KORTE, F. (1965). Metabolism of insecticides. X( 1). Detection of dieldrin metabolites by GLC-analysis. Life Sci. 4, 2027-203 I. MATSUMURA. F. (1975). Toxicology of Insecticides, pp. 165-25 I. Plenum Press, New York. MATTHEWS. H. B.. AND MATSUMURA, F. (1969). Metabolic fate of dieldrin in the rat. d. .-lgr. Food C’hem. 17.845-852. PARK;. K. S.. AND BRUCE. W. N. (1968). The determination of the water solubility of aldrin. dieldrin. heptachlor, and heptachlor epoxide. J. Ecor7. Entomol. 61, 770-774. SCHNEIDER. R. P. (1975). Mechanism of inhibition of rat brain. (Na + K)-adenosine triphosphatase by 2.2.bis(p-chlorophenyl)1. I. I-trichloroethane (DDT). Biochem. Pharmacol. 24. 939-946. SHROEDER, M. E.. AND SHANKLAND, D. L. (1975). Paper presented at the Ent. Sot. Amer. National Meeting. Minneapolis (Dec. 2. 1974) as referenced by: DESAIAH. D., AND Koctr. R. B. (1975). Inhibition of fish brain ATPases by aldrin-transdiol, aldrin. dieldrin. and photodieldrin. Biochem. Biophvs. Res. Cornman. 64, 13- 19. VAN DEN B~KIC~N. J.. AND ~IARAHASHL T. (1974). Effects of aldrin-transdiol-a metabolite of the insecticide dieldrin-on nerve membrane. Ear. J. Pharmacol. 27.255-258. WANG. C. M.. NARAHASHI, T., AND YAMADA, M. (1971). The neurotoxic action of dieldrin and its derivatives in the cockroach. Pest. Biochem. Phwiol. 1, 84-9 1.
Actions Metabolite.
of Dieldrin and Aldrin-transdiol, on Cat CNS Function R. M.
Depurltnent
of Physiological
Sciences.
Receil)ed
Unirersify California October
JOY
of California, 956 16
21, I976:
Its
accepted
School
April
of Vererinacv
Medicine,
Du~~;s,
6, I977
Contrasting Actions of Dieldrin and Aldrin-transdiol, Its Metabolite,on Cat CNS Function. R. M. (1977). Toxicol. App/. Pharmacol. 42. 137-148. Dieldrin and aldrin-transdiol were administered to cats to test whether dieldrin is directly active or whether it requires conversion IO aldrin-transdiol for CNS effects. In cats 2-4 mg/kg dieldrin produced convulsive activity in 215 min. This was preceded by changes in the EEG and in responses evoked by sensory stimuli consistent with changes produced by pentylenetetrazol-type convulsants. Aldrin-transdiol in doses up to 20 mg/kg was without effect in cats. No convulsions were observed. No evidence 01 CNS action could be discerned from examination of the EEG or from responses to sensory stimulation. The results are consistent with the idea that dieldrin is directly active in the mammalian CNS. Its metabolite. aldrin-transdiol. when injected directly into the blood stream is either inactive or is incapable of entering the CNS from this route. The former alternative appears most likely. JOY.
