O States
EPA-600/1-80-028
May 1980
»( j'' ' '
Toxicity, Interactions,
and Metabolism of
Formamidine Pesticides
in Mammals
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
ii
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FOREWARD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those already in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health
care and surveillance of persons having suffered imminent and substantial
endangerment of their health.
This report describes investigations of the mechanism(s) of acute
toxicity of formamidine pesticides in mammals. Formamidine pesticides are
a relatively new group of insecticide-acaricides which are particularly
useful for the control of Lepidoptera, Hemiptera, phytophagous mites,
and cattle ticks. Their widespread use for control of cotton insects and
cattle ticks make the investigation of their toxicity extremely important.
F. Gordon Hueter
Director
Health Effects Research Laboratory
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ABSTRACT
The overall goal of this research project was to investigate the
mechanism(s) of acute toxicity of formaroidine pesticides in mammals
using chlordimeform (^'-(M-chloro-o-tolyD-^NUdimethylformamidine) and
its metabolites as the primary model compound!?. The role of
blotransformations, particularly jN-demethylation reactions, in
generating potentially toxic metabolites was also studied.
By comparing the effects of hepatic microsomal mixed function
oxidase inducers and inhibitors administered Jin vivo on the toxicity,
metabolism, and distribution of metabolites in mouse tissues, it was
concluded that although NUdemethylation products are innately more toxic
than chlordimeform, they~are also less stable, and the best correlation
of toxicity was obtained with the total level of formamidines in the
brain, rather than with the level of any individual metabolite.
In a series of studies with dogs, rabbits, and cats, the cause of
death was found to be cardiovascular collapse accompanied by respiratory
arrest. Cardiovascular collapse resulted primarily from a peripheral
local anesthetic-like effect of chlordimeform. Monoamine oxidase
inhibition was not a major factor in lethality. Respiratory arrest was
central in origin. Several other central effects of the formamidines
were described, some of which may be local anesthetic actions, and a
behavioral profile for chlordimeform poisoning in the rat was developed.
The effectiveness of various drug treatments as potential therapeutic
aids for formamidine intoxication were studied. Finally, the
formamidines were found to possess a number of aspirin-like actions
which resulted from an ability to inhibit prostaglandln synthesis.
This report was submitted in fulfillment of Research Grant No.
R-803965 by Purdue Research Foundation under the sponsorship of the U.S.
Environmental Protection Agency. This report covers the period July
1st, 1975 to December 31st, 1978, and the work was completed as of July
1st, 1979.
iv
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CONTENTS
Foreword . . ill
Abstract iv
List of Figures . vl
List of Tables vlti
Abbreviations, ... x
Acknowledgments xl
1. General Introduction 1
2. Conclusions 4
3. Section 1 - Evaluation of Monoamine
Oxldase as a Target for Formamidlne Pesticides. ..... 6
4. Section 2 - Actions of Chlordimeform on the
Cardiovascular System ........ 20
5. Section 3 - The Central Actions of Chlordimeform. ..... 29
6. Section 1 - Local Anesthetic Properties of Chlordimeform
and Potential Means of Alleviating Toxicity 38
7- Section 5 - Behavioral Effects of Chlordimeform 48
8. Section 6 - Inhibition of Prostaglandin Synthesis
by Formamidines 57
9. Section 7 - Blotransformation of Chlordimeform and its
Relation to Toxicity 67
References 93
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FIGURES
Number
1 Recovery of mouse brain mitochondrial MAO from inhibition
by CDM after successive washes to remove the inhibitor. . . .
2 Time course of inhibition of MAO from mouse intestine
by chlordimeform, N-demethyl chlordimeform, and
j|I-formyl-4-chloro-o-toluidine ................ 12
3 Inhibition of MAO from mouse intestine by CDM assessed
with three MAO substrates .................. 13
M Inhibition of MAO from mouse intestine by NFT assessed
with three MAO substrates .................. 14
5 Typical records showing the effects of CDM and lidocaine
on heart rate, cardiac contractility, and blood pressure
in the pentobarbital-anesthetized dog ............ 21
6 Initial (1°) and secondary (11°) effects of CDM and
lidocaine on blood pressure and cardiac contractility .... 23
7 The effects of CDM and lidocaine on heart rate and cardiac
contractility in the isolated rabbit heart preparation ... 24
8 The effects of intraarterial CDM and lidocaine on the hind
limb perfusion pressure in the pentobarbital-anesthetized
dog ............................. 25
9 Typical record showing the effects of CDM on blood pressure
and spike discharge frequency recorded preganglionically
from the superior cervical nerve of the pentobarbital-
anesthetized cat ....................... 26
10 Pressor responses in the carotid artery after intraventric-
ular injections of lidocaine or CDM in a rat anesthetized
with urethane ......................... 31
11 Suppression of amygdala EEC upon raphe stimulation and
antagonism of this suppresison by intraventricular lidocaine
or CDM ............................ 32
12 Duration of suppression of amygdala EEC activity and
antagonism of this raphe-mediated EEC suppression by
lidocaine and CDM ....................... 32
vi
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13 Amygdala EEC spikes and blood pressure oscillation recorded
during overt CDM seizures 33
14 Antagonism by diazepam of the EEC and blood pressure effects
of intraventricular lidocaine and CDM 33
15 Respiratory and cardiovascular parameters of a urethane-
anesthetized rat infused with a lethal iv dose of lidocaine . . 34
16 Same as Fig. 15 except drug infused was chlordimeform 35
17 Chemical structure of chlordimeform and related local
anesthetics 38
18 Effect of chlordimeform, N-demethylchlordimeform, procaine,
holocaine, and lidocaine on the action potential in frog
sciatic nerve 40
19 Effect of chlordimeform at pHs 6.0 and 7.0 in the sheathed
and at pH 7.0 in the desheathed nerve on action potential
amplitude at 2 min intervals following drug application .... 41
20 Dose response effect of PCA, CDM, and quipazine in eliciting
serotonergic syndrome and additional symptoms 50
21 Effect of cinanserin pretreatment on the serotonergic syndrome
and additional symptoms elicited by PCA, CDM, and quipazine . . 51
22 CDM-induced depression of the electrically stimulated twitch
height in guinea pig ileum preparation 59
23 Effects of naloxone, tolazoline and phentolamine on twitch
depression by 3 x 10 M CDM. Effects of PGE. on twitch
depression induced by 10~ M indomethacin, CDM, or
lidocaine 60
24 Effects of drug free wash on twitch depression induced by
3 x 10"b M indomethacin or by 10 M CDM 60
25 Antagonism of yeast-induced fever in rats by acetylsalicylic
acid, indomethacin, and two formamidine pesticides,
chlordimeform and amitraz 61
26 Antagonism of the carrageenin-induced edema of rat paw by
the formamidines amitraz and chlordimeform 63
27 Metabolic map showing fate of chlordimeform in mammals 69
28 Rates of hydrolysis of chlordimeform and its N-demethylated
metabolites at pH 7.4 and 37° 77
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TABLES
Number
Page
1 Acute Toxicity and MAO Inhibition In Vitro by
Chlordimeform and its Major Metabolites 9
2 Reversibility of Inhibition of Mitochondrial MAO
From Mouse Brain by Chlordimeform and its Metabolites
on Washing 11
111
3 Recovery of Injected [ C] Tryptamine After Pretreatment
of Mice With MAO Inhibitors 15
4 The Effect of Time After Dosing on the Ability of
Chlordimeform to Increase the Recovery of Injected
[ C] tryptamine From Mouse Tissues 16
5 Time Course of the Primary and Secondary Blood Pressure
and Cardiac Contractility Responses in Chlordimeform-
Treated Dogs 22
6 Effects of Anticonvulsants on CDM Convulsions and
Lethality in Mice 42
7 Effect of Other Pharmacological Agents on CDM Convulsions
and Lethality in Mice 44
8 Toxicity in Mice of CDM Combined with Agents Possessing
Local Anesthetic Activity 45
9 Effect of Cinanserin on Serotonergic Syndrome Produced
by PCA, CDM and Quipazine 52
10 Effect of Cinaserin on Additional Symptoms Produced by
PCA, CDM and Quipazine 53
11 Effect of 5-HT on Serotonergic Syndrome and on Additional
Behaviors in the Rat 54
12 Inhibition of PGE Synthesis by Bovine Seminal Vesicle
Microsomes With Two Formamidines and Two Common
Non-steroidal Anti-Inflammatory Agents 62
13 Behavioral and Pharmacological Profile of CDM in the Rat . . 65
viii
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14 The Effect of Several Pretreatments Affecting Microsotnal
Oxidations on the Toxicity of Formamidines to Mice 76
15 Rate Constants and Half Lives for the Hydrolysis of
Chlordimeform and its N-demethylated Metabolites 78
16 Relative Activities of Mouse Tissue Microsomes and Other
Cellular Subfractions in Degrading Chlordimeform 79
14
17 Metabolites Formed in Reaction of C-Chlordimeform With
Mouse Liver Microsomes 80
18 ^n Vitro Metabolism of Chlordimeform and its N-demethylated
Metabolites by Mouse Liver Microsomes 81
19 Effect of Several Pretreatments Affecting Microsomal
Oxidations on the In Vitro Metabolism of Chlordimeform
by Mouse Liver Microsomes 82
20 Total Radioactivity in Tissues After an Oral Dose
of Chlordimeform 83
21 Effect of Several Pretreatments Affecting Microsomal
Oxidations on the ^n Vivo Metabolism and Distribution
of Chlordimeform in Mice 85
22 Effect of Several Pretreatments Affecting Microsomal
Oxidations in the Mouse on the III Vivo Metabolism
of Chlordimeform 86
23 Comparison of the Effect of Pretreatments on the Toxicity
of CDM to Mice and on the Levels of Selected Formamidines
in their Tissue 87
ix
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LIST OF ABBREVIATIONS
ABBREVIATIONS
ACh
CAA
CDM
CDU
CNS
CT
DCDM
DCDU
DDCDM
DDCDU
DFP
EGG
EDTA
EEC
G.C.
GSH
5-HT
ia
ip
iv
ivc
LSD
MAO
MFO
NADPH
NE
NFA
NFT
PCA
PG
PGE
PGE
1
sc
SD
SE(M)
TCA
TLC
acetylcholine
5-chloroanthranilic acid
chlordimeform
1 , 1-dimethyl-3-C*-chloro--o-tolyl)urea
central nervous system
4-chloro-o-toluidine
N-demethyl chlordimeform
1 -methyl-3- ( U-chloro-o-tolyl) urea
N^.II-didemethyl chlordimeform
M-chloro-o-tolylurea
diisopropylphosphorofluoridate
electrocardiograph
ethylenediamine tetr acetate
electroencephalograph
gas chromatography
glutathione
5-hydroxytryptamine (serotonin)
intraarterially
intraperitoneally
intravenously
intraventricularly
D-lysergic acid diethylamide
monoamine oxidase
mixed function oxygenase
reduced nicotinamide-adenine
dinucleotide phosphate
norepinephrine (noradrenaline)
N-formyl-5-chloroanthranilic acid
N-formyl-4-chloro-o-toluidine
parachloroamphetamine
prostaglandin
prostaglandin E
prostaglandin
subcutaneously
standard deviation
standard error (of mean)
trichloroacetic acid
thin-layer chromatography
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ACKNOWLEDGMENTS
The collaboration, advice, and technical assistance of the following
people is gratefully recognized: Dr. D. E. Blake, Dr. C. Chinn, Mr. M. P.
Holsapple, Mr. M. T. Lowy, Ms. V. Noland and Dr. W. R. Pfister and Mr. J.
Rolley of the Department of Pharmacology and Toxicology, Purdue University;
Dr. D. E. Nichols of the Department of Medicinal Chemistry, Purdue
University; and Mrs. K. Dersch, Dr. G. Ghali, Mr. J. Leister, Dr. A. E.
Lund and Dr. D. L. Shankland of the Department of Entomology, Purdue
University.
The following material is reprinted with the permission of the
copyright holders, to whom we express our appreciation. Some text
material, Figs. 1 through 4 and Tables 1 through 4 in Section 1 are
reproduced from Chemico-Biological Interactions, vol. 24, pages 35-49,
copyright 1979 by Elsevier/North-Holland Scientific Publishers Ltd.,
Shannon, Ireland. Some text material, Figs. 5 through 9 and Table 5 in
Section 2 are reproduced from Toxicology and Applied Pharmacology, vol. 44,
pages 357-365, copyright 1978 by Academic Press, Inc., New York. Some text
material and Figs. 10 through 14 in Section 3 are reproduced from Neuro-
pharmacology, vol. 16, pages 867-871, copyright 1977 by Pergamon Press,
Inc., New York. Some text material, Figs. 17 through 19 and Tables 6
through 8 in Section 4 are reproduced from Pesticide Biochemistry and
Physiology, vol. 9, pages 148-156, copyright 1978 by Academic Press, Inc»,
New York. Some text material, Figs. 20 and 21, and Tables 9 through 11 in
Section 5 are reproduced from Communications in Psychopharmacology, vol. 2,
pages 287-295, copyright 1978 by Pergamon Press, Inc., New York. Some text
material, Fig. 25 and Table 12 in Section 6 are reproduced from Life
Sciences, vol. 23, pages 2509-2515, copyright 1978 by Pergamon Press, Inc.,
New York. Some text material, and Figs. 22 through 24 in Section 6 are
reproduced from Prostaglandins and Medicine, vol. 2, pages 215-222,
copyright 1979 by Churchill Livingstone, Edinburgh, Scotland.
xi
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GENERAL INTRODUCTION
The formamidines are a relatively new group of insecticide-
acaricides which are particularly useful for the control of Lepidoptera,
Hemiptera, phytophagous mites, and cattle ticks (Hollingworth, 1976). In
the U.S. two compounds are of primary interest, chlordimeform (I;
£«_(4_chloro-£-tolyl)-N^N-dimethylformamidine)) and amitraz (II;
l,5-di-(2,4-dimethylphenyl)-3-methyl-1,3,5-triazapenta-1,4-diene).
N=C-N<
CH
Chlordimeform
(Fondol : Nor-Am/Schering)
(Galecron : Ciba-Geigy)
Amitraz
(BTS-27419: Boots)
(U-36059-.Upjohn)
During this grant period both compounds were reported to be
carcinogenic in mice at high doses (Johnson, 1977). This is probably due
to the metabolic release of carcinogenic anilines rather than because of
any innate carcinogenicity of the parent formamidines (Ghali, 1980).
Registration for amitraz has thus been limited to control of psylla on
pears (Johnson, 1979), although it is used widely outside the U.S., e.g.
for cattle tick control. By voluntary agreement, chlordimeform (CDM) is
confined to use for control of cotton insects, in which situation it
remains an important insecticide. It too has significant uses outside
the U.S. A number of other compounds related to these formamidines are
currently undergoing field testing.
The formamidines are structurally unlike other pesticides and their
mechanism(s) of toxic action in vertebrates and invertebrates appear to
be novel (Matsumura and Beeman, 1976; Lund et al., 1979b). At the start
of this project it was known only that the symptoms of poisoning by
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lethal doses of CDM in rodents were excitatory and included
hyperexcitation, tremors, and convulsions preceding death (Beeman and
Matsumura, 1973). The symptoms were described as sympathomimetic in
nature (Aziz and Knowles, 1973). No cholinomimetic responses have been
reported. The most likely site of action of formamidines was thought to
be monoamine oxidase (MAO). Despite evidence that MAO was inhibited in
v-'tro (Aziz and Knowles, 1973; Beeman and Matsumura, 1973), we felt that
tne rapid, excitatory poisoning symptoms were not congruent with the MAO
theory, nor were the symptoms predominantly sympathomimetic, though this
component was present. In particular it seemed important to determine
the degree, duration, and reversibility of MAO inhibition in vivo. Not
only is this crucial in order to interpret the role of MAO in poisoning,
but also to evaluate the likelihood of dangerous interactions with
dietary or medicinal amines (Sjoqvist, 1965) or of cumulative inhibition
of MAO on repeated exposure to formamidines.
A number of other biochemical actions of formamidines have been
described, such as mitochondrial uncoupling (Abo-Khatwa and Hollingworth,
1973) and inhibition of RNA synthesis (Murakami and Fukami, 1974), but
these are not plausible as the cause of the symptoms observed (Lund jit
al.. 1979b). At the start of the project the actual cause of death from
formamidines was unknown although Beeman and Matsumura (1974) noted
briefly that CDM caused a depression of blood pressure in rabbits.
Before and during the work presented here a number of publications
appeared describing the metabolism and fate of CDM in mammals (see
Section 7 for references). A major metabolic route for CDM in all
species studied is N-demethylation to yield N-demethyl chlordimeform
(DCDM) (Knowles and Sen Gupta, 1970; Sen Gupta and Knowles, 1970;
Morikawa j5t £l., 1975). Subsequently a second N-demethylation to give
N^N-didemethyl chlordimeform (DDCDM) was reported (Benezet and Knowles,
1976b). Our preliminary results indicated that DCDM was more toxic than
CDM to mice (Hollingworth, 1976) and more rapid in its actions. Also,
piperonyl butoxide, an insecticide synergist which inhibits many
j^-demethylations, strongly antagonized some of the toxic effects of CDM,
but not of DCDM, in cattle ticks, suggesting that conversion of CDM to
DCDM is crucial for such toxicity (Knowles and Roulston, 1972). If this
were true in mammals also, there are obvious implications regarding the
appropriate compounds to use in mode of action studies, and for
unexpected toxic interactions arising after exposure to mixed function
oxidase (MFO) inducers or inhibitors.
The goals of this project were therefore:
(a). To define the symptoms and mechanism(s) of toxicity of CDM in
mammals.
(b). To specifically examine the role of MAO in these processes and
to evaluate the possibility of interactions or cumulative
effects based on MAO inhibition.
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(c). To develop information leading to potential means of therapy
for formamidine overexposure.
(d). To study other pharmacological and behavioral actions of
formamidines which might have toxicological implications,
indicate potential sites of action, or have utility as an
index of exposure to these pesticides.
(e). To investigate the metabolism of CDM and the accumulation of
CDM and its metabolites jin vitro and ^.n vivo. Specific
objectives in this respect were to determine whether
N-demethylations are important activation reactions for
formamidines, and to assess the possibility of significant
interactions with MFO inducers and inhibitors.
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CONCLUSIONS
1. Chlordimeform is a moderately potent, reversible inhibitor of
monoamine oxidase. Inhibition of monoamine oxidase in vivo is ephemeral
and never reaches high levels even at near lethal doses. Several other
lines of evidence also indicate that monoamine oxidase inhibition is not
a major mechanism underlying acute toxicity, and the possibility of
dangerous interactions resulting from concurrent exposure to f ormamidines
and natural or medicinal biogenic amines is slight. No evidence of such
interactions was seen in cardiovascular studies performed with dogs.
Inhibition of monoamine oxidase is unlikely to be cumulative with
continued daily exposure to formamidines because of their rapid
metabolism and the reversibility of inhibition.
2. Animals poisoned with chlordimeform suffer concurrent cardiovascular
collapse and respiratory arrest. When given intravenously, chlordimeform
has a biphasic action on blood pressure i.e. an intial depression
followed (in survivors) by an elevation. Both actions are shared with
lidocaine and are attributable to a local anesthetic-like effect of
chlordimeform. The depressor effect results primarily from vasodilation
and reduced cardiac contractility while the pressor response results
centrally from enhanced sympathetic outflow. Respiratory arrest is a
secondary effect arising centrally, probably through depression of the
respiratory center following convulsions.
3. The effects of chlordimeform and lidocaine are also similar when
administered intraventricularly in rats and when applied to the isolated
frog sciatic nerve. Intraventricular chlordimeform induces EEC seizure
discharges in the amygdala and blocks amygdalar inhibition by the raphe.
Evidence for actions of formamidines on the nervous system other than as
local anesthetics exists e.g. biogenic amine accumulation through partial
inhibition of monoamine oxidase, a direct excitatory effect on some types
of axons, and a partial -adrenergic agonist effect of l£-demethylated
formamidines. However, on the basis of present evidence the local
anesthetic-like actions appear to explain well most of the symptoms and
signs observed, including the primary cardiovascular depression,
secondary increase in sympathetic outflow, and the convulsive and
respiratory effects of chlordimeform. Like other local anesthetics,
chlordimeform can induce "pharmacological kindling" i.e. precipitation of
convulsions and other behavioral effects after repeated subliminal doses.
4. Therapeutic approaches to local anesthetic overdose are limited.
Diazepam was clearly helpful in alleviating the central symptoms of
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chlordimeform poisoning in rats and dogs, but only at low doses of
diazepara. A combination of therapeutic steps is likely to be necessary
and probably should include:
(a). Artificial resuscitation to combat respiratory depression.
(b). Low doses of diazepam.
(c). Vasoconstrictors and cardiac stimulants (e.g. epinephrine and
calcium gluconate) for the cardiovascular depression.
5. In developing the "behavioral profile" for chlordimeform to serve as
an aid in detecting and diagnosing overexposure, it was found to induce
responses typical of the so-called "serotonergic behavioral syndrome".
Most of these and the other behavioral effects of chlordimeform are not
induced by known local anesthetics. However, further study casts doubt
on the existence of this 'serotonergic syndrome1 as presently defined and
on any general role for serotonin in the behavioral effects of
chlordimeform. The origins of these behavioral responses remains unclear
in most cases although some possibilities other than local anesthetic
action or presented in Conclusion 3.
6. Because of their ability to inhibit prostaglandin synthesis, the
formamidines were found to have antipyretic, analgetic, and
anti-inflammatory effects which are comparable to those of aspirin.
However, they lack the potent gastric ulcerogenicity typical of
aspirin-like agents.
7. The two successive metabolic N-demethylations of chlordimeform yield
compounds of increased acute toxicity. These N-demethylation products
are produced rapidly by mouse liver microsomes ^n vitro and iin vivo.
Metabolic reactions which result in the alteration of the formamidine
nucleus are detoxications, and, in part, appear to be catalyzed by
cytochrome P448 (3-methylcholanthrene inducible). Studies with several
mixed function oxidase inhibitors and inducers indicate that
N-demethylation in vivo tends to be a toxicologically 'neutral1 process.
Although the N-demethylation products are more toxic, they are also less
stable. After dosing mice with chlordimeform, the best correlation of
toxicity is found with the total level of all formamidines in the brain
rather than with the level of any individual metabolite.
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SECTION ONE
EVALUATION OF MONOAMINE OXIDASE
AS A TARGET FOR FORMAMIDINE PESTICIDES
INTRODUCTION
In considering the possible biochemical site of action of the
formamidines, much attention has been directed to the actions of
formamidines on monoamine-mediated neurotransmission in both mammals and
arthropods (Matsumura and Beeman, 1976; Knowles and Aziz, 1974). In
particular it has been reported that these compounds inhibit monoamine
oxidase (MAO) activity in rat liver (Beeman and Matsumura, 1973; Aziz
and Knowles, 1973) and in rat brain (Benezet .and Knowles, 1976a). The
symptoms of acute poisoning in the rat have been described as 'similar
to those elicited by sympathomimetic agents including known MAO
inhibitors' (Aziz and Knowles, 1973) and as sympathomimetic in nature
and lacking normal cholinomimetic features (Beeman and Matsumura, 1973).
Beeman and Matsumura (1973) also reported that treatment of rats with
chlordimeform (CDM) caused an elevation in the serotonin and
norepinephrine levels of the brain and also antagonized certain symptoms
of reserpine intoxication which are generally believed to arise by
release of biogenic amines from their presynaptic stores. These
observations are stimulating since no other pesticides have been shown
to exert their primary effect through aminergic mechanisms. Neumann and
Voss (1977), recently have cast doubt on the role of MAO inhibition in
the acute toxicity of CDM and some related toluidine derivatives on the
basis of a lack of correlation of anti-MAO potency and lethality to rats
among these compounds. Additionally, Robinson and Smith (1977),
selectively depleted rat brain serotonin (5-HT) and norepinephrine (NE),
or treated rats with the a-adrenergic agonist, phenylephrine. None of
these pretreatments altered the acute toxicity of CDM, and the authors
therefore concluded that MAO was probably not involved in its acute
toxicity. Previously they had shown that pretreatments with reserpine,
to deplete monoamines, or with a-adrenergic and serotonergic blockers
also did not alter the LD^_ of CDM (Robinson ^t _al., 1975). Meanwhile
the view that MAO inhibition in particular, and interference with
aminergic transmission in general, play a central role in generating the
poisoning syndrome has been reiterated (Matsumura and Beeman, 1976;
Knowles and Aziz, 1974; Knowles, 1976). Until now there has been no
consideration of the potency of CDM and its metabolites as MAO
inhibitors in other vertebrates than the rat, and no reports of their
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effectiveness as MAO inhibitors in vivo. In the investigation presented
here such studies were made and the results related to the hypothesis
that MAO inhibition is an important feature of the toxicology of the
formamidines in vertebrates.
MATERIALS AND METHODS
Chemicals
The radiolabelled MAO substrates were tryptamine bisuccinate (48.5
and 53 Ci/mol), 5-hydroxytryptamine (48.5 and 51 Ci/mol), and
3-phenylethylamine (9.9 Ci/mol) from New England Nuclear, and tyramine
hydrochloride (55 Ci/mol) from Amersham/Searle. All were labelled with
C in the side chain. MAO inhibitors, tranylcypromine hydrochloride
and harmaline hydrochloride, were obtained from Sigma Chemical Co.
Pheniprazine hydrochloride was the kind gift of Dr. C. Chinn. The
formamidines JN'-(4-chloro-£-tolyl)-N^N-dimethylformamidine (CDM),
N'-(4-chloro-o-tolyl)-Nrmethylformamidine (DCDM), and
IJ-(4-chloro-o-tolyl)fonnamidine (DDCDM), and their metabolites
N-formyl-4-chloro-o-toluidine (NFT) and 4-chloro-o-toluidine (CT) were
synthesized and purified as described in Section 7. The identities and
purity of these synthesized products were assessed by IR and NMR
spectroscopy and by TLC on silica gel developed in benzene/diethylamine
(19:1). Small amounts of NFT were sometimes present in the parent
formamidines, particularly after storage. The amount of NFT did not
exceed 2-3% and, when present, did not significantly affect the results
obtained. Particular care was taken before the MAO assays to reduce the
levels of NFT present in the formamidines.
Acute lethality
Male Swiss white mice (20-25 g) were obtained from Bellaire Acres,
Danville, IN. The mice were starved for 4 hr before administration of
the toxicants by gavage in a corn oil vehicle at 0.01 ml/g body wt. At
least 5 doses with at least 10 mice/dose were utilized and the tests
were replicated for each compound. Mortality was determined after 48 hr
and the LD was determined by the method of Litchfield and Wilcoxon
(1949). 5
Monoamine oxidase inhibition in vitro
MAO activity was assayed by the general radiometric method of
Wurtman and Axelrod (1963). Mouse tissues were dissected, rinsed clean
in saline, weighed after drying, and homogenized at 20 mg tissue/ml in
0.25 M sucrose containing 1 mM EDTA in a teflon-glass system. A
mitochondrial fraction was isolated by differential centrifugation,
initially at 600 g for 15 rain and with further centrifugation of the
supernatant at 12,000 g for 15 min. The resulting pellet was washed by
resuspension in the same solution and recentrifugation. The washed
mitochondria were then resuspended in the original volume of Tris buffer
(50 mM, pH 7.5) containing 1 mM EDTA.
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For the MAO assay, mitochondria (0.3 ml) were incubated with
inhibitor (^Q 1 in ethanol) for 30 min at 25° in a stoppered centrifuge
tube. The C-labelled amine substrate (tryptamine, tyramine,
e-phenylethylamine, or 5-HT at a final concentration of 10-30 PM) was
added in 10-20 yi water and the MAO reaction allowed to proceed for
exactly 2 min before addition of 0.2 ml 2N HC1. Toluene (5 ml) was
added, the tube shaken, and 3 ml of the toluene layer was taken for
scintillation counting of the amount of deaminated product present.
Controls were run in which only 10 Ul ethanol was added to the enzyme.
Blank values obtained with 0.3 ml boiled enzyme were used to correct
these data. In every MAO assay enzymatic rates were determined in
triplicate and averaged. Under these conditions the assay was linear
over several min with tissue concentrations up to 50 mg/ml. I5Q values
were calculated graphically from plots of percent inhibition or MAO
against log of inhibitor concentration. The I _ values reported are the
means of at least three such determinations.
The reversibility of the inhibition of MAO was examined by repeated
washing of the mitochondria to remove the inhibitor. Mitochondria from
mouse brain were incubated with 5 X 10 M COM.for 30 min. One portion
was removed to assay for MAO activity using [ C] tyramine, while the
rest was centrifuged, and the mitochondrial pellet resuspended to its
original volume in inhibitor-free buffer. After removing a further
portion for enzyme assay the centrifugation and resuspension in fresh
buffer was repeated several times more with samples removed for assay
each time. An inhibitor-free MAO sample was treated similarly as a
control. Protein was determined in each sample by a modified Lowry
method (Schacterle and Pollack, 1973) and the activities of MAO were
compared in the inhibited and control samples after correction for any
loss of protein during the washing procedures.
In order to study the relation of MAO inhibition to time of
exposure to the inhibitor, intestinal_or brain mitochondria were
incubated with CDM or DCDM at 5 X 10"5M, or NFT at 5 X 10 M at 25 .
Portions were removed at intervals from 20 sec-3 hr after addition of
the inhibitor and immediately assayed for MAO using tryptamine.
Activity was compared to a control sample incubated without inhibitor.
MAO inhibition in vivo
The effect of inhibitors on the activity of MAO in mouse liver and
small intestine in vivo was assayed indirectly from -the recovery of
injected C C]-tryptamine. The method was based on that of Wang Lu and
Domino (1976) for J<,N-dimethyltryptamine. Potential MAO inhibitors were
given orally as their hydrochlorides in water at sublethal doses except
for NFT which was administered in corn oil. Two hr later, labelled
tryptamine at 0.05 uCi/g body wt. was injected intraperitoneally. After
a further 5 min the mice were killed and the liver and intestine were
rapidly removed, rinsed with saline, and homogenized in 4 ml cold IN
HC1. The homogenate was centrifuged at 9000 g for 15 min, and the
pellet was washed twice by resuspension in 1.5 ml portions of 0.1N HC1
and centrifugation. The supernatants were combined and extracted with
8
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10 ml ethyl acetate/toluene (1/1) and the aqueous phase was made
alkaline by mixing with 2 ml 10 N NaOH. The residual tryptamine was
then extracted with three 10 ml portions of the ethyl acetate/toluene.
The organic phase was pooled and 5 ml samples taken for scintillation
counting. Chromatography of such extracts from liver on silica gel
plates developed with n-butanol/acetic acid/water (25/4/10) showed that
the major peak of radioactivity cochromatographed with authentic
tryptamine. When labelled tryptamine was added to the tissue during the
initial homogenization, recovery of the added tryptamine ranged from
86-91% with a mean of 89%. The results were not corrected for recovery
losses. Preliminary experiments showed that the tissue levels of
tryptamine peaked at about 5 min after injection in untreated mice.
Further initial experiments were conducted as described above but with
various intervals between the administration of CDM.HC1 and tryptamine
from 15 min-24 hr to follow the change in MAO activity with time after
exposure to the inhibitor.
RESULTS
Acute lethality of COM and its metabolites to mice
The LD values for CDM, its two N-demethylation products (DCDM and
DDCDM), and two hydrolysis products (NFT and CT) are presented in Table
1. It is notable that although CDM itself is only of medium potency
(LD,_0 = 267 rag/kg) successive N-demethylations yielded increasingly more
toxic metabolites with an LD Q for DDCDM of only 78 mg/kg. With CDM,
TABLE 1: ACUTE TOXICITY AND MAO INHIBITION IN VITRO BY CHLORDIMEFORM AND
ITS MAJOR METABOLITES
MAOa,I5()(yM)
LD__,mg/kg Liver Intestine Brain
(mouse,oral)
Chlordimeform (CDM) 267
N-Demethyl chlordimeform (DCDM) 163
N.N-Didemethyl chlordimeform (DDCDM 78.
N-Formyl-4-chloro-o-toluidine (NFT) 750
4-Chloro-o-toluidine (CT) >1000
47
21
133
4.5
88
121
61
73
2.8
185
122
69
104
3.2
253
substrate:tryptamine bisuccinate.
solvent: propylene glycol.
DCDM, and DDCDM, all of which contain the formamidine nucleus, symptoms
developed in less than 1 hr after dosage and were excitatory in nature.
-------
Hyperexcitability, marked tremors in the head and limbs, gasping, and
rapid death following one or more convulsive episodes were typical of
all three compounds. The other two metabolites (NFT and CT) were less
acutely toxic, slower-acting, and depressant in their effect. A gradual
loss of responsiveness to external stimuli and hypothermia were noted in
each case.
Innibition of MAO in vitro
The anti-MAO potency of COM and these metabolites was compared in
mitochondria from three tissues, the liver, brain, and small intestine. The
I values are presented in Table 1. Although the tissues differed somewhat
in their relative sensitivities to these agents, the differences were not
extreme and no regular pattern of differential sensitivity was apparent. Most
I50 values were in the range of 20-200 yM. Successive N-demethylations in
tnis case did not greatly increase biological potency since although DCDM was
about twice as effective as a MAO inhibitor compared to CDM, DDCDM was similar
to CDM in its overall effectiveness. However, one metabolite, NFT, was
notably better than any other compound as a MAO inhibitor with all three
tissues, having I,-0 values in the 3-5 UM range. In contrast, the parent
toluidine, CT, was somewhat less effective than CDM.
Reversibility of MAO inhibition
The recovery of mouse brain MAO from inhibition by CDM on repeated
washing is shown in Fig. 1. The initial level of inhibition was 72%, this was
reduced to 28% after one wash, 1% after the second, and activity returned to
the control level with subsequent washes. This behavior was repeated with
DCDM and the more potent inhibitor, NFT, as shown in Table 2. With these
compounds the mitochondria were washed twice after exposure to the inhibitor
for 30 min at 25°. In each case inhibition was reversed essentially to
control levels. The high variability in the data for the washed mitochondria
arises because of the substantial increase in MAO activity which occurred on
washing the mitochondria in both the treatments and controls.
The time course of inhibition of MAO
When incubated with MAO from brain or intestine for periods from
20 sec-3 hr, all three compounds tested (CDM, DCDM, and NFT) gave essentially
instantaneous inhibition. With NFT, inhibition after 20 sec was the same as
after 3 hr and at all intermediate times. However, the two formamidines
showed a more complex behavior. A large part of the final level of inhibition
achieved at 3 hr was obtained 'instantaneously1, but in each case a slow
progressive component also was observed so that the degree of inhibition rose
steadily about 7%/hr over the 3 hr observation period. This behavior is
illustrated for the intestinal mitochondria in Fig. 2.
10
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1OO
NO. WASHES
Figure 1. Recovery of mouse brain mitochondria! MAO from inhibition by CDM
(50 UM) after successive washes to remove the inhibitor.
TABLE 2. REVERSIBILITY OF INHIBITION OF MITOCHONDRIAL MAO FROM
MOUSE BRAIN BY CHLORDIMEFORM AND ITS METABOLITES ON WASHING
Mean % Inhibition (+SD)'
Unwashed
Washed
Chlordimeform (50
]}-Demethyl chlordimeform (50yM)
N-Formyl-4-chloro-o-toluidine (5yM)
68.5+4.9
86.1+0.7
84.1+2.1
2.4+ 2.7
8.9+14.2
-1.4+ 5.6
Mean of three replicates. Tyramine hydrochloride as substrate.
11
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Classification of CDM and its metabolites as MAO inhibitors
In order to determine whether these compounds are Type A or Type B
inhibitors, the inhibition of MAO from mouse intestine was assayed over a wide
1OO '
80
I
a 60
40
20
__— DCDM
— *
>„---*
CDM
3O
60
90
120
150
180
MINUTES
Figure 2. Time course of inhibition of MAO from mouse intestine by
chlordimeform (CDM, 50yM), N-demethyl chlordimeform (DCDM, 50yM), and
N-formyl-4-chloro-o-toluidine (NFT, 5yM).
range of inhibitor concentrations with three substrates, 5-HT (Type A),
tryptamine (Mixed A and B), and g-phenylethylamine (Type B). The results are
shown in Figs. 3 and 4, for CDM and NFT respectively, derived from three
independent experiments in each case. DCDM behaved similarly to CDM. For each
of the inhibitors, the MAO reaction with 5-HT is most sensitive to inhibition
and 3-pnenylethylamine is the substrate least effective for inhibition, with
tryptamine intermediate. The distinction between these three substances is
most evident with NFT. The I-Q values obtained for the three substrates,
5-HT, tryptamine, and 3-phenyIethylamine were 34,100, and 140 M respectively
for CDM, 12, 33, and 39 yM for DCDM, and 1.2, 3.0, and 12 yM for NFT. In
limited studies with tyramine, another mixed A and B type substrate, the
results were very similar to those obtained with tryptamine.
Inhibition of MAO in vivo
The assumption that the inhibition of MAO would allow more injected
tryptamine to survive intact in the tissues is confirmed by the results in
Table 3. Although this method proved effective for assessing MAO activity in
vivo in liver and small intestine, the amount of labelled tryptamine present
12
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in the brain was too small to allow accurate determinations for this tissue.
Tranylcypromine and pheniprazine are well-established and potent irreversible
MAO inhibitors, and with these compounds 4-6 times as much tryptamine was
recovered in the liver and intestine as in the untreated controls. Since
tranylcypromine is an irreversible inhibitor, MAO inhibition was also assayed
by homogenization of tissues from treated mice (15 mg^kg) followed by our
normal MAO assays with isolated mitochondria using [ C] tryptamine as
substrate. Under these conditions tranylcypromine caused 80% inhibition of
liver MAO and 92% inhibition of intestinal MAO in vivo 2 hr after dosage.