The convulsant properties of dieldrin are well known. In mammals the acute toxicity of dieldrin is primarily of central nervous origin (Gowdey et al., 1954), and its actions at that level are similar to pentylenetetrazol (Joy, 1973, 1974, 1975). Following iv administration in cats of 2-4 mg/kg, convulsions develop in 2-15 min, suggesting that dieldrin is directly acting in the CNS (Joy, 1976). However, studies of the dieldrin metabolite, aldrin-transdiol, have caused others to conclude that the metabolite is largely responsible for the actions of dieldrin. Wang et a/. (197 1) proposed that dieldrin is converted to an active metabolite, basing this conclusion on the relative dose required and the temporal development of effects of dieldrin and metabolites in vitro. Van den Berken and Narahashi (1974) reported that aldrin-transdiol suppressespeak transient current in voltage-clamped squid axons while dieldrin is ineffective. Similarly, Akkermans et al. (1975a,b) have reported that aldrin-transdiol is both more potent and more rapid acting than dieldrin in a number of in vitro nerve and muscle preparations. However, Shroeder and Shankland (1975) have reported that, in cockroaches in vivo. internal concentrations of lo-) M aldrintransdiol maintained for 1 week were not toxic whereas dieldrin at similar concentrations gave 80% mortality in 10 hr. Korte and Arent (1965) have also reported that the oral LD50 for aldrin-transdiol in mice is at least 10 times higher than for dieldrin. In this report the CNS actions of dieldrin and aldrin-transdiol are compared to test the hypothesis that dieldrin must be converted to its metabolite before it can produce CNS activity in mammals. Dieldrin produces clearly recognizable changes in the CopyrIght 0 1911 hy Academic Press. Inc. All rights of reproducrmn in any form reserved. Pnnlcd I” Great Britam
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electroencephalogram (EEG) and in evoked responses in sensory and motor pathways before precipitating tonic-clonic convulsions (Joy, 1973. 1974, 1976). If dieldrin must be converted to an active metabolite, then the metabolite would be expected to exert effects both at a lower dose and more rapidly. Aldrintransdiol was examined on these same systems to compare potency and temporal development against dieldrin. The results suggest that aldrin-transdiol is neither responsible for the convulsive properties ot dieldrin nor does this metabolite contribute significantly to it in mammals.
METHODS General Procedures Twenty-four male cats. 2.5-4 kg, were subjects. Eight received dieldrin. eight received aldrin-transdiol, and eight served as controls. All procedures followed were in accord with the Guiding Principles in the Care and Use of Animals as approved by the Council of the American Physiological Society. Subjects were fasted overnight, anesthetized, tracheotomized, and placed in a stereotaxic frame. Halothane was the anesthetic for all surgical procedures. Both brachiocephalic veins and a femoral artery were cannulated for drug injection and to record blood pressure. The skin and muscles overlying the cranium were dissected laterally and holes were drilled through the bone to insert electrodes into subcortical structures. The bone overlying the sigmoid gyri was removed, the dura was dissected away, and the exposed cortex was covered with 38°C mineral oil to prevent drying. In 12 experiments the sciatic nerve was exposed and cut, and a Sherrington electrode was placed on the central end. After these procedures, a rectal probe was inserted, and 0.25% dibucaine was infiltrated into all wound margins and pressure points. All wound margins were wrapped in gauze kept moist with saline and mineral oil, and the corneas were coated with mineral oil to prevent drying. Then anesthesia was discontinued. Gallamine (-7 mg/kg/hr) was infused to produce paralysis, and respiration was supported by a Harvard 620 respirator. End-expiratory CO, was continually monitored with a Beckman LB1 analyzer, and respiration was adjusted to maintain end-expiratory CO, between 3.2 and 3.5%. Temperature was kept at 38OC with a heating pad and wraps. During all subsequent procedures, EEG, EKG, blood pressure, CO,, and temperature were monitored to assess physiological state and to assure freedom from pain. After recovery from anesthesia, all subjects displayed cycles of alert and relaxed or drowsy periods as monitored by EEG and autonomic activity. EEG Recording EEGs were obtained with chlorided silver ball electrodes, 0.5 mm in diameter, placed directly on the cortex or with stainless-steel electrodes tapped firmly into small burr holes made in the cranium overlying cortex. All records were monopolar, the stereotaxic frame serving as reference. EEGs were recorded on a Grass Model 78 polygraph, and selected samples before and after drug administration were chosen for illustrative purposes. In all figures an upward deflection in the EEG indicates negativity at the electrode with respect to the reference. Half-amplitude frequencies for all records are I and 90 Hz.