1OO
80
60
4O
20
1O
-6
1O'
10
.-4
1O"
INHIBITOR CONC. (M)
Figure 3. Inhibition of MAO from mouse intestine by COM assessed with three
MAO substrates: 5-Hydroxytryptamine, 0 • ; Tryptamine, X x;
3-phenylethylamine, O O •
Harmaline, a reasonably potent reversible inhibitor of MAO, did not
protect tryptamine in the liver but did increase the amount of tryptamine
surviving in the intestine. All of the standard MAO inhibitors were given at
doses which caused no mortality and little if any external sign of toxicity.
CDM at 100 mg/kg also caused no strong poisoning symptoms and gave no
significant increase in tryptamine recoveries. At 200 mg/kg poisoning symptoms
were clear and an occasional death occurred. In this case a significant
increase in tryptamine levels were seen, but this was much less than the
increase caused by tranylcypromine and pheniprazine at their symptomless
13
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100
INHIBITOR CONG. (M)
Figure 4. Inhibition of MAO from mouse intestine by NFT assessed with three
MAO substrates: 5-Hydroxytryptamine, • *; Tryptamine.B •;
3-Phenylethylamine x— -/.•
doses. NFT as a more potent MAO inhibitor than CDM caused a greater
accumulation of tryptamine at 100 mg/kg than did CDM despite difficulties with
getting the dose of NFT into solution in corn oil at this concentration.
The justification for the choice of 2 hr as the time delay after CDM
dosage before administration of the tryptamine is illustrated by the data in
Table 4. In this case varying times were left between the doses of
chlordimeform (150 mg/kg) and tryptamine in order to evaluate the dynamics of
MAO inhibition in vivo. The increase in tryptamine levels was not great at
this dose but appeared to peak around the 2-4 hr period after dosage and to be
declining by 6-12 hr with a return to normal levels within 24 hr.
14
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1U
TABLE 3. RECOVERY OF INJECTED [I4C] TRYPTAMINE AFTER PRETREATMENT OF MICE
WITH MAO INHIBITORS
(pmol tryptamine recovered/g tissue) —SD (n)
a
Dose Liver Intestine
(mg/kg)
Tranylcypromine
Pheniprazine
Chlordimeform
Chlordimeform
N-Formyl-4-chloro-c>-tol uid ine
Harmaline
Control
15
20
200
100
100
50
562 + 183(8)°
536 + 90(8)
237 + 59(10)
118 +37(7) „
198 + 36(9)
83 + 24(9)
86 + 27(10)
632 + 126(8)C
482 + 84(8) h
233 + 47(10)°
152 + 50(7)
218 + 84(9)
359 + 155(9)
131 + 39(10)
S.D.(n) = standard deviation (number of observations).
Significantly different from control at P = 0.05.
Significantly different from control at P = 0.01.
DISCUSSION
The data presented in the initial section dealing with MAO inhibition j.n
vitro establishes that with mitochondrial MAO from several mouse tissues, CDM
is a readily reversible inhibitor of medium potency, ^n vivo it is rapidly
metabolized by successive N-demethylations to yield DCDM and then DDCDM.
These formamidines are susceptible to hydrolysis to NFT and then CT (see
Section 7). These metabolites are in general rather similar to CDM in potency
against MAO and, in the case of DCDM and NFT at least, are also reversible
inhibitors. The only exception to these generalizations was the 10 to 40-fold
greater potency of NFT compared to CDM as a MAO inhibitor jln vitro. Thus the
metabolism of CDM to NFT is potentially an activation reaction as far as MAO
inhibition is concerned. The I^Q values here are generally similar to those
obtained by other investigators working with MAO from rat liver (Beeman and
Matsumura, 1973; Aziz and Knowles, 1973) and rat brain (Benezet and Knowles,
1976). Benezet and Knowles (1976) in their more detailed study of the
mechanism of inhibition of MAO by formamidines also noted the greater potency
of NFT against rat brain, although in their earlier study with rat liver MAO,
NFT was not found to be more effective than CDM. In our work with the mouse,
NFT was clearly more potent than CDM against MAO from all tissues examined
including liver. Otherwise MAO from the rat and the mouse responds to CDM and
its metabolites similarly.
15
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From the studies reported here it is evident that NFT inhibits MAO with
no progressive component when enzyme and inhibitor are preincubated in the
absence of substrate. However, superimposed on the 'instantaneous phase' with
CDM and DCDM was a slow progressive increase in the degree of inhibition
observed. This effect was also noted by Benezet and Knowles (1976a) for CDM
and DCDM with their rat brain MAO. They suggested that this might be due to
tho slow generation of NFT by hydrolysis of the formamidines. Since NFT is a
more potent inhibitor, the level of inhibition would thus slowly rise. This
is a plausible explanation and in accord with the observed rates of hydrolysis
of these formamidines (Section 7). Our observation that NFT itself lacks a
progressive component of inhibition is compatible with this explanation.
TABLE 4. THE EFFECT OF TIME AFTER DOSING ON THE ABILITY OF CHLORDIMEFORM TO
INCREASE THE RECOVERY OF INJECTED [C]TRYPTAMINE FROM MOUSE TISSUES
Tryptamine recovered
(pmol/g tissue) + SD (n)
Hours after
CDM dose3
0
0.25
0.50
1.0
2.0
4.0
6.0
12.0
24.0
Liver
65 + 6(8)
95 + 34(6)
81 + 24(6)
85 + 16(5)°
125 + 32(5)°
106 + 28(5)°,
104 + 28(41°
92 + 8(4)a
64 + 6(5)
Intestine
105 + 20(8)
121 + 42(5)
97 + 21(6)
139 + 57(6)
136 + 16(5)°
163 •»• 66(6)c
142 + 39(5)°
90 + 15(8)
108 + 11(5)
.Dose was 150 mg/kg, oral.
SD (n) = standard deviation (number of observations)
°Significantly different from control at P = 0.05.
Significantly different from control at P = 0.01.
It is known that mitochondrial MAO exists in several forms which may be
distinguished by their differential responses to inhibitors, thermal
inactivation, and differing substrate specificities (Squires, 1968 and 1972;
Neff and Yang, 1974). Particular attention has been focused on the division
of MAO enzymes into two classes, type A (with 5-HT and NE as preferred
substrates) and type B (with 3-phenylethylamine and benzylamine as preferred
substrates). Several substrates are oxidized with similar efficiency by both
types of MAO e.g. tryptamine, tyramine, dopamine, and kynuramine. Similarly
some inhibitors such as tranylcypromine and pheniprazine are not particularly
selective between the two forms of MAO while others are quite specific for one
16
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of the types. Thus harmaline and clorgyline are considerably more potent as
inhibitors of type A MAO while deprenyl is a relatively specific inhibitor of
the B type. Different tissues vary widely in their relative contents of types
A and B MAO and such variations are not constant among different species. The
data in Figs. 3 and 4 show clearly that CDM, DCDM, and particularly NFT are
more potent inhibitors when 5-HT is the substrate than when 3-phenylethylamine
is the substrate. Thus they appear to be selective inhibitors of type A MAO,
although the degree of selectivity is quite low. As would be expected with
tryptamine or tyramine as substrate, the potency is intermediate since these
compounds act as efficient substrates for both types of MAO. However, if the
situation were as simple as the assumptions above indicate, one would predict
that a plateau in the inhibition curve should be observed using tryptamine,
since the activity of the type A MAO against this substrate should be
inhibited before that of the type B also present. Such plateaus are commonly
seen with more highly selective inhibitors such as deprenyl, clorgyline, and
harmine (Squires, 1972; Neff and Yang, 1974; Yang and Neff, 1974). However
from these results it may be concluded that, like other selective inhibitors
of type A MAO such as clorgyline (Yang and Neff, 1974), CDM and its
metabolites jji vivo should cause the preferential accumulation of the
substrates for this form of MAO i.e. 5-HT, NE and dopamine if any high degree
of inhibition were induced. Beeman and Matsumura (1973)t report a 22%
increase in NE and a 70% increase in 5-HT in the brains of rats 1 hr after an
intraperitoneal dose of 200 mg/kg of CDM (LD Q). The results discussed above
were obtained with mouse intestinal mitochondria. Recently Neumann and Voss
(1977) obtained K. values for CDM against rat liver MAO using the same
substrates as in our study plus kynuramine. No trends in the K. values with
substrate were found which would allow classification on the type A/type B
system. However, using rat brain MAO, Benezet and Knowles (1976) obtained
results rather similar to ours with the same substrates.
The data presented in this section offer several lines of evidence
regarding the probable contribution of MAO inhibition to the acute lethality
of CDM. We believe that this evidence strongly disfavors the idea that MAO is
the primary target. Despite the fact that CDM and its metabolites show some
degree of potency as MAO inhibitors ^n vitro, there is no correlation between
the ability to inhibit MAO and either the lethality or symptoms of poisoning.
Sequential J4-demethylations decrease the LD_Q values in the formamidines (CDM,
DCDM, DDCDM), but there is no corresponding trend in the I values for MAO
from any of the tissues examined. On the other hand NFT is easily the best
MAO inhibitor, but is not particularly toxic. Even the poorly toxic
toluidine, CT, is an inhibitor with activity in about the same range as the
formamidines. This suggests that MAO inhibition is more dependent on the
presence of the substituted toluidine moiety than on the presence of an intact
formamidine nucleus. A similar conclusion was reached by Neumann and Voss
(1977) with a series of substituted chlorotoluidines including CDM and NFT.
Furthermore, although CT is comparable to the formamidines in its potency
against MAO, and NFT is more effective, their speed of action and symptoms are
completely different from those of the formamidines. Rapid mortality
accompanied by marked excitation was seen only for compounds with an intact
formamidine nucleus. Since MAO is not an enzyme which is immediately
essential to the integrity of nervous functions and for survival of the
17
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organism, as compared for example to acetylcholinesterase, the rapid onset of
symptoms and strong excitatory effects of CDM and the related formamidines is
not in keeping with MAO as the site of action. Many potent MAO inhibitors
such as tranylcypromine and the hydrazines exert their acute toxic actions
through mechanisms other than MAO inhibition (Pletscher et al., 1966) and CDM
probably resembles them in this respect.
The results obtained here with MAO inhibition in vivo are entirely
consistent with this conclusion. Since CDM is a readily reversible inhibitor
of MAO, estimation of the degree of inhibition _in vivo presents severe
problems because iin vitro assays of MAO which involve tissue homogenization
and dilution before assay will partially reverse the inhibition. Thus an
indirect but potentially more realistic method of assessing the status of MAO
JJ1 vivo was adopted which measured the ability of injected [ C]tryptamine to
survive in the tissues. Judging by the observed actions of the known MAO
inhibitors, tranylcypromine, pheniprazine, and harmaline, this method is an
appropriate one for assessing the status of MAO in. vivo* The first two
compounds are potent, irreversible MAO inhibitors which are not selective
between type A and type B MAO (Neff et^l., 1974). In both liver and
intestine they cause a substantial increase in the amount of tryptamine
recovered. Harmaline is a reversible inhibitor which is strongly selective
for type A MAO (Neff et_ al., 197M). It was found to preserve tryptamine in
the intestine but not in the liver. This is reasonable since while the MAO of
mouse intestine is about 70% of the A type, this enzyme class is virtually
absent in mouse liver (Squires, 1968 and 1972). Thus, in the liver the
tryptamine is destroyed by type B MAO which is insensitive to harmaline.
Assayed in this way, CDM causes only a moderate degree of MAO inhibition
in_ vivo even at near-lethal doses. This is a degree of inhibition which other
established MAO inhibitors, such as tranylcypromine and pheniprazine, easily
exceed without any apparent signs of poisoning. It seems quite unlikely that
the level of MAO inhibition after an oral dose of CDM as high as 200 mg/kg
approaches the 85% or more necessary to cause significantly damaging
accumulation of biogenic amines in the brain and other organs (Pletscher gt
al., 1966. Tranylcypromine at 15 mg/kg does cause this degree of inhibition,
but also leads to a far greater accumulation of tryptamine than does CDM at
200 mg/kg.
From the I_n data of Table 1, one would not expect CDM to be a more
effective inhibitor of brain MAO in vivo compared to liver or intestinal MAO.
However, this could not be assessed by the tryptamine recovery method and
further data are needed regarding the status of the MAO in the brain during
CDM poisoning.
The data of Table M reveal that the MAO inhibition which does result from
dosage with CDM is not long-lasting, being essentially reversed within 12-2*1
hr after exposure. This is entirely what would be expected from a rapidly
metabolized reversible inhibitor such as CDM. There thus seems to be little
risk of the cumulative effects on MAO and possible interactions with natural
or medicinal biogenic amines which are seen with many of the irreversible
inhibitors (Sjoqvist, 1965). even with repeated exposure to rather large
amounts of CDM.
18
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Although our work does not exclude the possibility that under some
circumstances, in some species, the MAO-inhibitory actions of COM may present
some hazard or contribute to the symptoms observed, it does indicate that this
risk is probably not great and that the biochemical lesion underlying the
acute lethality of the fortnamidines to mammals must be sought elsewhere. It
has been suggested that other effects on the regulation of biogenic amines may
be important in the lethality of COM (Matsumura and Beeman, 1976; Knowles and
Aziz, 1974; Knowles, 1976). Although this cannot be ruled out, there is
little direct evidence to support this view, and other workers have shown that
blockade of serotonergic and a-adrenergic receptors, and prior depletion of
tissue stores of these amines did not reduce the lethality of CDM to rats
(Robinson and Smith, 1977; Robinson ^t jl., 1975). A more plausible
hypothesis on the basis of current information relates the lethal actions of
CDM and related formamidines to their local anesthetic-like actions (See
Section 4) with a consequent depressor effect on the cardiovascular system
(See Section 2) and marked central stimulation with enhanced sympathetic
outflow which may account for the sympathomimetic symptomatology reported by
other investigators (See Sections 3 and 4). A local anesthetic-like action
could also account for the depression in motor end-plate sensitivity and
inhibition of the depolarization-contracture process observed with the frog
neuromuscular junction (Wang^t^., 1975; Watanabe jjt al., 1976). In fact
Watanabe et aL., (1975) briefly noted some similarities between CDM and the
local anesthetic, procaine, in their actions on the acetylcholine-stimulated
contraction of the frog rectus abdominis muscle. However the formamidines are
compounds with actions on multiple biochemical systems, and it is clearly
premature to ascribe all observed effects of these compounds to any single
type of biochemical action.
19
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SECTION TWO
ACTIONS OF CHLORDIMEFORM ON THE CARDIOVASCULAR SYSTEM
INTRODUCTION
As a first step in attempting to define the cause of death and toxic
mechanism with chlordimeform we started with the observation of Beeman and
Matsumura (1974) that 200 mg/kg of COM, ip, decreased the mean arterial bood
pressure in the pentobarbital-anesthetized rabbit. The purpose of this
investigation was therefore to analyze further the effects of CDM on the
cardiovascular system. Parallel studies in these laboratories (Sections 3 and
4) showed that CDM shared many of the actions of local anesthetic agents,
hence the cardiovascular actions of CDM were compared to those of lidocaine to
determine if these agents had common sites and modes of action.
MATERIALS AND METHODS
Blood pressure, heart rate, and cardiac contractility
Fourteen dogs of both sexes, 7-12 kg each, were anesthetized with 30-35
mg/kg of pentobarbital sodium iv. The left femoral arterial bood pressure was
monitored via a Statham pressure transducer with a Grass polygraph. Drugs
were administered via a cannula in the cephalic vein. Lead II of the ECG and
beat-to-beat heart rate were recorded on an E & M Physiograph. Artificial
respiration was provided to the dogs using a Harvard Apparatus respirator, and
right ventricular contractile force was monitored by means of a Walton-Brodie
strain gauge sewn onto the right ventricle.
Blood pressure, heart rate, and peripheral resistance
Fifteen dogs of both sexes, 7-12 kg each, were anesthetized with 30-35
mg/kg of pentobarbital sodium iv. The right carotid artery and the left
external jugular vein of each animal were cannulated and arterial and venous
pressures were recorded. Lead II of the ECG was monitored along with the
heart rate on a cardiotach. The dogs were given 600-700 units/kg of heparin.
The right femoral artery was cannulated both toward and away from the heart.
Blood was pumped from the femoral artery by a Harvard Apparatus
constant-perfusion pump and back into the leg via the femoral artery.
Perfusion pressure was monitored with a Statham pressure transducer distal to
the pump and was displayed on the polygraph. The flow rate through the pump
was adjusted (usually 27 ml/min) so that the perfusion pressure approximated
the carotid pressure. The volume of the perfusion apparatus was 34 ml. Drugs
were administered via a cannula in the cephalic vein or intraarterially (1 ml
volume) into the perfusion apparatus distal to the pump.
20
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Contractility of isolated rabbit hearts
Eighteen conventional Lagendorf rabbit heart preparations were perfused
with 38°, oxygenated Locke-Ringer solution. Contractility was displayed on a
Physiograph using a Type B, E & M myograph, and heart rate was monitored on
another channel via a cardiotach. Drugs (1 ml volume) were injected into the
perfusate as it entered the aorta.
Sympathetic outflow in the cat
Six cats, 2.6-3.4 kg, were anesthetized with 30 mg/kg of pentobarbital
sodium. Both cervical sympathetic nerves were exposed and placed on bipolar
electrodes for recording, and the cervical region was filled with warm mineral
oil. Spike activity was amplified by a Tektronix 122 preamplifier and spike
discharge frequency was recorded on the polygraph. Drugs were administered
via a cannula in the cephalic vein.
RESULTS
Blood pressure
Chlordimeform or lidocaine at doses of 3-30 mg/kg iv. in dogs, caused
dose-dependent decreases in mean arterial blood pressure within 1 min. A
secondary increase was observed after COM injection which returned to predrug
values in less than 1 hr (Fig. 5, Table 5). Hyperventilation, tremors,
particularly in the forelimbs, and occasionally clonic convulsions were
associated with the transition from depressor to pressor responses. Animals
under light anesthesia, as determined by the presence of active toe pinch and
corneal reflexes, exhibited tremors and large secondary pressor responses
after 10 or 30 mg/kg of COM iv, while animals under deep anesthesia showed
neither tremors nor pressor responses, indicating that these were of central
HI Rate 1<
Beals/min ft
Blood
Pressure
mm Hg
f 30 mg/kg
COM, i.v.
30 sec
TlOmg/kg
I Lido iv
Figure 5. Typical records showing the effects of (a) CDM and (b> lidocaine on
heart rate, cardiac contractility (in arbitrary units), and blood pressure in
the pentobarbital-anesthetized dog.
origin. Secondary pressor responses were smaller and less frequently observed
following lidocaine. Similarly, somatic manifestations of CNS stimulation by
21
-------
TABLE 5. TIME COURSE OF THE PRIMARY (1°) AND SECONDARY (11°) BLOOD PRESSURE AND CARDIAC CONTRACTILITY
RESPONSES IN CHLORDIMEFORM-TREATED DOGS
Time (min) to:
t-0
NJ
Dose
( mg/kg iv)
3
10
30
n
9
9
6
1°
Depressor
response
0.7 + 0.2a
0.8 + 0.1
0.8 + 0.1
1°
Decreased
contractility
response
0.9 + 0.2
0.7 + 0.1
0.7 + 0.1
11°
Pressor
response
5.6 - 1.3
10.5 + 1.6
14.6 + 0.5
11°
Increased
contractility
response
3.8 + 1.3
9.5 + 1.6
14.2 + 0.5
Recovery
to control
12.8 + 0.7
29.5 + 4.0
53.3 + 4.3
Mean + SE.
-------
lidocaine were less intense and less frequently observed than after CDM.
Dose-response data were collected on animals that were "light" by the
above criteria. Level of anesthesia appeared to have no effect on the blood
pressure depression caused by CDM or lidocaine. Blood pressure responses to
CDM in four unanesthetized but restrained rabbits were qualitatively identical
and quantitatively similar to those shown in Fig. 5.
Lethal doses of lidocaine (>30 mg/kg) or CDM (>50 mg/kg) caused an
irreversible decrease in blood pressure. Although respiratory arrest occurred
almost simultaneously, cardiovascular collapse was assumed to be the primary
cause of death since artificial respiration did not protect the dogs against
death. Lead II of the ECG showed that neither CDM nor lidocaine caused
cardiac arhythmias at the doses used in these experiments.
Heart Rate and Cardiac Contractility
Figure 5 and Table 5 show that CDM caused a depression of cardiac
contractility concurrent with the decrease in mean blood pressure, as well as
a secondary increase concurrent with the increase in blood pressure. The
dose-response curves for these events are shown in Fig. 6. The inotropic
effects did not appear to result from changes in diastolic filling of the
heart since vena cava pressure recordings at the level of the right atrium
(six experiments) showed no decrease or increase in venous pressure associated
with the decrease or increase in contractility, respectively. Therefore, CDM
must have some effect either on the autonomic nervous system or on the
myocardium itself.
£ -
o
a
20
-40
-60
3 10
mg/kg, i.v.
80
60 «-
I
-20
o
a.
tn
&
:-40
20
W
o
u
°M-60
30
3 10
mg/kg. i.v.
80
60 s
o
a
VI
41
at
40 ~
u
O
O
20°H
30
Figure 6. Initial I°(» •), and secondary, 11° (I
CDM and lidocaine on blood pressure and cardiac contractility.
expressed as a percentage of predrug control (mean +_ SE, number of
observations).
), effects of
Results are
23
-------
Bilateral vagotomy (five experiments) or 1 mg/kg of atropine (three
experiments) had no effect on the cardiac depression caused by 10 or 30 mg/kg
of COM. The same CDM doses had no effect on the positive chronotropic and
inotropic effects of the ganglionic stimulant DMPP (1 yg/kg, two experiments)
or the 3-adrenergic agonist isoproterenol (1 yg/kg, three experiments).
Furthermore, CDM caused a decrease in contractility very similar to that of
lidocaine in the isolated rabbit heart (Fig. 7). Therefore, CDM depressed
cardiac muscle independently of the autonomic nervous system in a way similar
to that of lidocaine.
The secondary increase in cardiac contractility was observed following CDM in
the dog but not in the isolated rabbit heart preparation and therefore it is
not a direct cardiac effect. The observations in the dog that propranolol (1
mg/kg, three experiments), hexamethonium (10 mg/kg, three experiments), and
diazepam (1 mg/kg, five experiments) all blocked the increase in contractility
and blood pressure caused by 10 mg/kg of CDM further suggests that the
positive inotropic effect of CDM results from sympathetic activity of central
origin.
M ml.ol
I I mM COM
30 sec.
lOOr
10
10-* IO'J
Molar Concentration
Figure 7. The effects of CDM (I
I) and lidocaine (<
on heart
rate and cardiac contractility (in arbitrary units) in the isolated rabbit
heart preparation (7b). Doses are expressed as the concentration of 1 ml
injected into the perfusate as it enters the aorta. Results are expressed as
a percentage of predrug control (mean + SE, number of observations). A
typical record is shown in la.
24
-------
Peripheral Resistance
Chlordimeform or lidocaine injected into the femoral artery caused a
decrease in perfusion pressure in the perfused hind limb of the dog (Fig. 8a).
Figure 8b shows that CDM was approximately 10 times more potent in this action
than lidocaine. Propranolol at 2 mg ia (two experiments) or 5 mg of atropine
ia (two experiments) blocked the decrease in perfusion pressure caused by 2 yg
of acetylcholine (ACh) ia, respectively, but neither blocking drug attenuated
the response to 10 mg of CDM ia. The increase in perfusion pressure caused by
2 yg of norepinephrine ia was not altered by 10 mg of CDM ia (three
experiments). Tripelennamine at 2 mg ia blocked the decrease in resistance
caused by 5 yg of histamine ia, but not that caused by CDM (two experiments).
a
70
90
J
10
0.1
SOllM
I mg COM
10
30 sec
CDM
1.0
mg-i.a.
10
0 and lidocaine
Figure 8. The effects of intraarterial CDM (•
(• •) on the hind limb perfusion pressure in the pentobarbital-
anesthetized dog (8b). Results are expressed as a percentage of the predrug
control (mean +_ SE, number of observations). A typical record is shown in 8a,
Finally, denervation of the perfused limb by the ligation of the femoral and
sciatic nerves had no effect on the intraarterial CDM response. These
findings suggest that the decrease in perfusion pressure caused by
intraarterial CDM is caused by nonspecific relaxation of vascular smooth
muscle and is not the result of adrenergic blockage or of cholinergic,
3-adrenergic, or histaminergic stimulation.
25
-------
When CDM or lidocaine was injected iv at 10-30 mg/kg, hind limb perfusion
pressure decreased simultaneously with the systemic blood pressure. The
secondary rise in systemic pressure caused by CDM was accompanied by a rise in
perfusion pressure. This rise was abolished by ligation of the femoral and
sciatic nerves (six experiments), by 10 mg/kg of hexamethonium iv (one
experiment), and by 5 mg of phentolamine ia (one experiment). Therefore, it
appears that CDM given iv in addition to its cardiac effects, caused a
decrease in peripheral resistance that contributed to the depression of
orterial blood pressure, and a secondary increase in resistance apparently
mediated by the sympathetic nervous system which probably contributes to the
secondary pressor response.
Central Sympathetic Involvement
CDM administered at doses of 1-30 mg/kg iv to cats produced an initial
decrease in blood pressure followed by a secondary increase as in dogs.
Recordings of spike discharge frequency from the cervical sympathetic nerves
in six cats showed that 10 mg/kg of CDM caused a M6 +_ 28% increase in mean
discharge rate over the predrug control level (seven observations). This
increase in rate was concurrent with the secondary blood pressure response,
thereby providing further evidence that the secondary pressor response results
from a central sympathetic discharge (Fig. 9). Surprisingly, the initial
depressor response was accompanied by a decrease in cervical sympathetic
discharge rate, suggesting that some central nervous system action of CDM
might contribute to the initial blood pressure depression. Another
observation lending support to this idea is that 10 mg/kg of CDM iv blocked
the pressor response caused by bilateral carotid occlusion (five dogs).
Blood
Pressure
(mm Hg) 100
50
t
10 mg/kg
CDM. i.v.
2 min.
Figure 9. Typical record showing the effects of CDM on blood pressure and
spike discharge frequency (in impulses per second) recorded preganglionically
from the superior cervical nerve of the pentobarbital-anesthetlzed cat.
26
-------
DISCUSSION
The experiments reported here show that cardiac depression and
vasodilation resulting in hypotension can account for death in animals exposed
to COM. Respiration is depressed by COM but artificial respiration does not
alleviate or delay toxicity.
Both the cardiac and vascular depression involve direct effects of CDM on
cardiac and vascular smooth muscle since neither denervation of these
structures nor autonomic pharmacological manipulations reduced the depressor
responses. Cardiovascular effects of toxic doses of local anesthetics have
been well studied (for a review see Covino and Vassalo, 1976) and have been
found to be identical to those described here for CDM. Although qualitatively
identical effects are observed in this investigation with CDM and lidocaine,
interesting quantitative differences are present. In concurrent studies
(Section 4) we found that lidocaine is 10 times more potent than CDM in
blocking the action potential of the frog sciatic nerve. Figures 6 and 7 show
that CDM and lidocaine depress the dog heart, isolated rabbit heart, and blood
pressure with similar potency while Fig. 8 shows that CDM is 10 times more
potent than lidocaine in dilating peripheral blood vessels. The lack of any
quantitative correlation between intrinsic anesthetic potency, as defined
using nerve conduction blockade, and potency of effects on other excitable
tissues is well documented for local anesthetics (Covino and Vassalo, 1976).
It is therefore not particularly surprising that no such correlation is
observed between CDM and lidocaine.
The increased contractility and vasoconstriction, which were responsible
for the secondary pressor response, were blocked by sympatholytic agents and
were absent in the isolated rabbit heart and denervated perfused hind limb
preparations. Other observations also indicate that the secondary pressor
response results from massive sympathetic discharge, which in turn is an.
autonomic component of CDM-induced seizures. Indeed, the blood pressure
oscillations and discharge patterns of the superior cervical sympathetic nerve
were similar to those observed during picrotoxin and strychnine seizures
(Polosa jet al., 1969). Further, the CDM-induced secondary pressor response
was observed under conditions which favored the appearance of tremors and
clonic limb movements. Both pressor and seizure responses were observed
following the injection of 3 to 4 mg of CDM into the lateral ventricles of
rats (Section 3). Finally, diazepam, which antagonizes local anesthetic
seizures (de Jong, 1972), blocked both responses of intraventricular CDM
(Section 3) as well as the secondary pressor response of intravenous CDM.
Even the mydriasis induced by CDM in mice could be explained better by central
sympathetic discharge than by monoamine oxidase inhibition, as suggested by
Beeman and Matsumura (1973)f since the present studies indicated that CDM had
little or no direct sympathomimetic or parasympatholytic activity.
Furthermore, CDM did not potentiate the action of the biogenic amines assayed
on the cardiovascular system, an action which would be expected if CDM
significantly inhibited MAO.
27
-------
Centrally mediated pressor responses were also observed following
intraventricularly administered lidocaine (Section 3). The smaller and less
frequently observed secondary pressor response to lidocaine may be related to
the weaker convulsive action of lidocaine as compared to CDM (Prince and
Wagman, 1966; Pfister, W. F., Noland, V., and Yim, G. K. W., unpublished
observations). The stronger convulsive action of CDM may also be due in part
to direct neuronal excitation by CDM, which has been observed in cockroach
nerve cord preparations (Beeman and Matsumura, 1973; Yamamoto and Fukami,
1976; Lund^t^l., 1979a).
Clearly these studies indicate that the cardiovascular toxicity of CDM is
similar to that of local anesthetics. This implies that diazepam,
vasoconstrictors, cardiac stumulants, and artifical respiration should be
useful in treating cases of CDM poisoning. Observations in these laboratories
with the dog suggest that this regimen is effective in reversing the
hypotension caused by doses of CDM that otherwise would be marginally lethal,
but is ineffective in antagonizing the severe cardiovascular depression caused
by larger doses. Additional studies on this topic are presented in detail in
Section 4.
28
-------
SECTION THREE
THE CENTRAL ACTIONS OF CHLORDIMEFORM
INTRODUCTION
In rats and mice, chlordimeform produces hyperactivity, respiratory
depression and convulsions. The mechanisms behind these central nervous
system effects have not been elucidated. Since the chemical structure of
CDM is similar to that of the local anaesthetic phenacaine, and since CDM
exhibits a local anesthetic effect on the isolated frog sciatic nerve
(Section U) and cardiovasular system of several species (Section 2). the
possibility was entertained that it may have actions on the central nervous
system similar to that of local anaesthetics.
Lidocaine has been shown to produce an action on the amygdala which
initiates seizures in the cat (Wagman _et al., 1967). In dogs, lidocaine
increases arterial blood pressure by a central mechanism (Kao and Jalar,
1959). This Section describes some central actions of chlordimeform in the
urethane-anaesthetized rat and compares them to those of lidocaine. The
arterial blood pressure and electrical activity in the amygdala were
examined after intraventricular injections of chlordimeform or lidocaine.
Since the raphe nucleus and serotonin exert inhibitory effects on seizures
and on arterial blood pressure (Boggan and Seiden, 1973; Baum and
Shropshire, 1975), the effects of raphe stimulation were also examined.
Local anesthetics and CDM (Wang and Narahashi, 1975) depress
neuromuscular transmission although relatively high concentrations of CDM
are needed (10 -10~^M). Thus, studies were also initiated to identify
whether the respiratory depression caused by CDM was of central or
peripheral origin.
MATERIALS AND METHODS
Central responses to intraventricular drugs
Male Sprague-Dawley rats weighing 260-350g were used in all
experiments. Rats were anaesthetized with pentobarbital sodium, 35 mg/kg,
given intraperitoneally and fixed in a Kopf stereotaxic apparatus. Bipolar
stainless-steel electrodes having diameters of 0.010 in. (303 series.
Plastic Products Co.) were implanted in the dorsal raphe nucleus and left
basal lateral part of the amygdaloid nucleus according to the stereotaxic
atlas of Pelligrino and Cushman (1967). A right lateral ventricular cannula
29
-------
was also implanted for drug injections. The lateral ventricular cannula was
cut from a 23-gauge stainless-steel needle and fitted with an indwelling
stylet. The electrodes and cannula were fixed to the calvarium by pouring
cranioplastic cement around them and over two stainless-steel screws which
had been inserted into the skull. After a minimum of 7 days recovery
period, the animals were tested.
Rats prepared as described above were anaesthetized with urethane, 1.25
rf/kg, given intraperitoneally. The external jugular vein was cannulated
with polyethylene tubing for intravenous injections, and the carotid artery
cannulated for recording of blood pressure, using a Statham P 23 pressure
transducer. Electrical activity from the basolateral nucleus of the
amygdala was monitored on a Grass polygraph. Using a Grass S-88 stimulator,
the raphe nucleus was stimulated, usually with a 10 sec train of square wave
pulses (0.1-1.5 V, 0.5 msec duration, 10-50 Hz). Intraventricular drug
injections were made from a needle fitted to polyethylene tubing connected
to a microsyringe. Injection volumes were 10-20 ul. Body temperature was
maintained at 37-38° with a thermostatically controlled heating pad.
Drugs used in these experiments were chlordimeform, lidocaine HC1
(Astra Pharmaceuticals, Inc.), and diazepam (commercially available Valium,
Roche, Inc.)
Respiratory arrest
Rats were lightly anesthetized with urethane (1.2 g/kg, ip), and the
test chemicals were infused over a period of 20-30 min via the femoral vein
until respiratory arrest occurred. During the progression of symptoms, a
Grass polygraph was used to monitor the following: Femoral arterial blood
pressure (via a Statham P23A transducer); ECG via transthoracic electrodes;
diaphragmatic contractions (via a Grass FT03 force displacement transducer);
and end tidal pCO,, levels (via a catheter in a tracheal tube and Bee km an LB
medical gas analyzer). The phrenic nerve was dissected free in the cervical
region and an oil pool formed. Phrenic nerve discharges were detected via a
pair of platinum electrodes, amplified (filter settings: 0.1-10 KHz),
displayed on a Tektronix storage oscilloscope, and fed to a Grass AM7 audio
monitor. The output was also fed to a Grass P7 Integrator and the
integrated phrenic burst pattern was recorded on the polygraph. Displays of
the phrenic discharges on the storage oscilloscope were also photographed
using a Tektronix C5 oscilloscope camera.
When respiratory arrest occurred the status of the phrenic nerve-
diaphragm system was assessed by delivering single and 2-4 sec long trains
of square wave pulses (0.1 msec duration, 10 Hz, 0.2-5V) to the phrenic
nerve via the pair of platinum electrodes, in order to monitor neuromuscular
function.
RESULTS
Blood pressure responses to intraventricular injection of lidocaine or
chlordimeform
30
-------
In a total of 21 rats, the intraventricular injection of 300-1000 yg
lidocaine produced variable responses ranging from pure pressor or depressor
responses to biphasic ones. Of 21 rats, 7 exhibited pressor, 10 depressor
and 4 biphasic responses. Because of the variable responses, it was not
feasible to quantify the data. However, in any particular animal, the
effect produced was dose related. A dose-dependent pressor response is
illustrated in Figure 10. In 17 rats, the effects of COM were also
variable: 9 being pressor, 3 depressor and 5 biphasic. As with lidocaine,
for a particular response, the effect was dose-dependent. Figure 10
illustrates the dose-dependent pressor response to COM.
BP 200'
mm Hg 100 •
LI DC
O.lSmt
BP 200
mm Hg 100"
1MIN
COM
0.3 mg
CDM
0.6 mf
Figure 10. Pressor responses in the carotid artery after intraventricular
injections of lidocaine or CDM in a rat anaesthetized with urethane.
Effects of lidocaine and CDM on changes in amygdaloid electrical activity
induced by raphe simulation.
When the dorsal raphe was stimulated, the amplitude of the spontaneous
electrical activity of the amygdala was suppressed. This suppression was
antagonized by lidocaine or CDM, and the degree of antagonism was
dose-dependent. These effects are illustrated in Figure 11. The duration
of the EEC suppression was dependent on the frequency of raphe stimulation
and was antagonized by lidocaine or CDM given intraventricularly. Such an
effect is illustrated in Figure 12.
31
-------
CONTROL
CONTROL
I t
\ \
AFTER LIOOCAINE O.J mg
AFTER COM l.o mg
' I
II
100 HV
S SEC
AFTER CDM Z.O mg
VV^VVAfvVV*"-M/Wr^^
t t
Figure 11. Suppression of amygdala EEC upon raphe stimulation and
antagonism of this suppression by intraventricular lidocaine or CDM.
Between arrows: 10 sec of dorsal raphe stimulation (1.5V. 0.5 msec. 20 Hz).
120
100
80
60
40
20
120
IOO
80
60
40
20
10 20
30
Hz'
40
50
Figure 12. Duration of suppression of amygdala EEC activity and antagonism
of this raphe-mediated EEC suppression by lidocaine and CDM. Panel A:
duration of suppression at varying frequencies of raphe stimulation, control
(• •) and after 0.5 mg intraventricular lidocaine (O o). Panel
B; control (• •) and after 1.0 mg intraventricular CDM (o o).
Vertical bars = SEM; n = 5.
32
-------
AMY
IIO
SEIZUIfS
A.-^V^VvY*^
JOB-
10O-
0-
Figure 13. Amygdala EEC spikes and blood pressure oscillation recorded
during overt CDM seizures. Dose: 3 successive 1 mg intraventricular doses
Between arrows, the dorsal raphe was stimulated electrically at 1.5V, 0.5
msec duration at 60 Hz.