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Responses along Sensoty and Motor PathwaJts To provide stimuli for sensory pathway analysis, the sciatic nerve was stimulated in 12 experiments with supramaximal rectangular pulses (one per 2 set, 0.1 msec duration, 0.5-l mA). The stimuli were generated by a WPI Series 800 stimulator and were led to the electrode through a photon-coupled isolator. Stimuli were initiated at least 1 hr before data collection began to reduce the possibility of habituation during experii mental periods. Usually responses stabilized within 30-40 min (2.5 hr after termination of anesthesia). Responses were recorded at locations along the primary sensory pathway including the medial lemniscus (ML), coordinates from the stereotaxic atlas of Jasper and Ajmone-Marsan (1954), A 4.0, L 5.0. V -2.0: nucleus ventralis posterolateralis (VPL) of the thalamus (A 9.0, L 7.5, V 1.0); and from the posterior sigmoid gyrus (PSG) which, in the cat, represents primary somatosensory cortex. Bipolar stainless-steel electrodes were used to record responses in ML and VPL, and an Ag-AgCl ball electrode was used to record from the cortex. Subcortical electrodes were lowered to just above the desired recording site under stereotaxic control. Final placements were made while stimulating to produce a maximal amplitude evoked response. Cortical electrodes were positioned at the location yielding the largest response to stimulation. Responses during the experiment were recorded on tape (Hewlett-Packard 755C, O-2.5 kHz bandwidth) and analyzed at a later time. In 12 other experiments carried out in a similar manner, the VPL was stimulated at twice threshold intensity (one per 2 set, 0.1 msec, 0.6-1.5 mA). Responses were obtained from the posterior sigmoid gyrus (PSG). anterior sigmoid gyrus (ASG), and from corticofugal axons at the level of the cerebral peduncle (PED) (A 4.0, L 4.0, V -6.0). In every experiment 3&40 min of control data were collected which included 20 min of evoked-response data and l&20 min of spontaneous activity. Then specified amounts of solvent, dieldrin, or aldrin-transdiol were injected. Evoked responses were continuously elicited for 20 min; then additional compound was administered. This sequence was continued for the duration of the experiment. In four experiments with each compound, the time interval between injections was increased to 25 min to gather spontaneous activity. All doses indicated in the results are cumulative amounts and indicate the total amount given up to that point. At the end of the experiments the cats were sacrificed with pentobarbital, and the electrode locations were marked by passing current through them. The brains were perfused with formol-saline containing potassium ferricyanide and removed. Frozen sections, 50 ,um thick, were taken at the electrode locations and stained with Luxol fast blue. Actual locations were determined with the atlas of Jasper and Ajmone-Marsan (1954). In all cases, the locations of the electrodes were within the desired structure. Administration
of Dieldrin and Aldrin-transdiol
Dieldrin (>99% HEOD) and aldrintransdiol (>95% pure) were provided by the Shell Chemical Company. Both compounds were verified for content and purity by gas chromatographic and mass spectral analyses. The chemicals were dissolved in ethanol at concentrations of 10 or 20 mg/kg/ml. Administration was accomplished by first injecting 0.05 ml of ethanol into the cannula, and then administering 0.1-0.5 ml of ethanol containing the cyclodiene derivative. The cannula was cleared with an additional 0.05 ml of ethanol followed by 2 ml of saline. Injection took l-2 min. In all
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cases except the administration of aldrin-transdiol at 20 mg/kg, the total volume of ethanol administered was no greater than 1.1 ml, Ethanol in doses over 1 ml can produce transient signs of depression, but such effects are opposite to nature to and clearly distinguishable from the CNS actions of dieldrin.
Data Analysis In experiments where spontaneous EEGs were obtained, at least 5 min of data were gathered before and after each injection of chemical. EEGs were visually evaluated by two independent observers, and samples considered representative of a particular state were selected for illustration. Evoked responses in sensory and motor pathways were analyzed after the experiment. The taped responses were played back, and the average evoked response at each locus was determined at every minute interval with a computer of average transients. The peak-to-peak amplitude of the primary response was measured. These amplitudes provided a minute to minute measure of the evoked activity at various locations. For statistical analysis, the evoked-response amplitude during the last 10 min of data collection at each dose of solvent, dieldrin, or aldrin-transdiol were averaged for each animal. The results for four different subjects experiencing identical experimental conditions were used to generate means and SE for each condition. Finally the significance of the dieldrin and aldrin-transdiol groups as compared to the solvent control groups was determined by Student’s t test.