AMY
EEO
Control
»°"*
MtoJL
If 200
,00 •
LIDO
MINUTES
t
COM
I -t-«
I I
OIAZIMM UDO
LO«t/M o.(«a
fe
MINUTES
I
CDM
1-0 ma
Figure 14. Antagonism by diazepam of the EEC and blood pressure effects of
intraventricular lidocaine and CDM. Panels A and B: before; Panels C and
D: after intravenous diazepam, 1.0 mg/kg.
Seizure activity induced by CDM and blockage by raphe stimulation
The successive injections of 3 to M intraventricular 1 mg doses of CDM
resulted in clonic limb movements. This seizure activity was accompanied by
oscillations in the arterial blood pressure, and the amygdala EEC was
characterized by slow spikes (Fig. 13). spindle bursts (Fig. 14A) or
high-amplitude rhythmic discharges (Fig. 14B). During seizure activity,
33
-------
high frequency electrical stimulation of the dorsal raphe nucleus inhibited
seizure activity along with its EEC and autonomic concomitants. This
inhibition persisted during the entire period of electrical stimulation.
Upon cessation of raphe stimulation, spiking of the EEC and oscillation of
the blood pressure reappeared. Seizures did not return in the 5 rats
observed. These effects are illustrated in Fig. 13.
Effects of diazepam
Intravenous diazepam, 1.0 mg/kg, blocked the effects of intra-
ventricular lidocaine or CDM on the electrographic activity of the amygdala
of the 5 rats. This dose of diazepam also antagonized the centrally
mediated blood pressure responses produced by lidocaine or CDM. These
results are illustrated in Figure 14.
Mechanism of respiratory arrest
The decreasing order of lethality and dose resulting in respiratory
arrest were: cocaine (35.3 +. 11 mg/kg), lidocaine (35.4 + 9.6 mg/kg), CDM
(62.3 _+ 6.0 mg/kg), and morphine (93.5 +_ 11.0 mg/kg). In lidocaine-treated
rats, the amplitude and rate of diaphragmatic movements and of phrenic nerve
bursts gradually decreased until respiratory arrest (Figure 15).
LIDOCAINE
B
( 5 mg/ml - 0 I ml/mln )
C
CONTROL
PHRENIC 50_
RATE
counts/sec
0
DIAPHRAMg
FORCE
orbit units
B P
mm Kg
25 MV
MIN
5 SEC
Figure 15. Respiratory and cardiovascular parameters of a urethane-
anesthetized rat infused with a lethal iv dose of lidocaine. Measurements
include: End-tidal pCO levels; integrated discharge rate recorded from cut
central end of phrenic nerve; diaphragmatic contractions; carotid blood
pressure; oscilloscope tracings of phrenic bursts and diaphragmatic
contractions.
-------
In contrast, as shown in Figure 16, respiratory rate was initially
increased by cocaine and COM. Diaphragmatic contractile force and the
amplitude of phrenic nerve bursts remained near control values, but
disappeared abruptly upon respiratory arrest. The profile of morphine on
the phrenic nerve activity was unique in that continous inter-burst
discharge preceded the abrupt decrease in phrenic nerve amplitude and
respiratory arrest. Naloxone reversed both actions of morphine but did not
reverse the respiratory depression induced by the other agents. Following
respiratory arrest produced by all of these agents, the diaphragm and
gastrocnemius muscle still contracted following electrical stimulation of
the phrenic and sciatic nerves, respectively. In contrast, central output
in the phrenic nerve declined and disappeared at the time of respiratory
arrest. These results indicate that respiratory arrest induced by these
agents is central in origin, and not a result of neuromuscular blockade.
CHLORDIMEFORM ( 10 mg/ml - 0 I ml/mm)
ABC
CONTROL L025 LD50
i 19 mg/Kg • 23 mg/Kg
P X
C°2 0.
L09S
• 36 mg/Kg
LlL <,:..,,, L
PHRENIC
RATE 50
counts/see
OL
3r-
DIAPHRAUt
FORCE
orbit units '
0.
200p
B P
mmHgioo)- I
25 MV
t INFUSION
START
^|^^^L|,
INSPIRATION
I MIN
YYTmrmrv
YVYYYYVTVYYV
5 SEC
Figure 16. Same as Fig. 15 except drug infused was chlordimeform.
DISCUSSION
As previously found in the frog sciatic nerve (Section U), the isolated
rabbit heart, and dog blood pressure preparations (Section 2), the central
actions of chlordimeform that were examined in the present study were
qualitatively indistinguishable from those produced by the local anaesthetic
lidocaine. Thus, when injected intraventricularly in the anaesthetized rat,
both agents produced similar responses on blood pressure and electrical
activity of the amygdala. The antagonism by intravenous diazepam of the
effects of CDM and lidocaine were anticipated in view of diazepam's well
established effectiveness in supressing local anaesthetic seizures (de Jong,
1970).
35
-------
The studies of Wagman ^t _al. (1967), and Eidelberg et _al_. (1963) have
implicated the amygdala as the probable focus of seizure activity induced by
the local anaesthetics, lidocaine and cocaine. The present study also
points to the importance of the amygdala in the action of lidocaine as well
as chlordimeform. The generally held scheme for explaining local
anaesthetic seizures is that local anaesthetics selectively block tonic
inhibitory mechanisms, thereby releasing excitatory mechanisms (de Jong,
19/0). The observed block by lidocaine or chlordimeform of the suppression,
induced by raphe stimulation, of the electrical activity in the amygdala
raises the possibility that local anaesthetics may produce seizures by
blockade of raphe inhibitory input to the amygdala.
Blockade by lidocaine and CDM of raphe inhibition of the amygdala is an
attractive possibility in view of previous findings of antagonism of
5-hydroxytryptamine (5-HT) inhibition of cortical neurones by cocaine
(Phillis, 1970) and antagonism of 5-HT-mediated rhythmic activity of
cockroach malphighian tubules (Hollingworth, unpublished observations).
Spontaneous firing of amygdala neurones is reduced following systemic
5-hydroxytryptophan (Eidelberg ^t jl., 1967), iontophoretic 5-HT or dorsal
raphe stimulation (Wang and Aghajanian, 1977). Moreover, electrical
stimulation of the midbrain raphe inhibits seizures induced by electrical
stimulation of the amygdala or by pentylenetetrazole (Kovacs and Zoll,
1974), and 5-hydroxytryptophan raises the threshold for audiogenic seizures
and restores the reserpine-induced hypersensitivity to noise in mice (Boggan
and Seiden, 1973). Although reduction of brain 5-HT levels facilitates, and
elevation of brain 5-HT levels antagonizes, pentylenetretrazole or
electrically induced seizures (Killian and Frey, 1973). brain 5-HT levels
are elevated in CDM-treated rats (Beeman and Matsumura, 1973). The elevated
5-HT levels have been attributed to MAO inhibition by CDM, but more recent
work suggests that the elevation is slight compared to standard MAO
inhibitors (Benezet ^t al., 1978). Since the 5-HT antagonist, LSD, also
causes an increase in [^f]5-HT levels following [^H]tryptophan injection
(Diaz and Huttunen, 1971). it is apparent that the effectiveness of CDM and
lidocaine in antagonizing 5-HT and raphe inhibition of amygdala neurones
must be directly examined in order to better assess the role of the raphe
system in seizures induced by CDM and lidocaine.
The blood pressure oscillations occurring during seizures induced by
CDM are similar to those induced by convulsants such as picrotoxin and
strychnine in the cat and described in detail by Polosa ^t al. (1969) and
Polosa e£ al. (1972). This oscillatory behavior has been observed to be
associateid~with synchronized periodic activity of sympathetic preganglionic
neurones and appears to be generated in the central nervous system. Similar
discharges have been recorded from peripheral sympathetic efferent nerves
during the pressor response following intravenous CDM (Section 2). The
pressor and convulsant effects of intraventricularly applied lidocaine and
CDM might involve similar mechanisms, since both agents also blocked raphe
inhibition of pressor responses evoked by stimulation of the amygdala. If
the raphe system exerts a tonic inhibitory influence on amygdala and other
nuclei responsible for vasomotor tone, then blockade of such inhibitory
input could result in pressor responses such as those seen after
36
-------
intraventricular lidocaine and chlordimeform. Since serotonergic neurones
also exert an inhibitory influence upon spinal sympathetic neurones
(Neurmayr _et _al., 1974), blockade of this descending inhibitory pathway may
also contribute to the increase in blood pressure observed after CDM and
lidocaine.
In regard to the mechanism underlying respiratory arrest induced by
CDM, the results presented here clearly support the CNS rather than a
blockade of neuromuscular junctions as the site of action. In CDM-poisoned
rats, neuromuscular function appears to be relatively unimpaired, while CNS
output via the phrenic nerve is diminished and disappears, indicating a
probable block of the respiratory center in the brain stem.
37
-------
SECTION FOUR
LOCAL ANESTHETIC PROPERTIES OF CHLORDIMEFORM
AND POTENTIAL MEANS OF ALLEVIATING TOXICITY
INTRODUCTION
In the preceding two Sections, it was shown that COM in both its
peripheral and central actions is qualitatively indistinguishable from the
local anesthetic, lidocaine. In addition, CDM, when injected into the
lateral ventricle, caused a delayed respiratory arrest that was consistent
with slow passage of CDM via the third ventricle and subsequent depression of
the brain stem respiratory "center" by a local anesthetic-like action of CDM.
CDM and local anesthetics have common toxic symptoms consisting of
hyperexcitability, tremors, convulsions, and respiratory arrest (Beeman and
Matsumura, 1974; deJong, 1970). Furthermore, the chemical structure of CDM
is similar to that of the local anesthetics, phenacaine and guanicaine (Fig.
17).
The first objective of this study was to determine if CDM did have a
local anesthetic action on the frog sciatic nerve preparation. Since
diazepam is an effective antagonist of convulsions and lethality induced by
local anesthetic agents (deJong, 1970; Richards et al., 1968), and was found
to reverse the hypotensive effects of CDM in dogs (Section 2), a second
objective of this study was to examine diazepam and other agents for their
effects on CDM-induced convulsions and lethality.
3 Q
DJ\ II /C7H5
}-N-C-CH,-N(C
2*5
CH
v_n.
N=C-N/
PHENACAINE
C,H50-
-N=C( NH
GUANICAINE
Figure 17- Chemical structure of chlordimeform and related local
anesthetics.
38
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MATERIALS AND METHODS
Frog sciatic nerve preparation
The hydrochloride salts of COM, DCDM, and of the local anesthetic agents
phenacaine (Holocaine, Winthrop-Stearns, Inc., New York), lidocaine
(Xylocaine, Astra Pharmaceutical Products, Inc., Worcester, Massachusetts),
and procaine (Mallinckrodt, Inc., St. Louis, Missouri) were dissolved in frog
Ringer's solution (Wesseling et al., 1971) and the pH was adjusted to 7.0 _+
0.1 with 0.1 M NaOH. Sheathed and desheathed sciatic nerves dissected from
frogs (Rana pipiens) measuring 3 to 3.5 in. in length were placed into
Harvard nerve chambers with pairs of platinum wire stimulating and recording
electrodes spaced at 1 cm intervals. Compound action potentials were evoked
using supramaximal stimulation (0.6-4.0 V, 0.15 msec, 1 Hz) delivered from a
Grass Model SD-5 stimulator. The action potential amplitude was monitored on
a Tektronix 565 oscilloscope. Conduction rate was estimated as the ratio of
the change in latency to the distance between two sets of recording
electrodes. During twin pulse stimulation, the interval between pulses that
resulted in 50% depression of the response to the second pulse was used as a
measure of the relative refactory period. Drug solutions were applied to a 5
mm segment of the nerve between the stimulating and recording electrodes.
Each nerve was used only once and six to eight nerves were run per drug
concentration. Three or four concentrations of each agent enabled the
calculation of the ED__ and standard error values by reverse regression
analysis (Aldrete and Daniel, 1972).
CDM-drug interactions in vivo
White mice (25-30 g; Cox-Swiss, Laboratory Supply Co., Indianapolis,
Indiana) were used in the studies of the convulsant and lethal actions of
COM. The mice were housed in groups under a 10 hr light-dark cycle for at
least 1 week prior to testing. They were pretreated with the potential
antagonists usually 30 min prior to being injected with approximate LDQ(. dose
of COM (100 mg/kg, ip). When assessing agents for possible enhancement of
COM toxicity, the pretreated mice were challenged with the approximate LD
dose of CDM (80 mg/kg COM, ip). Comparisons were made using Fisher's metnod
of calculating exact probabilities (Schwartz, 1974).
Diazepam (Valium: Hoffman-LaRoche Inc.) and the other pretreatments
were injected intraperitoneally in a volume of 10 ml/kg. Saline was the
solvent for diphenylhydantoin sodium, librium HC1 (Hoffman-LaRoche Inc.),
pentobarbital sodium (Nembutal, Abbott Laboratories), atropine sulfate
(Nutritional Biochemicals Corp.), imipramine HC1 (Geigy Pharmaceuticals),
pyribenzamine HC1 (Ciba Pharmaceutical Co.), phenoxybenzamine HC1 (Smith,
Kline & French), chlorphenoxamine HC1 ( Pitman-Moore Co.), neostigmine
methylsulfate (Hoffman-LaRoche, Inc.), physostigmine (Eserine Salicylate,
Mallinckrodt Chemical Works), cycloheximide (Calbiochem), and cinanserin (SQ
10,643: Squibb and Co.). Trimethadione (Tridione: Abbott Laboratories).
mephenesin (K & K Laboratories, Inc.), and chlorpromazine HC1 (Thorazine:
Smith, Kline & French) were dissolved in 10? ethanol, 20% propylene glycol,
70% saline mixture.
39
-------
RESULTS
Effects of CDM and DCDM on the frog sciatic nerve preparation
Figure 18 shows the effect of CDM, DCDM, phenacaine, procaine, and
lidocaine on the amplitude of the compound action potential of the frog
sciatic nerve. The slopes of the dose-response relationship for all
co .pounds were similar: no significant departure from parallelism was
observed between the regression lines for holocaine, procaine, DCDM, or
lidocaine compared to that of CDM. The order of increasing potency for the
compounds studied was found to be DCDM < CDM < procaine < phenacaine <
lidocaine. The ED 's + SE were 35+6.2 mM (DCDM); 15.0 + 3.7 mM (CDM); 8.7
+_ 2.5 mM (procaine?; 4.0 +_ 1.5 mM (phenacaine), and 1.9 +_ 0.3 mM (lidocaine).
Hence, CDM and its demethylated metabolite, DCDM, were 0.6 and 0.3 times as
potent as procaine respectively. The effects of CDM and DCDM on all other
parameters studied were similar to those observed with procaine, phenacaine,
and lidocaine. After 15 min of exposure to either CDM or procaine, threshold
stimulus voltages and relative refractory periods were increased by
approximately 30 and 20%, respectively. Conduction velocity was decreased by
15 and 20? after 15 min of exposure to CDM (30 mM, pH 6.0) and procaine (10
mM, pH 7.0).
IOO
O 80
O 60
b
UJ
UJ
-------
Repetitive or spontaneous firing was never observed. After complete
blockage of the nerve by CDM, partial recovery was obtained following
repeated washing of the nerve. When the degree of ionization of CDM was
increased by lowering the pH from 7.0 to 6.0, the nerve-blocking
effectiveness of 30 mM CDM was reduced by more than 5058 (Fig. 19). With
desheathed nerves, 1 mM CDM (pH 7.0) caused 20% depression (Fig. 19). Note
that with intact nerves (Fig. 18) approximately 5 mM CDM was required to
cause 20% depression. Thus CDM was about five times more active on the
desheathed nerve.
Sheathed Nerve COM
pH 6.0,90 mM
pHTO.30 mM
Desheathed Nerve
O O pH70 , ImM
6 8 10 12 14 16
TIME IN CHLORDIMEFORM (CDM)MINUTES
IB
20
Figure 19. Effect of chlordimeform (30 mM) at pHs 6.0 and 7.0 in the
sheathed and at pH 7.0 (1 mM) in the desheathed nerve on action potential
amplitude at 2 min intervals following drug application. Abscissa: time
(min) following application of chlordimeform to the nerve. Ordinate: action
potential amplitude (percentage of control). Each point represents the mean
+ SE (30 mM, pH 6.0; n = 7; 30 mM, pH 7.0; n = 6; 1 mM, pH 7.0; n = 6).
Diazepam and other agents on CDM convulsions and lethality
The approximate LD dose of CDM, 100 mg/kg, ip, resulted in periodic
convulsive jerks (mean latency: 4 min—see Table 6). The frequency,
severity, and duration of the convulsive episodes increased until a
continuous seizure bout resulted in repiratory arrest and death (mean
latency: 6-12 min for groups of 10 mice). The LD,-0 (and 95? confidence
interval) for CDM was 85.5 (87.9-83-3) mg/kg). The threshold dose for
inducing convulsions was well below 50 mg/kg, a dose at which no acute
lethality was evident.
41
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TABLE 6. EFFECTS OF ANTICONVULSANTS ON COM CONVULSIONS AND LETHALITY IN MICE
Pretreatments'
Dose No. convulsed/
Cr n pound (mg/kg) No. injected
Saline controls
Saline controls
Saline controls
Saline controls.
Vehicle control
Diphenylhydantoin
Trimethadione
Mephenesin
Mephenesin
Diazepam
Diazepam
Diazepam
Librium
Librium
Pentobarbital
Pentobarbital
40
400
100
200
15
15c
30°
15
40
15n
25°
10/10
10/10
10/10
10/10
10/10
10/10
10/10
8/10
10/10
10/10
10/10
10/10
10/10
10/10
10/10
4/10*
Mean
latency No. deaths/
(min) No. injected
3.6
4.3
3.7
2.3
3.0
2.2
2.8
4.6
3.8
4.7
5.1
3.2
4.3
___
10.0*
10/10
9/10
10/10
10/10
10/10
10/10
10/10
7/10
10/10
1/10*
3/10*
7/10
5/10**
10/10
9/10
6/10**
Mean
latency
(min)
6.0
12.7
8.0
6.2
14.6
6.2
10.2
8.7
8.7
15.0*
20.0*
25.4*
10.4
24.2*
11.6
Mice were pretreated 30 min prior to 100 mg/kg COM, ip (LE> dose).
Exceptions: mephenesin (15 min), diphenylhydantoin (120 min?, trimethadione
(60 min).
iO% ethanol, 20% propylene ^lycol, 70% saline.
°Resulted in ataxia and decreased motor activity.