RESULTS
Electroencephalograms Intravenous administration of dieldrin induces a consistent sequence of EEG alterations in the cat (Fig. 1). Early signs of intoxication include the development of hypersynchronous activity distributed symmetrically across the cortical surface (Fig. 1B). This may occur with doses of 1 mg/kg or more. Later, epileptiform spikes or groups of spikes begin to appear (Figs. IC and 1D). These are distributed symmetrically between hemispheresand tend to be of larger voltage over anteriorly placed electrodes. They may occur spontaneously or may be evoked by sensory stimulation. The electrical correlates of convulsions (Fig. 1D) develop shortly thereafter. Seizures most often begin with the appearanceof high-amplitude synchronous waves. which are of largest amplitude from frontal cortical locations. These spread rapidly across the cortex and slow with time to generate the clonic phaseof the seizure. These events have beendescribed in detail in previous reports (Joy, 1973. 1976). The administration of aldrin-transdiol in dosesof l-20 mg/kg did not evoke any of the EEG actions characteristic of dieldrin (Fig. 2). No effects were observed at doses from 1 to 5 mg/kg. At 10 and 20 mg/kg there was an increase of slow waves and a disruption and slowing of spindles in the EEG. These changes are not typical of convulsive agents, but rather are more characteristic of sedative or CNS depressive agents. As similar changes occurred when equal volumes of solvent without aldrin transdiol were given, these changes are solvent related and can not be attributed to aldrin-transdiol. The important finding, however, is that aldrin-transdiol in doses 5-10
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times greater than the convulsant dose of dieldrin does not induce any evidence of hyperexcitability or preconvulsive changes in the EEG. In two subjects which had received 10 mg/kg aldrin-transdiol with no effect, dieldrin was administered. The EEG changes and convulsions reported for dieldrin occurred when 2 mg/kg were injected, indicating that the subjects were not unusually resistant to convulsive agents.
FIG. I. EEG changes developing after dieldrin administration. (A) Before injecting 2 mg/kg dieldrin. (H) Five minutes after dieldrin injection. This record demonstrates the high-amplitude hypersynchrony. which is the first indication of developing hyperexcitahility. (C) Ten minutes later the EEG shows intermittent bursts of hypersynchrony with single or multiple superimposed spikes. It is from this background that convulsions most often develop. (D) Single or grouped spikes occurring occasionally before convulsions. (E) Twenty-five minutes after dieldrin administration a convulsion develops spontaneously on a background of intermittent hypersynchrony. Seizure is followed by a period of post-ictal depression. Lower right: Amplitude calibration. A and B. 100 ,uV: C-E. 200 pV. Time marks indicate I and 5 sec.
Changes in cardiovascular function were seen with 10 and 20 mg/kg aldrintransdiol. All subjects developed transient hypotension accompanied by transient or sustained arrythmias. One subject died of ventricular fibrillation after 20 mg/kg. Evoked Responses to Sciatic Nerzle Stimulatiorl The effects of the various treatments upon the amplitudes of responses evoked by sciatic stimulation are given in Table 1. Administration of ethanol solvent in doses up to 0.8 ml/kg did not alter these responses. With dieldrin. responses along the somatosensory pathway in ML and VPL were reduced. The cortical responses. however, were increased in amplitude. The degree of enhancement was dose dependent. A more complete analysis of these effects (Joy, 1974) has demonstrated that they are due to an increased response of cortical neurons to afferent stimuli. In contrast to dieidrin, aldrin-transdiol has no effect upon these responses in doses up to IO mg/kg.
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FIG. 2. EEG activity during aldrintransdiol administration. EEG activity was unaffected at doses of 220 mg/kg aldrintransdiol. The slowing of the EEG and the disruption of EEG spindle waves at IO and 20 mg/kg are due to the ethanol employed as a solvent. The straight line segment in the bottom trace at 20 mg/kg is an artifact. Lower right: Amplitude calibrations. all records 50 pV. Time marks represent I and 5 sec.
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FIG. 3. Effect of dieldrin on evoked-response amplitudes in sensory and motor pathways, Graphs indicate the changes in amplitudes. as percentage of control amplitudes, during dieldrin administration. Amplitudes were averaged for every 2-min period and are plotted continuously over time from IO min before dieldrin injection until appearance of seizures. (A) Evoked-response amplitudes along the somatosensory pathway to sciatic nerve stimulation. (B) Evoked-response amplitudes in sensorimotor pathway to VPL stimulation.
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R.M..lOY TABLE
AMPLITUDES
OF
EVOKED
RESPONSES
1
IN SOMATOSENSORY STIMULATION Amplitude
PATHWAY
of evoked
TO SCIATIC
response”
NERVI:
(X -t SE)
Dose Compound Ethanol
Dieldrin
administered 0.1 ml/kg 0.2 ml/kg 0.4 ml/kg 0.8 ml/kg 1 mg/kg
2 mg/kg 3 mg/kg Aldrin-transdiol
1 mg/kg 2.5 mg/kg 5 mg/kg
10 mg/kg
N 4 4 4 4 4 3’ 4 4 4 4
ML 98i 3 96+ 5 95+ 3 901 9 86 * 4h 16 + lh 69& 13 952 5 952 4 862 I 80F 5
VPL 935 4 92k 7 901 IO 91+ 9 82 k 4h 7li 7fi 74+ 10 92+ 3 855 4 872 2 81+ 4
PSG 98+ 995 97& 99t 147 + 177 + 210 & 108 & 101 * 103 * 98+
3 9 IO 9 12” IS” 36:’ I I 5 16 3
” Expressed as percentage of preinjection control amplitude. “p < 0.05 as compared to solvent group. V One subject had a seizure before sufficient data could be collected.
In Fig. 3A the temporal onset of the changes dieldrin produces in these evoked responses are shown. Response amplitudes were determined every 2 set throughout the experiment, and the average amplitude for each successive 2-min period was calculated. The graphs indicate the changes occurring in amplitude over a 5%min period starting 10 min before injecting dieldrin and ending at the onset of the first convulsive seizure. The actions of dieldrin develop rapidly upon administration. The growth in amplitude of the cortical response was steady throughout the experiment. No sudden or abrupt transitions in the responses occurred, suggesting that cortical excitability increases progressively with time to finally reach a level at which spontaneous seizures can occur. A similar experiment with aldrin-transdiol is shown in Fig. 4A. Data were obtained as described above and plotted over time. The figure indicates evoked-response amplitudes over a 2-hr period. These results demonstrate that even over this long interval of time. there is no indication of alteration in evoked responses by aidrin-transdioi.
Evoked Responses in Sensory and Motor Cortex to Stimulation Thalarnic Somatosensory RelaJT Nucleus, VPL
of the Spec$c
The action of dieidrin on cortical excitability can be more clearly demonstrated by stimulating the thalamic nuclei conveying somatosensory activity to the cortex. Such stimuli reach the PSG, which contains the primary somatosensory receiving area of the cortex, and the ASG, which serves as a multimodality sensory integration area for the cat. Both of these areas provide the bulk of corticofugal fibers that project from the cortex to activate subcortical and spinal motor systems. A comparison of the effects of dieldrin and aldrin-transdiol administration on evoked responses at these loci are given in Table 2. Ethanol in doses up to 0.8 ml/kg was without effect. Dieldrin increased the amplitude of evoked responses at all levels. At each locus the extent of the increase was
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dependent upon the amount of dieldrin administered. Dieldrin consistently increased the responses evoked in ASG and PED to a greater extent than responses in PSG. This effect is predictable from the fact that the pathway from VPL to ASG is largely multisynaptic, whereas that from VPLto PSG is monosynaptic. These results are in agreement
200
CONTROL ALORIN
TRANSDIOL
- mg/hg
OIELDRIN
FIG. 4. Effect of aldrintransdiol on evoked-response amplitudes in sensory and motor pathways. Graphs indicate the changes in amplitudes, as percentage of control, during aldrin-transdiol administration. Amplitudes were averaged for every 2-min period and are plotted continuously over time beginning 20 min before aldrin-transdiol administration. (A) Evoked-response amplitudes along the somatosensory pathway to sciatic nerve stimulation. (B) Evoked-response amplitudes in sensorimotor pathway to VPL stimulation,
with the concept that dieldrin produces a generalized increase in cortical responsiveness. In contrast, aldrin-transdiol had no effect on these responses in doses up to 10 mg/kg. Figure 3B shows the temporal onset of changes in evoked-response amplitudes to VPL stimulation after injecting dieldrin. The graph shows changes developing over a 60-min period. In this particular subject, changes were not evident until 2 mg/kg had been administered. Subsequently, evoked-response amplitudes at all loci increased progressively until the hyperexcitability reached a level where convulsions developed.