* P <_ 0.001
** P < 0.01
42
-------
Pretreatment with the anticonvulsants diphenylhydantoin, trimethadione,
or mephenesin did not result in prevention of either the convulsions or
lethality that was observed following the LDq(. dose of COM (Table 6). At the
15 mg/kg dose, diazepam pretreatment reduced the severity and duration but
did not appreciably reduce the incidence nor prolong the onset time of the
CDM convulsions. However, lethality was reduced about 20%, and the onset
time to death was lengthened to about 20 min. The 30 mg/kg dose of diazepam
was also ineffective in preventing CDM convulsions, and it was less effective
than the 15 mg/kg dose in preventing CDM lethality. Mice receiving this
higher dose of diazepam exhibited motor impairment, ataxia, decreased motor
activity, decreased responsiveness to sensory stimulation, and, in some
cases, loss of righting reflex. Similar results were obtained with librium:
the 15 mg/kg dose attenuated CDM lethality, whereas the 40 mg/kg dose
prolonged the latency to death, but was ineffective in preventing CDM
lethality. Pentobarbital reduced the incidence of convulsions and death, but
only at the 25 mg/kg dose which was accompanied by marked sedation and
depression.
The effects of other agents that were tested as possible antagonists of
CDM are summarized in Table 7. Chlorpromazine (20 mg/kg) increased the
latency to convulsions and the latency to death, but provided little
protection against CDM lethality. Agents that did not reduce either the
severity of CDM convulsions or the incidence of CDM convulsions and lethality
included haloperidol, chlorphenoxamine, cycloheximide, neostigmine, and
physostigmine. Physostigmine increased the latency to death. Cinanserin
increased the onset time and decreased the severity and incidence of
convulsions as well as increased the latency to death and decreased the
incidence of lethality in mice treated with CDM. Animals treated with the 50
mg/kg cinanserin were ataxic and exhibited ptosis and decreased muscle tone.
During preliminary cardiovascular studies with CDM, it appeared that atropine
and pyribenzamine intensified the hypotensive action of CDM. Hence, possible
enhancement of CDM lethality by atropine and other agents that possess "local
anesthetic" activity was tested by pretreating mice with these agents, and
then challenging them with 80 mg/kg CDM (approximate LD_ dose). This dose of
CDM caused convulsions in 100? of the saline-pretreated mice (mean latency:
5.2 ± 0.2 min - see Table 8). As expected, no pretreatment reduced the
incidence of convulsions. The onset time for CDM convulsions was reduced in
the animals treated with imipramine, phenoxybenzamine, and atropine but not
with the mice dosed with pyribenzamine. CDM lethality was enhanced following
pretreatment with pyribenzamine, imipramine, and phenoxybenzamine, but not
after atropine or lidocaine pretreatment.
DISCUSSION
The acute toxic symptoms of CDM are similar to those observed following
local anesthetic overdose, i.e., clonic convulsions, respiratory depression,
and cardiovascular collapse followed by death (dejong, 1970). The
effectiveness of diazepam and pentobarbitol in reducing lethality by toxic
doses of CDM also parallels the effectiveness of these agents in decreasing
lethality from local anesthetic agents such as lidocaine (Richards et al.,
1968; Wesseling et al.t 1971; Aldrete et al., 1972). The ineffectiveness of
43
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TABLE 7. EFFECT OF OTHER PHARMACOLOGICAL AGENTS ON COM CONVULSIONS AND
LETHALITY IN MICE
Pretreatments
Compound
Saline controls
Saline controls
Saline controls
Chlorpromazine
Chlorpromazine
Haloperidol
Haloperidol
Chlorphenoxamine
Cyloheximide
Neostigmine
Physostigmine
Physostigmine
Cinanserin
Cinanserin
Dose No.
(mg/kg) No.
5
20
1
5
5
0.7
0.1
0.25
0.50
25 K
50b
convulsed/
injected
10/10
10/10
10/10
9/9
5/10
9/10
10/10
10/10
8/10
10/10
10/10
8/8
8/10
6/10»*
Mean
latency
(min)
2.8
2.3
3.6
3.5
6.0*
3.1
3.6
2.4
3.8
2.0
2.9
3.6
6.0*
7.2*
No. deaths/
No. injected
10/10
9/10
10/10
9/9
7/10
9/10
10/10
9/10
8/10
10/10
10/10
6/8
9/10
6/10**
Mean
latency
(min)
6.4
6.2
6.0
15.3*
9.8
8.1
9.5
9.5
5.9
18.1*
14.0*
9.0*
13.8*
Mice were pretreated 30 min prior to 120 mg/kg COM, ip (LD dose)
Exceptions: cycloheximide (2 hr), Chlorpromazine (3 hr).
Resulted in ataxia and decreased motor activity.
* P <_ 0.001
** P < 0.01
44
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TABLE 8. TOXICITY IN MICE OF COM COMBINED WITH AGENTS POSSESSING LOCAL ANESTHETIC ACTIVITY
•o
Ln
—
Pretreatment
Saline
Pyribenzamine
Pyribenzamine
Atropine
A tr opine
Imipramine
Imipramine
Phenox yben zam ine
Phenox yben zam ine
Lidocaine
Lidocaine
Dose
(mg/kg)
10
30
1
10
10
30
1
10
10
20
Latency to convulse
(min)
5.2+0.2
4.4+0.3
4.2+0.6
3.6+0.2*
3.0+0.2*
4.5+0.2
3.2+0.5*
3.1+0.3*
3.6+0.4**
5.2+0.4
4.0+0.2
No. deaths/
No. injected
7/70
5/10*
8/10*
2/20
3/20
4/10**
4/10**
4/10**
4/10**
4/30
3/20
aMice were pretreated 15 min prior to 80 mg/kg COM ip (LDe
30 mg/kg (30 min).
* P _< 0.001
** P < 0.01
dose) except lidocaine (3 min);.pyribenzamine
-------
diphenylhydantoin, trimethadione, and mephenesin in reducing COM lethality
was anticipated in view of the previously reported inability of these agents
in antagonizing local anesthetic toxicity (deJong, 1970). The observed
enhancement of CDM toxicity by the antihistaminic, pyribenzamine, the
a-adrenergic blocker, phenoxybenzamine, and the tricyclic antidepressant,
imipramine, could have been due to the local anesthetic actions of this
diverse group of pharmacologic agents (Crescitelli and Geissman, 1951;
Guerrero and Molgo, 197*0. However, CDM toxicity was not enhanced by
atropine, which also has comparable local anesthetic activity (Sheu et al.,
1969), or by lidocaine.
In the frog sciatic nerve preparation, CDM and its metabolite DCDM
produced effects similar to those of the local anesthetic agents studied:
amplitudes and conduction velocities of the compound action potential were
depressed, and threshold voltages and refractory periods were increased.
Reversibility of the nerve block, increased effectiveness in desheathed
preparations, and decreased potency at pHs favoring ionization of the
compounds (pKa of CDM is near 7.0) are additional characteristic actions
shared by CDM and the local anesthetics (deJong, 1970; Ritchie and Greengard,
1966).
In our comparisons of the actions of CDM and local anesthetics in vivo
(Sections 2 and 3), we found that both CDM and lidocaine caused similar
biphasic cardiovascular responses when injected intravenously in dogs, and
similar amygdala EEC spike discharges, overt seizures, and blood pressure
responses when injected into the lateral ventricle of rats. Further, like
lidocaine and cocaine (Post est^l., 1975), CDM induced "pharmacological
kindling" in rats i.e. a type of reverse tolerance in which repeated daily
subconvulsant doses eventually precipitated generalized convulsions (Yim, et
al.. 1977). These results indicate that some of the pharmacological and
toxic actions of CDM are due to actions that are shared by local anesthetic
agents.
Unlike the situation with local anesthetic seizures, which are readily
antagonized by diazepam (deJong, 1970), CDM seizures could not be prevented
even at doses higher than that which reduced CDM lethality. This was
especially surprising since we found that diazepam did antagonize seizures
and amygdala EEC spike discharges that resulted from intraventricular CDM
(Section 3). We also observed that diazepam blocked the secondary pressor
response of CDM, an autonomic consequence of CDM-induced seizures (Section
2). The latter studies were carried out in anesthetized preparations and
perhaps an additive anticonvulsant effect of the anesthetic? might explain
the lack of protection observed in this study with unanesthetlzed mice. The
general CNS depressant actions of pentobarbital and cinanserin might likewise
explain their effectiveness against the CDM seizures and lethality.
The resistance of CDM convulsions to diazepam might also reflect
manifestations of direct excitatory actions of CDM that are not shared by
local anesthetics. Indeed, Beeman and Matsumura (1974), reported repetitive
firing from the cockroach ventral nerve cord treated with CDM. Moreover,
repetitive firing in cockroach giant axons (Lund _et al., 1979a) and increased
46
-------
firing of caudate neurons (Pfister, W. and Yim, G. K. W., unpublished) have
been noted following the application of CDM but not lidocaine. The greater
intensity of the CDM convulsions, as compared to lidocaine seizures, is also
consistent with this possibility. These indications of a direct excitatory
action of CDM on neuronal membranes suggest that CDM may have effects on
sodium and potassium conductances beyond the depression characteristic of
local anesthetics such as lidocaine and procaine. The acute symptoms of CDM
toxicity are qualitatively similar to those produced by DDT, which also
elicits repetitive firing in the cockroach giant axon (Narahashi and
Yamasaki, 1960). Although cycloheximide is effective in preventing acute
lethality and decreasing the severity of convulsions induced by DDT (Hrdina
and Singhal, 1972). cycloheximide failed to protect the mice from convulsions
and lethality induced by CDM.
An understanding of the mechanisms of CDM-induced lethality must precede
the analysis of the diazepam-CDM interactions observed in this study. In the
lightly anesthetized dog intravenous CDM can result in simultaneous
respiratory arrest and cardiovascular collapse, and the irreversible
hypotension is mediated primarily by direct depressant effects of CDM on the
heart and blood vessels (Section 2). It seems unlikely that diazepam would
prevent these direct cardiovascular depressant actions. The report by Wang
£t al.., (1975) of CDM block of acetylcholine depolarization at the frog
neuromuscular junction raises the question of whether CDM-induced respiratory
arrest involves peripheral blockade of neuromuscular transmission. The
observed inability of neostigmine and physostigmine to reduce CDM lethality
does not support the possibility of a peripheral site of action, and is in
keeping with the conclusion already reached in Section 3. It is more likely
that CDM-treated mice succumbed from postconvulsive depression of the
respiratory "centers." Diazepam at 15 mg/kg appeared to have sufficiently
attenuated the severity of the CDM convulsions to prevent the secondary
postical respiratory arrest and consequent death. The ineffectiveness of 30
mg/kg of diazepam in reducing CDM lethality could possibly be due to additive
central respiratory depressant actions of CDM and the higher dose of
diazepam. Block by diazepam of the secondary pressor response of CDM could
also unmask an irreversible hypotensive action by CDM. The interactions of
diazepam with CDM and local anesthetic on central respiratory mechanisms
deserve further study. An unequivocal interpretation of these results is not
presently possible.
47
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SECTION FIVE
BEHAVIORAL EFFECTS OF CHLORDIMEFORM
INTRODUCTION
Recently, Jacobs described a behavioral model in the rat consisting of
a spectrum of symptoms termed the "serotonergic syndrome" which is purported
specifically to reflect "increased central serotonergic transmission".
Regimens that induced the syndrome include release of serotonin (5-HT) by
parachloroamphetamine (PCA), or direct stimulation of postsynaptic 5-HT
receptors by L-tryptophan plus monoamine oxidase (MAO) inhibition (Jacobs,
1976).
Interestingly, sub-toxic doses of CDM elicit some of the component
symptoms characteristic of the 5-HT syndrome in rats. The symptoms of CDM
behavioral toxicity have previously been likened to serotonin poisoning in
the presence of a MAO inhibitor (Beeman and Matusumura, 1973). In addition,
CDM possesses moderate MAO inhibitory potency (Section 1) and brain levels
of both 5-HT and norepinephrine are elevated in rats receiving toxic doses
of CDM (Beeman and Matsumura, 1973), although recent work suggests that the
elevations are low compared to those induced by other MAO inhibitors (Beeman
et al.t 1978).Since CDM inhibits MAO and elevates brain 5-HT levels, while
PCA (Jacobs, 1976) indirectly, and quipazine (Hong^t^., 1969) directly,
stimulate postsynaptic 5-HT receptors, each should be capable of eliciting
the 5-HT syndrome.
The first aim of this study was to determine if the serotonergic
syndrome could be produced by CDM, quipazine and by intraventricular 5-HT.
The second objective was to determine if the serotonergic syndrome produced
by these agents could be blocked by 5-HT antagonists (cinanserin and
methysergide). An additional objective was to develop a "behavioral
profile" for the effects of lethal and sublethal doses of CDM in the rat.
MATERIALS AND METHODS
Adult, male Sprague-Dawley derived albino rats (350-450 g) (Laboratory
Supply Co., Indianapolis, IN) were housed in groups (8-10/cage) except
animals with lateral ventricular cannulas which were housed individually in
air-conditioned rooms (24 ) in a quiet environment, with lighting between
07:00 and 18:00 hours. Animals were supplied with food (Wayne Lab Blox,
Allied Mills, Inc., Chicago, ID and water ad libitum for approximately one
week prior to use.
48
-------
For the intraventricular injection of 5-HT, stainless steel cannulas
were implanted 0.5mm above the lateral ventricles. The stereotaxic
coordinates were: L = 1.5 mm; AP = 1.0 mm: H = 3.0 below dura. 5-HT,
dissolved in saline, was j.njected in a volume of 10 pi over a period of 30
sec through a stainless steel needle which protruded 1 mm below the tip of
the cannula. Doses are expressed as the total salt.
The presence (or absence) of the following six components of the
"Serotonergic syndrome" was assessed: rhythmic reciprocal forepaw treading;
slow lateral head weaving; hindlimb abduction (splayed out posture);
rigidity (manifested by increased resistance of the hindlimbs to passive
extension and flexion); Straub tail, and resting tremor (especially of the
head and forelimbs). Positive control regimens for inducing the 5-HT
syndrome included PCA (5 mg/kg), and L-tryptophan (200 rag/kg, ip) 1 hr
following pargyline (50 mg/kg, ip). Animals were injected with appropriate
drugs and observed at 30, 60, and 90 min, except animals injected with 5-HT
which were observed at 4 x 15 min intervals following injection. The
animals were also observed for the presence of slow periodic backing
movements; periodic circling; hyperactivity (periodic movement about the
cages); hyperreactivity (startle reaction and/or vocalization accompanying
handling); hyperreflexia (periodic clonic spasms) and salivation.
All drugs were prepared immediately prior to use and administered
intraperitoneally to rats in volumes of 1 ml/kg of body weight.
Chlordimeform HC1 (CDM); parachloroamphetamine HC1 (PCA) (Regis Chemical
Co., Chicago, IL); quipazine, 2-(l-piperazinyl)quinoline maleate (Miles
Laboratories, Elkhart, IN); cinanserin HC1 (E. R. Squibb and Sons, New
Brunswick, NJ); and pargyline (Eutonyl , Abbott Laboratories, North Chicago,
IL) were dissolved in physiological saline. L-Tryptophan (Calbiochem, Los
Angeles, CA) and methysergide (Sandoz Inc., Hanover, NJ) were made up in a
1.5% suspension of methocellulose (USP) and acidified with a few drops of
0.1 N HC1. 5-Hydroxytryptamine creatine sulfate complex (5-HT, Sigma
Chemical Co., St. Louis, MO) was dissolved in physiological saline and
administered intraventricularly (ivc).
Where applicable, data were analyzed for significance between treatment
groups by using Fisher's method for calculating exact probabilities (Fisher,
1970), calculating X for 2x2 contingency tables, or using Student's £
test. Three doses of each agent enabled the calculation, by reverse
regression analysis (Brownlee, 1965), of threshold doses and standard errors
for eliciting 4 out of 6 serotonergic or additional symptoms.
RESULTS
Rats treated with both PCA and CDM exhibited the serotonergic syndrome
in a dose-related manner (Fig. 20). The calculated threshold PCA and CDM
doses (+ SE) for eliciting 4 of the 6 symptoms were 4.7 + 0.8 and 94.8 + 6.7
mg/kg, respectively. In addition, the slopes of the dose response lines of
PCA and CDM were not significantly different (F = 0.72; p > 0.5). Since
pargyline plus L-tryptophan elicited behaviors indistinguishable from those
of PCA, all further comparisons were made between non-lethal sub-threshold
doses of PCA (5 mg/kg). CDM (80 mg/kg), and quipazine (20 mg/kg).
49
-------
m
x
X
UJ
en
co 3
UJ
QD
SEROTONERGIC ADDITONAL
COM O—--O
PCA
QUIPAZINE0
I I
6 8 K) 20 40 60
DOSE mg/Kg
100 200
Figure 20. Dose response effect of PCA, COM, and quipazine in eliciting
serotonergic syndrome (closed symbols) and additional symptoms (open
symbols). Each point represents the mean + SE number of symptoms observed (n
= 6-12/dose).
The mean number of symptoms exhibited per rat and the frequencies with
which each of the 6 different component symptoms of the 5-HT syndrome were
exhibited by the groups of rats treated with 5 mg/kg PCA, 80 mg/kg COM and
20 mg/kg quipazine are given in the first three columns of Table 9. Thus
the eleven animals treated with 5 mg/kg of PCA exhibited an average of 3.7 +
0.5 of the 6 possible "5-HT-like" responses. CDM at 80 mg/kg also ~
effectively elicited the 5-HT syndrome, as the 12 animals averaged 4.3 + 0.3
responses. The onset of PCA and CDM-induced behavioral alteration was rapid
(5-10 min), and of long duration (up to M hours at the highest doses
tested). Forepaw treading, head weaving and tremor were the most frequently
observed symptoms, whereas Straub tail responses were infrequently observed.
Since the quipazine response changed from an excitatory to a depressant
phase after about 30 min, the quipazine-treated rats were observed several
times during the first 30 min. In contrast to PCA and CHM, quipazine did
not effectively elicit the serotonergic syndrome in rats; no dose response
relationship was evident (Fig. 20). The mean number of symptoms exhibited
by the 12 rats that received 20 mg/kg quipazine was only 0.8 + 0.3 of the 6
50
-------
possible (Table 9). The three responses seen most often with PCA and CDM
(forepaw treading, head weaving, and tremor) were absent in the 12
quipazine-dosed rats. Forepaw treading and tremor could be elicited only
with doses of quipazine producing acute lethality (HO and 80 mg/kg). Even
with the 40 and 80 mg/kg dose (Fig. 20), the overall incidence of the 6
serotonergic symptoms was far below the 4 of 6 criteria of Jacobs (1976).
Cinanserin (20 mg/kg) effectively antagonized the 5-HT syndrome induced
by PCA in rats (Fig. 21). with the total incidence of the components of the
syndrome halved (3.7 + 0.5 vs 2.0 + 0.6, t = 2.19, P < 0.05). The dose of
cinanserin employed (20 mg/kg) produced no overt behavioral symptoms of its
own, except a decrease in muscle tone. Cinanserin also completely prevented
quipazine from inducing any signs of the 5-HT syndrome (Table 9 and Fig.
21). In contrast, the incidence of the components of the 5-HT syndrome
elicited by CDM was unchanged by pretreatment with cinanserin (20 mg/kg)
(4.3 + 0.5 vs 3.8 + 0.4, t = 1.05, p < 0.05)- Higher doses of cinanserin
(40-60 mg/kg) could not be tested for their effectiveness in antagonizing
the CDM-induced 5-HT symptoms, since these higher doses of cinanserin alone
induced hindlimb abduction as well as marked signs of depression (decreased
motor activity, diminished responsiveness to external stimuli and
respiratory depression).
Q 6
Ui
£5
o
12
CO I
PRETREATMENTS
SALINE B CINANSERIN 20 mg/Kg
SEROTONERGIC SYNDROME
ADDITIONAL BEHAVIORS
PCA CDM QUIP PCA CDM QUIP
Figure 21. Effect of cinanserin (20 mg/kg) pretreatment (30 min) on the
serotonergic syndrome and additional symptoms elicited by PCA (5 mg/kg). CDM
(80 mg/kg), and quipazine (10 mg/kg). Values = X +_ SE; (n = 6-12).
* p < 0.05, ** p < 0.01.
51
-------
TABLE 9. EFFECT OF CINANSERIN ON SEROTONERGIC SYNDROME PRODUCED BY PCA, CDM AND QUIPAZINI
S3
Treatments
Dose
PCA
Controls
After
Cinanserin
(20 mg/kg)
CDM Quip-
PCA
CDM
azine
mg/kg
//Symptoms X
Exhibited/Rat +SE
Symptoms
Forepaw Treading
Head Weaving
Hindi imb Abduction
Rigidity
Tremor
Straub Tail
Totals
Percent Incidence
5
3.7
+0.5
7/11
10/11
6/11
6/11
10/11
2/11
41/66
62$
80
4.3 0
+0.3 ±0
12/12
12/12
8/12
6/12
12/12
1/12
51/72
71*
20
.8
.3
0/12
0/12
2/12
6/12
0/12
2/12
10/72
14*
5
2.0*
+0.6
5/11
5/11*
1/11*
2/11
7/11
1/11
22/66
33*
80
3.8
+0.4
8/8
8/8
4/8
3/8
8/8
1/8
32/48
67*
Quip-
azine
20
0.0*
+0.0
0/8
0/8
0/8
0/8*
0/8
0/8
0/48
0*
After
Methysergide
(10 mg/kg)
PCA
5
3.0
+0.6
0/5*
1/5*
4/5
5/5
4/5
1/5
15/30
50*
CDM
80
3.6*
+0.2
0/10*
2/10*
10/10
10/10
4/10*
10/10
36/60
60*
* P < 0.05 when compared to corresponding controls (Fisher, 1970).
-------
TABLE 10. EFFECT OF CINANSERIN ON ADDITIONAL SYMPTOMS PRODUCED BY PCA, COM AND QUIPAZINE.
to
Treatments
Dose
mg/kg
No. Symptoms X
Exhibited/ Rat +SE
Symptoms
Backing
Circling
Hyperactivity
Hyperreac tiv ity
Hyperreflexia
Salivation
Totals
Percent Incidence
Controls
PCA
5
4.3
+0.4
4/11
6/11
11/11
9/11
7/11
11/11
48/66
72%
CDM
80
3.8
+0.3
4/8
6/8
8/8
8/8
3/8
0/8
29/48
60}
Quip-
azine
20
2.0
+0.4
0/8
8/8
8/8
1/8
2/8
1/8
20/48
422
After
Cinanserin
(20 mg/kg)
PCA
5
0.7**
+0.3
0/11
1/11*
1/11*
2/11*
0/11*
2/11*
6/66
9%
CDM Quip-
azine
80 20
2.3*
+0.4
2/8
1/8*
7/8
7/8
2/8
0/8
19/48
40*
0.3**
+0.3
0/8
1/8
1/8*
0/8
0/8
0/8
2/48
4*
After
Methysergide
(10 mg/kg)
PCA
5
2.8*
+0.4
0/5
3/5
5/5
2/5
0/5*
4/5
14/30
47?
CDM
80
2.7
+0.2
2/10
0/10*
6/10
10/10
9/10
0/10
27/60
45*
* p < 0.05 when compared to corresponding controls (Fisher, 1970).
** p < 0.01 when compared to corresponding controls.
-------
Methysergide (10 mg/kg) pretreatment (30 min) significantly reduced the
incidence of the components of the serotonergic syndrome in CDM (80 mg/kg)
treated rats (4.3 ± 0.3 vs 3.6 ± 0.2, t = 2.31. P < 0.05) but not
following treatment with PCA (Table 9). Methysergide alone did not elicit
any overt behavioral effects.
In addition to the 5-HT syndrome, PCA, CDM, and quipazine elicited other
Affects which are categorized in Table 10. Only PCA and CDM exhibited these
effects in a dose related manner (Fig. 20). The calculated doses (X + SE)
for eliciting 4 of these 6 additional effects were 4.7 + 1.0 and 91.5 + 6.9
mg/kg. The fact that these doses are not significantly different from doses
that elicit the serotonergic syndrome raises the possibility that these
symptoms have common underlying mechanisms.
As illustrated in Figure 21 and detailed in Table 10, cinanserin
pretreatment also reduced the overall incidence of the 6 additional
behaviors elicited by PCA (4.3 vs 0.7, t = 10.2, p < 0.01); by CDM (3.8 vs
2.3, t ^ = 3.23, p < 0.01); and by quipazine (2.0 vs 0.3, t., = 3.5, p <
0.01). CDM-induced hyperactivity was noticeably resistant to antagonism by
cinanserin. In contrast, hyperactivity and circling induced by PCA or
quipazine were almost completely prevented by cinanserin. Methysergide (10
mg/kg) pretreatment did attenuate the total incidence of the additional
symptoms following PCA (4.3 ± 0.4 vs 2.8 + 0.4, t = 2.48, p < 0.05) but
not following CDM. 1^
When administered into the lateral ventricles, 5-HT depressed the
spontaneous activity of the rat but did not elicit the serotonergic syndrome
(Table 11). In particular, Straub tail, tremor and rigidity were not .
observed. Of the additional symptoms, circling and head twitches (pinnial
reflex) were present.
TABLE 11. EFFECT OF 5-HT ON SEROTONERGIC SYNDROME AND ON ADDITIONAL
BEHAVIORS IN THE RAT3
Mean (+ SE) number of symptoms and percent incidence
Dose ( g)
0
50
100
200
400
Serotonergic
Syndrome
0.3 + 0.2 ( 450
0.8 + 0.5 (13%)
2.0 + 0.4** (25*)
1.5 + 0.5* (25%)
1.5 -i- 0.3***(37%)
Additional
Symptoms
1.0 + 0.4 (17%)
0.5 + 0.3 (18%)
0.8 + 0.4 (17%)
0.3 + 0.3 (4%)
1.0 + 0.4 (17%)
aSee Methods for classification of Serotonergic Syndrome and
Additional Symptoms. Significant differences from controls:
* p < 0.05; ** p < 0.01; *** p < 0.001.
54
-------
DISCUSSION
The demonstrated ability of COM to induce the "5-HT syndrome" and the
cinanserin block of the PCA-induced "5-HT syndrome" seem compatible with
Jacobs' view that "the 5-HT syndrome" is a model of increased 5-HT
transmission, since CDM is an MAO inhibitor and elevates brain 5-HT levels
(Beeman and Matsumura, 1973; Benezet jt jal., 1978) while cinanserin is a
5-HT antagonist (Salas jit al., 1966).
Clearly in our study central injections of 5-HT did not produce
hyperactivity but rather depressed motor activity, and this is consistent with
previous reports that both intraventricular (Green et jal., 1976a) and systemic
(Jacobs and Eubanks, 1974) administration of 5-HT depresses motor activity and
produces sedation.
Even though 5 yl injections into the lateral ventricle rapidly reach the
fourth ventricle, brain stem nuclei distant to the ventricular surfaces and
the spinal area would be inaccessible (Myers and Yaksh, 1968). Hence, the
ineffectiveness of intraventricular 5-HT in inducing the complete serotonergic
syndrome could well be due to 5-HT not reaching all the appropriate sites of
action. In the case of quipazine, however, no explanation is apparent for the
inability of this direct 5-HT agonist to induce the 5-HT syndrome. Although
Green ^t al. (1976b) reported that quipazine (25 and 50 mg/kg) exhibited a
motor syndrome similar to that induced by L-tryptophan following
tranylcypromine, they did not provide data detailing the frequencies with
which the component symptoms were observed.
The ineffectiveness of the two 5-HT antagonists in blocking the 5-HT
syndrome induced by CDM likewise cannot be easily explained. Since PCA was
about 20 times as potent as CDM in its ability to induce the syndrome, it is
unlikely that these results are due to greater affinity of CDM (as compared to
PCA) for the 5-HT receptors involved. CPP, 6-chloro-2-(piperazinyl)-2-pyra-
zine, is a new agent which also induces the 5-HT syndrome (Clineschmidt ^t
al., 1977). In this case also the 5-HT syndrome was not antagonized by
cinanserin. One other confounding finding was that the higher doses of
cinanserin alone elicited hindlimb abduction, one of the components of the
"5-HT syndrome"! Clineschmidt et al. suggested that the 5-HT motor syndrome
could be due to central tryptaminergic activation to explain the
ineffectiveness of the specific 5-HT antagonist cinanserin. However, this
possibility was not supported by the effective antagonism by cinanserin of the
PCA-induced motor syndrome observed in this study. Thus, these latter
findings are difficult to explain by the simple concept that the serotonergic
syndrome specifically reflects increased serotonergic transmission.
The behavorial symptoms other than the components of the 5-HT syndrome
(Table 11) deserve further comment. Although the stereotypic behavior
observed with PCA, CDM and quipazine is usually associated with dopaminergic
agonists, this stereotypic activity was readily antagonized by cinanserin.
Quipazine-induced stereotypy had previously been shown to be abolished by
antiserotonergic agents as well as by dopamine receptor antagonists, leading
Grabowska ^t a±. (197*0 to suggest that serotonergic as well as dopaminergic
55
-------
pathways are essential for the full expression of the stereotypy. Our
preliminary observation of a block of CDM-induced stereotypy by both
cinanserin and by haloperidol is consistent with Grabowska's suggestion.
In view of these contradictions and uncertainties, the concept of a
spf.oific 5-HT syndrome is questionable and needs further validation. CDM
certainly has a number of behavioral effects in rats at sublethal doses, as
described above and summarized in Table 13, but whether they are wholly due to
serotonergic actions is dubious in view of the ineffectiveness of 5-HT
blockers in preventing their expression in most instances. In those cases
where methysergide was effective i.e., forepaw treading, head weaving, and
tremor, even if 5-HT is involved, it remains to be shown whether this response
is due to an increase in brain 5-HT through CDM's MAO inhibitory action or
whether other direct or indirect serotonimimetic actions are occurring.
56
-------
SECTION SIX
INHIBITION OF PROSTAGLANDIN SYNTHESIS BY FORMAMIDINES
INTRODUCTION
As presented in the previous Sections, CDM possesses a number of actions
(i.e. blockade of nerve conduction, inhibition of MAO, psychomotor
stimulation, and induction of stereotypic behavior) which are known to be
shared by cocaine. This encouraged us to make a further comparison between
these compounds in regard to their relative antinociceptive (analgesic)
activity in rats. CDM (40 mg/kg, ip) and cocaine (20 mg/kg, ip) both
increased tail flick latencies and raised the threshold for vocalization
induced by electrical stimulation of the tail. The narcotic antagonist,
nalorphine (5 mg/kg, so), did not block CDM- or cocaine-induced
antinociception. Lidocaine produced analgesic responses only at doses which
resulted in loss of the righting reflex (i.e. 60-80 mg/kg). Because of this
non-narcotic analgesic effect of CDM, which is probably not related to its
local anesthetic actions, further studies of the mechanism of analgesia were
conducted to detect any aspirin-like analgesic properties of CDM using the
isolated guinea pig ileum. Aspirin, a prostaglandin synthesis inhibitor,
cause depression of the electrically-induced ileum twitch which is reversed by
prostaglandin E.. (PGE.) but not by the morphine antagonist, naloxone. PGE.. is
believed to stimulate the release of the actual transmitter, acetylcholine, in
this preparation (Ehrenpreis et _al.f 1976). When CDM was found to block the
ileum twitch, further studies were performed to define other aspirin-like
actions of CDM (antipyretic, anti-inflammatory) and its activity as a PG
synthesis inhibitor.
MATERIALS AND METHODS
Male Sprague-Dawley rats (300-400 g) were obtained from Laboratory Supply
Co., Indianapolis, Indiana. Chlordimeform and amitraz were kindly provided by
Ciba-Geigy Corporation and the Upjohn Co. respectively. They were
recrystallized before use to a final purity of over 98J.
The drugs included: acetycholine iodide (Calbiochem); clonidine HC1
(Catapres, Boehringer Ingelheim, Ltd. Dist.); indomethacin (Indocin, Sigma
Chemical Co.); morphine sulfate (Mallenkrodt); naloxone HC1 (Narcan, Endo
Laboratories); 1-norepinephrine bitartrate (Sigma Chemical Co.); phentolamine
mesylate, USP (Regitine, Ciba Pharmaceutical Co.); tolazoline HC1 (Priscoline,
Ciba Pharmaceutical Co.); and prostaglandin E* (PGE., Sigma Chemical Co.).
Indomethacin and PGE. were dissolved in 95% ethanol and stored in the freezer
until needed. All other compounds were prepared fresh in distilled water.
57
-------
Guinea pig ileuro preparation
Strips of longitudinal muscle were obtained from the guinea pig ileum as
described by Rang (1964). The strips were equilibrated for one hour in 37°
Krebs solution aerated with 95/S 0-5% CO , and were "field" stimulated as
described by Paton (1957). A Grass S88 stimulator was used to deliver square
wave pulses (60 V, 2-4 msec, 0.1 Hz) and the submaximal (approximately 80%
maximum) contractions were recorded on a Grass Model 5 polygraph via Grass
FT-03 transducers. Postsynaptic effects were assessed using cumulative ACh
concentration responses according to the method of Ariens ^t al^., (1964).
Antipyretic and anti-inflammatory assays
Fever was induced in 24-hour fasted rats by the subcutaneous injection of
a 15% suspension of dried brewer's yeast (10 mg/kg). Fifteen hours later the
test drugs, suspended in 1.5J methylcellulose, were injected intraperitoneally
and body temperature was monitored rectally over a further 7 hours. Edema
was induced by injection of 0.05 ml of 1% suspension of carrageenin
(Viscareen, Marine Colloids) into the plantar tissue of the left hind paw
(Winter et^a^., 1962). The formamidines were administered as in the
antipyretic assay, 30 min before the carrageenin. The resultant edema was
expressed as the percent difference in paw volume, measured by mercury
displacement, compared to the volume of the untreated right hind paw.
Assay of prostaglandin synthesis
PG synthesis was assayed using bovine seminal vesicle microsomes (Flower
et aL., 1973). Lyophilized microsomes (1.5 mg) were incubated with
^i-arachidonic acid (5 Ci per mmole) at pH 8.2 in the presence of glutathione
(5 mM) and epinephrine (5 mM) for 5 min. The reaction (0.51 ml) was
terminated with 0.25 ml 1 N HC1, and extracted with 2 ml ethyl acetate.
Authentic PGE? was added to a 1 ml portion of the extract and the ethyl
acetate was removed. The residue was taken up in 0.1 ml ethanol and
chromatographed on silica gel plates in ethyl acetate:acetone:acetic acid
(90:10:1). The PGE_ spot was visualized by exposure to iodine vapor, scraped
off, and counted in a scintillation counter. Control reactions with a boiled
enzyme sample were run similarly, and the PGE2 counts from the other
incubations were corrected accordingly. Corrected rates of synthesis of PGEp
(arachidonate at 0.4 yM final concentration) in the absence of inhibitors
ranged from 15-20 pmoles per 5 min per mg protein. At least five
concentrations of each inhibitor were used, without preincubation with the
enzyme before addition of 0.4 or 50 yM arachidonic acid. From two to five
replicates of each assay were run. I^Q (50% inhibitory) concentrations were
determined graphically from plots of % inhibition of PGE^ synthesis against
log of inhibitor concentration.
RESULTS
COM produced a dose-related inhibition of the twitch responses of the
guinea-pig longitudinal muscle preparation induced by electrical stimulation
(Figures 22 and 23). The ED50 as determined by reverse regression analysis
58
-------
was 2.9 x 10 J* (7 trials, Figure 22A), with a 95% confidence interval of
2.4-3.6x 10 M. At this concentration, ACh-induced contractions were
reduced by approximately 15%.
As illustrated in Figure 22B, ACh-induced contractions were reduced by
approximately 30% in the presence of 10" M CDM (8 trials), a concentration
that depressed twitch responses by approximately 20* (Figure 22A). ACh
contractions were enhanced by the presence of 10J7 M PGE1 (8 trials. Figure
22B). ACh responses, in the presence of both 10 M CDM and 10 M PGE., were
not appreciably different from control ACh responses (8 trials, Figure 22B).
B
-i
.--'*
I CONTROL (N-8)
COM ICT4*) (N«8)
PGE, ICT*M (N-8)
CDM IOT*M +
PGE, ICT*M (N-8)
3xKT« lO* 3»0r»
CHLORDIMEFORM (M)
—I
3xl(T»
icrT
ACETYUCHOUNE (M)
Figure 22. A: CDM-induced depression of the electrically stimulated twitch
height. Ordinate: mean + SE (n = 7) percent depression from control height
spaced on a probit scale. Abscissa: log cumulative concentration of CDM. B:
ACh responses in the presence of CDM and PGE^ Ordinate: mean + SE (n = 8)
percent of maximum control ACh contraction. Abscissa: cumulative ACh
concentration.
_„ Twitch depression induced by CDM was not antagonized by naloxone (3 x
10"' M to 10" M, 3 trials, Figure.,23A). In contrast, twitch depression
produced by morphine (ED = 3 x 10"' M), was readily reversed by naloxone (3 x
10" M, 2 trials). This concentration of naloxone did not affect control
twitch height (2 trials). The depression induced by 3 x 10 M CDM (45.6
4.7% of control) was reversed by 3 x 10 " M tolazolipe (117 +^ 5.3% of control,
n = 5, Figure 23B), but not by phentolamine (3 x 10 M, 3 trials. Figure
23C). Twitch depression produced by norepinephrine or by clonidine was
readily reversed^by either tolazoline (2 trials) or by phentolamine (3
trials). At 10~ M (3 trials), phentolamine alone had only a slight
depressant effect on the twitch response. In contrast, tolazoline alone (3 x
10~ M) increased the twitch response to 143 +_ 4.5% of control (n = 6).
59
-------
COM 3XJO-* M
NALLINE 3*IO'TM
B
COM
2.5»IO'*M
TOLAZOLINE
l.8xl
-------
PGE.