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K.M.JOY TABLE
AMPLITUDES
2
OF EVOKED RESPONSES IN SENWRIMOTOR PATHWAY -1'0 SUMULAUON THALAMICSOMATOSENSORYRELAYNUCLEUS, VPL Amplitude
Compound Ethanol
Dieldrin
Aldrin-transdiol
Dose administered 0.1 0.2 0.4 0.8
ml/kg ml/kg ml/kg ml/kg
I mglkg
2 mg/kg 3 mg/kg I w/kg 2.5 mg/kg 5 mg/kg
IO mg/kg
N
PSG
4 4 4 4 4
4 2’ 4 4 4 4
IOOi 4 972 4 102-t 4 95 L IO 132-t 7h
l96k 15h 212 97i 112-t lO4i lO5i
7 3h 3 6
ofevoked (A’ f SE)
OI
response”
ASG
PED
107-t II 91-t 9 102 * 12 89i I3 I55 * l2h 293 rfr 25” 286 -95i 4 99+ 6 103+ 5 1035 8
93-t 8 8x+ 6 84 i IO 79* II 216 f 12” 430 t 48/’ 391 86& 6 x5, 5 882 4 83+ 6
‘I Expressed as percentage of preinjection control amplitude. h p < 0.05 as compared to solvent group. c Two subjects had a seizure before sufficient data could be collected
Figure 4B shows the temporal development of evoked-response amplitude after aldrin-transdiol administration. The graph covers 160 min. Aldrin-transdiol did not significantly alter the responsesduring an 80.min period in which a total of 10 mg/kg were injected. When dieldrin was given, however, the amplitude of the evoked responses did increase as previously described. This indicates that subjects not responding to aldrin-transdiol were responsive to dieldrin.
DISCUSSION
There is no evidence from these studies that aldrin-transdiol contributes to the toxicity seen following dieldrin exposure. Intravenous administration of it produced no indication of CNS actions, whereas dieldrin in much lower doseswas effective. Cardiovascular effects, somewhat like those reported for aldrin (Gowdey and Stavraky, 1955), were seen with aldrin-transdiol but only at dose levels well above the convulsive dose 100 for dieldrin in cats. In this respect, the data are consistent with the findings of Shroeder and Shankland (1975) in insects where continuous maintenance of internal concentrations of 1O-3 M aldrin-transdiol was without effect. They are also in accord with previous data that the LDSO for orally administered aldrin-transdiol in mice is l215 times higher than for dieldrin (Korte and Arent, 1965). The data indicate that aldrin-transdiol either has little or no CNS activity or that it cannot gain accessto the CNS upon iv administration. Although specific data about the penetration of aldrin-transdiol is unavailable, other evidence suggeststhat it should enter the CNS readily. Aldrin-transdiol is soluble in N-hexane (Ludwig and Korte,
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1965) readily extracted from aqueous solutions by ether (Korte and Arent, 1965; Matthews and Matsumura, 1969), and chromatographs in systems employing various mobile phases in a manner consistent with a compound expected to cross the bloodbrain barrier (Korte and Arent, 1965: Matthews and Matsumura, 1969). These data are very difficult to reconcile with the theory proposed by Wang et al. (1971) and supported by others (van den Berken and Narahashi, 1974; Akkermans et a/.. 1975a,b) that dieldrin is first converted to aldrin-transdiol before it can exert its neurotoxic action. If aldrin-transdiol does penetrate into the CNS, then the theory is incompatible with the findings reported herein. Even if aldrin-transdiol does not gain entry into the CNS, it would be necessary to assume that dieldrin reaches the CNS and then is rapidly converted to aldrin-transdiol. Such conversion would have to be rapid, as convulsions can occur within minutes of dieldrin administration. Although specific data are lacking, the brain is not considered as a significant metabolic site of dieldrin (Matsumura, 1975). Certainly, the bulk of evidence, particularly in intact mammals, is much more consistent with the belief that dieldrin is a direct acting neurotoxicant. A satisfactory explanation for the activity of aldrin-transdiol and dieldrin in in vitro systems is not presently available. However, certain possibilities merit consideration. Typically, dieldrin and aldrin-transdiol are applied in vitro as an alcoholic solution. When added to aqueous bathing solution, they form opalescent suspensions. The extremely low water solubility of dieldrin of 0.18 ppm (Park and Bruce, 1968) makes solutions in physiological media of > lo-’ M unattainable. In most reports in the literature “solutions” of 10mm4to 10ms M are described indicating they are at best dispersions. Schneider (1975) has demonstrated that for DDT, the amount of DDT bound to membranes is limited by the amount of DDT present, not by its concentration. The membranes behave more as a separate phase containing a solvent for DDT than as a protein with a limited number of binding sites. As a similar phenomenon can be expected to occur for dieldrin and aldrin-transdiol, concentrations used in in vitro studies become meaningless and cannot be correlated with effective in vioo concentrations. The effective membrane concentrations of organochlorine insecticides are likely to be greatly underestimated in in uitro studies, and it is questionable whether changes observed in such systems can be considered relevant to toxicity developing in intact organisms. A second distinction between in uivo and in vitro experiments relates to the quality and quantity of nervous tissue present. It is known for pentylenetetrazol, a water-soluble convulsant. that the brain is more sensitive to its actions than is the spinal cord (Lewin and Esplin, 196 1). Peripheral nerves are even less responsive (Eyzaguirre and Lilienthal, 1949). A similar progression of sensitivity has been found by Gowdey et al. (1954) for dieldrin. Dieldrin is comparatively inactive in vivo on peripheral nerves and striated muscle. For aldrin-transdiol, no activity comparable to dieldrin in mammals has yet been reported. In conclusion, it appears unlikely that aldrin-transdiol contributes significantly to the toxic CNS properties of dieldrin in mammals. For these species. dieldrin seems directly active and does not require metabolism for effect. On the basis of the evidence presented by Shroeder and Shankland (1975), it also seems unlikely that the metabolic conversion of dieldrin to aldrin-transdiol contributes significantly to the insecticidal actions of dieldrin.
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R. M. JOY ACKNOWLEDGMENTS
The author thanks Mr. R. Zimmerman and Mr. D. Wong for their technical assistance. 1 thank the Shell Chemical Company for their generous gift of dieldrin and aldrin-transdiol. This work was supported by a UCD Faculty Research Grant. REFERENCES AKKERMANS. L. M. A., VAN DEN BERKEN, J., AND VAN DER ZALM, J. M. (1975a). Effects of aldrin-transdiol on neuromuscular facilitation and depression. Eur. J. Pharmacol. 3 I. I66175. AKKERMANS, L. M. A., VAN DEN BERKEN. J., AND VEKSLUIJS-HELDER, M. (1975b). Excitatory and depressant effects of dieldrin and aldrin-transdiol in the spinal cord of the toad (Xer7opus laevis). Eur. J. Pharmacol. 34, 133- 142. EYZAGUIRRE. C.. AND LILIENTHAL, J. C., JR. (1949). Veratrinic effects of pentamethylenetetrazol (Metrazol) and 2.2.bis(p-chlorophenyl)1.1 ,l -trichloroethane (DDT) on mammalian neuromuscular function. Proc. Sot. Exp. Biol. Med. 70, 272-275. GOWDEY. C. W.. GRAHAM, A. R., SEGUIN, J. J.. AND STAVRAKY, G. W. (1954). The pharmacological properties of the insecticide dieldrin. Canad. J. Biochem. Phwiol. 32. 498503. GOWDEY. C. W.. AND STAVRAKY. G. W. (1955). A study of the autonomic manifestations seen in acute aldrin and dieldrin poisoning. Canad. J. Biochem. Physiol. 33, 272-282. JASPER, H. H.. AND AJMONE-MARSAN, C. (1954). A Stereotaxic Atlas of the Diencephalon oJ‘ the Cat. National Research Council of Canada, Ottawa, Canada. JOY. R. M. (1973). Electrical correlates of preconvulsive and convulsive doses of chlorinated hydrocarbon insecticides in the CNS. Neuropharmacologj~ 12,63-76. JOY. R. M. (1974). Alteration of sensory and motor evoked responses by dieldrin. Neuropharmacology 13.93- 110. JOY. R. M. (1975). Comparative effects of convulsants on the antidromic cortical response to pyramidal tract stimulation. Neuropharmacology 14. 869-88 1. JOY, R. M. (1976). Convulsive properties of chlorinated hydrocarbon insecticides in the cat central nervous system. Toxicol. Appl. Pharmacol. 35, 95- 106. KORTE. F.. AND ARENT, H. (1965). Metabolism of insecticides, IX(l). Isolation and identification of dieldrin metabolites from urine of rabbits after oral administration of dieldrinmiJC. Lij2 Sri. 4. 2017-2026. LEWIN, J.. AND ESPLIN, D. W. (1961). Analysis of the spinal excitatory action of pentylenetetrazol. J. Pharrnacol. Esp. Therap. 132. 245-250. LIIDWIG. G.. AND KORTE, F. (1965). Metabolism of insecticides. X( 1). Detection of dieldrin metabolites by GLC-analysis. Life Sci. 4, 2027-203 I. MATSUMURA. F. (1975). Toxicology of Insecticides, pp. 165-25 I. Plenum Press, New York. MATTHEWS. H. B.. AND MATSUMURA, F. (1969). Metabolic fate of dieldrin in the rat. d. .-lgr. Food C’hem. 17.845-852. PARK;. K. S.. AND BRUCE. W. N. (1968). The determination of the water solubility of aldrin. dieldrin. heptachlor, and heptachlor epoxide. J. Ecor7. Entomol. 61, 770-774. SCHNEIDER. R. P. (1975). Mechanism of inhibition of rat brain. (Na + K)-adenosine triphosphatase by 2.2.bis(p-chlorophenyl)1. I. I-trichloroethane (DDT). Biochem. Pharmacol. 24. 939-946. SHROEDER, M. E.. AND SHANKLAND, D. L. (1975). Paper presented at the Ent. Sot. Amer. National Meeting. Minneapolis (Dec. 2. 1974) as referenced by: DESAIAH. D., AND Koctr. R. B. (1975). Inhibition of fish brain ATPases by aldrin-transdiol, aldrin. dieldrin. and photodieldrin. Biochem. Biophvs. Res. Cornman. 64, 13- 19. VAN DEN B~KIC~N. J.. AND ~IARAHASHL T. (1974). Effects of aldrin-transdiol-a metabolite of the insecticide dieldrin-on nerve membrane. Ear. J. Pharmacol. 27.255-258. WANG. C. M.. NARAHASHI, T., AND YAMADA, M. (1971). The neurotoxic action of dieldrin and its derivatives in the cockroach. Pest. Biochem. Phwiol. 1, 84-9 1.