(10 M) readily reversed twitch depression induced by
*__ / f\ M i_i_ r»j r\*\r\ \ 11 4 n"" U *T\M 10*7 *7 -L O
10~4 M
indomethacin (2 trials, Figure 23D), and by 10"* M COM (27.7 ± 2.3* of control
with COM: 86.9+3.6% of control with COM plus PGEJt n = 9, Figure 23E). The
depression by 3 x 10~5 M CDM (49.M +. 1.8* of control) was readily reversed by
PGE (10~° M, 102.4 ± 2.1% of control, n = 6). Washing the strip with fresh
drug-free Kreb's buffer readily reversed twitch depression induced by CDM, but
not that induced by the irreversible PG synthesis inhibitor, indomethacin
(Figure 24). Depression of the twitch by lidocaine (50% at 10 M) was only
partially and erratically reversed by PGE., (10~ M, 5 trials, Figure 23F).
The antipyretic activities of the formaraidines, CDM and amitraz, were
assessed against yeast-induced fever in rats and compared to the effects of
two known antipyretic inhibitors of PG synthesis, indomethacin and aspirin.
Examination of the resulting dose responses presented in Fig. 25 yields the
following rank-order of the compounds according to their antipyretic activity:
indomethacin > chlordimeform > amitraz > aspirin. The reduction by
chlordimeform of the rectal temperatures to 37° in the yeast-treated rats is
consistent with the hypothermia effects of chlordimeform which we previously
observed in normal rats (Pfister and Yim, 1977).
40
39
K
o
00
36
40
,39
I
i 38
O
CD
0 SALINE
• ASA 20
o 40mg/Kg
A 160
A 320
i—3-
D SALINE
• CDM Sing/Kg
O 10
A 20
A 40
-i 6-
INDOMETHACIN
B
M=^=*~4
a SALINE
• INDO 0.23mg/Kg
o o.S
A I.O
A £0
a SALINE
• AMITRAZ 20mg/Kg
O 40
A 80
^T 2 3 4
TIME FOLLOWING INJECTION (HR)
ir\
2345
TIME FOLLOWING INJECTION (HR)
i-
Figure 25. Antagonism of yeast-induced fever in rats by acetylsalicylic
acid (aspirin), indomethacin, and two formamidine pesticides, chlordimeform
and amitraz. Normal body temperature at_time of administration of yeast
(-15 hr) is shown in A and C. Values = X + ; n = 6.
61
-------
TABLE 12. INHIBITION OF PGE SYNTHESIS BY BOVINE
SEMINAL VESICLE MICROSOMES WITH TWO FORMAMIDINES
AND TWO COMMON NON-STEROIDAL ANTI-INFLAMMATORY AGENTS
Inhibitor I (yM + SD) at
arachidonic acid
concentration of:
0.4yM 50 yM
Chlordimeform 34+19 145 +_ 36
Amitraz 880 + 170 >1500
Indomethacin 0.4+0.08 1.0+0.2
Aspirin 790 3900
To assay the inhibition of PG synthetase we used a standard method
based on the conversion of H-arachidonic acid to PGEp by bovine seminal
vesicle microsomes. The values in Table 12 reveal that chlordimeform is an
inhibitor of PG synthesis of some potency while amitraz is less active,
approximating aspirin in its activity under these conditions. Figure 26
demonstrates that chlordimeform and amitraz are anti-inflammatory agents.
The control curves show the time course of the edema produced by the
injection of carrageenin into the plantar tissue of the hind paw. Paw
swelling was reduced by approximately 5Q% in the chlordimeform-treated rats
(40 mg/kg). and by 25% in those treated with amitraz (80 rag/kg). Aspirin at
140 mg/kg reduced paw swelling by 60-65%. The ability of aspirin and
indomethacin to reduce the edema induced by carrageenin and other irritants
is well documented (Winter, 1965).
DISCUSSION
CDM produced a dose-related depression of the electrically-stimulated
twitch responses of the guinea-pig longitudinal muscle preparation. In this
study, the concentration of CDM which depressed the twitches by 50?
(3 x 10 M) and 70% (10~ M) reduced ACh-induced contractions by 15% and
30% respectively. In comparison, twitch depression by morphine (Paton,
1957), by NE (Paton and Vizi, 1969), by clonidine (Kroneberg and Oberdorf,
1974), and by indomethacin (Ehrenpreis eit _al., 1974) primarily involve
decreased ACh release, with minimal or no postsynaptic depression of ACh
responses.
62
-------
CONTROL
ASA I60mg/Kg
o-o CONTROL
*-• AMITRAZ 80mg/Kg
o—o CONTROL
•-•COM 40mg/Kg
o—o CONTROL
•-• COM 40mg/Kg
012345678 26
TIME AFTER CARRAGEENIN INJECTION (hr)
012345678
TIME AFTER CARRA6EENAN INJECTION (hr)
Figure 26. Antagonism of the carrageenin-induced edema of rat paw by the
formamidines amitraz and chlordimeform and aspirin (ASA). ^Compounds were
administered 30 minutes before the carrageenin. Values = X _+ SE; n = 6.
The CDM-induced twitch depression was not like that produced by
narcotic-type analgetics, because it was not antagonized by naloxone, which
readily reversed the twitch depression by morphine (Paton, 1957). In the
rat tail flick preparation, we had already found that CDM-induced
antinociception is likewise not antagonized by naloxone.
The CDM-induced twitch depression was also not like the depression
produced by appetite stimulants, clonidine, or another alpha-adrenergic
agonist, norepinephrine, since phentolamine, which is an alpha antagonist,
reversed the depression induced by either clonidine or norepinephrine, but did
not antagonize the CDM-induced effect. The ability of tolazoline, another
alpha-adrenergic antagonist, to readily reverse the depression of the twitch
height by CDM may possibly involve the previously reported anticholinesterase
action of tolazoline (Dzoljic, 1967). It is also unlikely that the local
anesthetic action of CDM is primarily responsible for the depression of the
63
-------
ileal twitch responses since PGE was much more effective in antagonizing
CDM-induced than lidocaine-induced depression.
Thus, the present studies indicate that the twitch depression produced by
CDM was most similar to that produced by the nonsteroidal anti-inflammatory
agent, indomethacin; both drugs were consistently reversed by exogenously
add ,d PGE . Indomethacin is a well-known irreversible inhibitor of
r.rostaglandin synthesis (Gryglewski, 1974). The results above show that
chlordimeform is also an effective inhibitor of prostaglandin synthesis. The
ICQ values reported here for aspirin and indomethacin with the bovine seminal
vesicle synthetase fall within the range of existing values in the literature
(Gryglewski, 1974). The mechanism and kinetics of inhibition by the
formamidines remain to be established, but preliminary results indicate that
chlordimeform is a reversible, non-progressive inhibitor. In each case in
Table 12 the inhibitory potency decreased at the higher substrate
concentration. Normal concentrations of arachidonate at the active site of
the synthetase jlri vivo are hard to estimate since it is believed that the
fatty acid precursors for PG are stored as phospholipids and are released as
needed by the action of phospholipases. Tissue levels of the free acid are
very low (Hinman, 1972). It is significant that the potencies of the four
compounds assayed as synthetase inhibitors are directly related to their
antipyretic activities as shown in Fig. 25.
The washing studies with the guinea pig ileum uncovered an important
difference between the actions of CDM and indomethacin. The persistence,
after washing with fresh Kreb's buffer, of the twitch depressant action of
indomethacin, has been attributed by Ehrenpreis et al., (1976), to
irreversible inhibition of PG synthesis (Ku and Wasvary, 1975). The
reversibility of the twitch depression induced by CDM is in keeping with the
conclusion that it is a reversible PG synthesis inhibitor. Thus, the guinea
pig ileal preparation may be a convenient screen to distinguish reversible
from irreversible inhibitors of PG synthesis.
We have shown that CDM has several aspirin-like actions (antinociception,
anti-inflammation, antipyretic, and block of guinea pig ileum twitch). It is
likely that aspirin achieves most or all of these actions by inhibition of
prostaglandin synthesis (Vane, 1971; Flower, 1974; Robinson and Vane, 1974),
an inhibition which we have found also to be shared by the formamidines.
Since aspirin and indomethacin are progressive, irreversible inhibitors of PG
synthetase (Gryglewski, 1974), and chlordimeform appears to be reversible and
non-progressive, a direct comparison of !,.« values can be misleading.
However, we feel that inhibition of PG synthesis can provide a reasonable
explanation of the several aspirin-like pharmacological actions of the
formamidines.
Our present results are of interest from a number of aspects: (i) PG
synthetase inhibitors with the general structural characteristics of the
formamidines have not been previously reported. Most known inhibitors are
acids rather than bases (Robinson and Vane, 1974). Further, the ability of
pesticides to interfere with prostaglandin-dependent processes has not
apparently been the subject of previous study, (ii) Early death from single
64
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high doses of the formamidines has been attributed to direct depression
myocardial and vascular muscle and accompanying respiratory depression, which
are local anesthetic-like actions. It is intriguing to note in this regard
the recent report that PG can antagonize the block of nerve conduction induced
with local anesthetics, and that the PG synthetase inhibitor indomethacin also
caused a conduction block which could be reversed by the addition of PGE..
(Horrobin et al_., 1977). Thus there may be a connection between the PG
synthetase inhibiting and local anesthetic actions of the formamidines. (iii)
Finally, the gastric ulcers, intestinal hemmorhaging, and death in 2-5 days
observed in animals treated with indomethacin and other non-steroidal
TABLE 13. BEHAVIORAL AND PHARMACOLOGICAL PROFILE OF COM IN THE RAT
Dose (mg/kg, ip)
Effects 1-20 21-40 41-60 61-80 81-100 200
HYPERPHAGIA +++ +
HYPERGLYCEMIA + -M- +-M-
ANTIPYRESIS + -M-
ANALGESIA + -M-
ANTIINFLAMMATORY + -H- -M-+
ANOREXIA +
STRAUB TAIL -
HYPERTONICITY + ++
HIND LIMB ABDUCTION + -H-
HYPERREACTIVITY + + -n- +++ +++ +-M-
INCREASED MOTOR ACTIVITY + + +++++ +++
CIRCLING - - + ++ +-M-
BACKING
TREMOR
HEAD WEAVING
SPASTICITY-HYPERREFLEXIA
CONVULSIONS
RESPIRATORY ARREST - -M-+
CARDIAC ARREST
ELEVATED BRAIN 5-HT
ELEVATED BRAIN NE
DEATH
Effects are rated subjectively according to intensity: + = present (low
intensity); ++ = moderate intensity; +++ = marked intensity; - = not present;
empty space = not known. The effects produced by CDM are arranged in
decending rank order of appearance over the dose range indicated.
65
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anti-inflammatory agents has been attributed to the inhibition of PG synthesis
(Robert, 1976). This raises the question of whether the formamidines also may
initiate these toxic consequences of the inhibition of PG synthesis.
Preliminary studies have shown that COM in fact is much less potent than
aspirin in inducing gastric ulcers. This may be attributable to the
reversibility of the inhibition of PG synthesis by CDM (Yanagi and Komatsu,
1/T6) and the fact that it is a base rather than an acid like aspirin (Brune
e;t ^L., 1976). Thus, CDM and related formamidines comprise a new and
interesting class of nonsteroidal anti-inflammatory agents which lack some of
the undesirable side-effects of current compounds.
A summary of the behavioral and pharmacological actions of CDM in the rat
as observed in the studies reported in Sections 2 through 6, and in other
studies in our laboratories, is provided in Table 13.
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SECTION SEVEN
BIOTRANSFORMATION OF CHLORDIMEFORH AND ITS RELATION TO TOXICITY
INTRODUCTION
Clearance and metabolism of chlordimeform in mammals.
The metabolism of COM and some of its intermediate metabolites has been
investigated in dogs and goats (Sen Gupta and Knowles, 1970), rats (Knowles
and Sen Gupta, 1970; Ahmad and Knowles, 1971a, 1971b; Morikawa et _al., 1975;
Benezet and Knowles, 1976b) and mice (Knowles and Benezet, 1977). In each
case, when CDM labelled with C in the ring methyl was orally administered,
it was readily metabolized and rapidly eliminated in the first 24 hours,
primarily in the urine. About 85% of the C-CDM equivalents were eliminated
in the rat urine (Knowles and Sen Gupta, 1970), while 70-80% was eliminated
in the dog urine in the first 24 hours. Slightly less than 5% of the
administered dose was accounted for in the bile of dogs with biliary cannulas
in 72 hours (Sen Gupta and Knowles, 1970). The clearance of CDM was also
rapid in goats. Excretion was faster in males, since 80% of the administered
dose was eliminated in the urine of male goats compared to only 65%
eliminated in the urine of lactating female goats in the same experimental
period of 24 hours. The elimination of chlordimeform in feces after an oral
dose is considered a minor route, since it accounted for only 7.556, 0.6% and
1.8% of the administered dose in 72 hours in rats, dogs and goats
respectively.
The pathway of excretion seems to be dependant on the^route of
administration since, after ip administration to mice of C-CDM, 39% of the
administered radiocarbon was eliminated in the urine and 45% in the feces in
24 hours (Knowles and Benezet, 1977), compared to about 81% recovered in the
urine of mice in the same experimental period when C-CDM was given orally
(Ghali and Hollingworth, unpublished).
The elimination of two chlordimeform metabolites after oral
administration was also rapid, but the relative^importance of the routes of
excretion were changed compared to CDM. When C-labeled DCDM or CT were
orally administered to rats, 35% and 71% of the radiocarbon from the two
compounds were eliminated in the urine and 64% and 24% were eliminated in the
feces respectively in 24 hours (Knowles and Sen Gupta, 1970; Ahmad and
Knowles, 1971 a).
In all of these studies, the majority of the radioactive materials in
urine were unidentified water soluble materials, most likely glucuronide and
sulfate conjugates, which yielded to arylsulfatase/g-glucuronidase cleavage.
67
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No individual conjugates have been identified, but some of the radiocarbon-
containing aglycones were identified after deconjugation as COM, DCDM, NFT,
and CT, in addition to other unidentified materials (Sen Gupta and Knowles,
1970). The organosoluble metabolites in the urine account only for 10-25J of
the total radiocarbon. This relatively wide range of variation in the amount
of organosoluble metabolites indicates a difference in the rates of
degradation of COM by different mammalian species. The major organosoluble
ratabolites recovered were NFT, CT and N^fortnyl-5-chloroanthranilic acid
(NFA). In addition to these major metabolites, others such as DCDM, DDCDM,
and 5-chloroanthranilic acid (CAA) were also present in all mammalian species
studied. Recently, Knowles and Benezet (1977) added three urea derivatives
to this list of metabolites, i.e. 1, l-dimethyl-S-CJ-chloro-o^tolyDurea
(CDU), 1-methyl-3-(M-chloro-£-tolyl)urea (DCDU) and U-chloro-c>-tolylurea
(DDCDU).
Iri vitro studies using various hepatic cellular subfractions have
indicated that the maximum activity in degrading CDM in rats was associated
with the microsomal subfraction (Ahmad and Knowles, 1971a; Morikawa et al.,
1975). The metabolites detected in vitro were well correlated qualitatively
with those found in vivo. With the elucidation of the molecular structure of
these metabolites, the sites of enzymatic attack on the formamidine molecule
can be tentatively established and a metabolic pathway (Fig. 27) has been
postulated (Ahmad and Knowles, 1971a; Morikawa jit al^., 1975; Knowles and
Benezet, 1977).
The initial metabolism of CDM is thought to be catalyzed exclusively by
mixed function oxidases (MFC). One major pathway involves successive
N-demethylations to yield DCDM and DDCDM. The initial Nkdemethylation
reaction is NADPH and 0 dependent, inhibited by SKF 525-A, and therefore is
probably catalyzed by microsomal MFC (Ahmad and Kowles, 1971a). However, it
is not known whether the second Nj-demethylation reaction is catalyzed by the
same enzymes. Oxidative iN-demethylation usually proceeds through an
N-hydroxymethyl intermediate (McMahon, 1966). This intermediate, however,
was not isolated with CDM in vitro (Ahmad and Knowles, 1971a; Morikawa eit
.al.. 1975).
Both of the J^-demethylated metabolites (DCDM and DDCDM), as well as CDM
itself, can be cleaved to NFT in a reaction which overall appears to be a
hydrolysis. The mechanism of this cleavage is an unresolved issue. Ahmed
and Knowles (1971 a) suggested a nonenzymatic hydrolytic mechanism for
formamidine cleavage. Morikawa jet al. (1975), although not entirely
excluding this possibility, provided evidence that this reaction is probably
mediated by an oxidative mechanism. This conclusion was based on the fact
that production of the .N-formyl derivative in hepatic microsomal incubation
was significantly dependent on the concentration of NADPH, and was inhibited
by SKF 525-A, a known inhibitor of microsomal drug oxidation. However, since
the N-formyl derivative can be produced non-enzymatically even more readily
from the two JY-demethylated derivatives than from CDM itself, and since the
_N-demethylation reaction is also impaired by microsomal inhibition, then the
decrease in the rate of production of the J^-formyl derivative might also be
an indirect result of the decrease in the rate of NU-demethylation.
68
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cm J>-N=C-N
CH3
-a
X
X
COM
V
CH
N.
DC DM
N=C-N
CH3
DDCDM
NFA
Cl
Cl
V 9 /CH3
N-C-N,
CDU
? 9 XCH3
N-C-NX
H
OCOU
9
H
x.
CH3
DDCDU
CH3
CT
2
COOH
CAA
Figure 27. Metabolic map showing fate of chlordimeform in mammals.
69
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NFT can be deformylated to CT. This reaction is inhibited by the
organophosphate DFP, but not by SKF 525-A, and is probably carried out by the
soluble liver formamidase which normally catalyses the deformylation of
N^formylkynurenine, and is inhibited by DFP (Mehler and Knox, 1950; Shinohara
and Ishiguro, 1970; Ahmad and Knowles, 1971b). This enzyme was also capable
of deformylating the corresponding anthranilic acid (NFA) to CAA, a common
metabolite from CDM.
CDU, one of the three urea metabolites recently reported by Knowles and
Benezet (1977), may be formed by hydroxylation of the central (amidine)
carbon of CDM followed by an enol-keto rearrangement. The other two urea
metabolites could be formed by a similar mechanism from their respective
formamidines or by sequential oxidative NN-demethylation of CDU. Oxidative
Nj-dealkylation is known to be an important pathway in the mammalian
metabolism of substituted phenylurea herbicides (Geissbuhler «it al^., 1975).
CT could also be produced by a direct cleavage of the ureas. Direct cleavage
of substituted phenylureas in mammals to yield substituted anilines has been
postulated but apparently is not a major pathway (Geissbuhler €it a±., 1975).
Therefore, it seems likely that most of the 4-chloro-£-toluidine is derived
by deformylation of NFT. In addition to the production of CT and NFT, which
with their conjugates, are major terminal metabolites, an additional reaction
has been observed. This involves oxidation of the ring methyl moiety
yielding various anthranilic acids (Knowles and Benezet, 1977). It is not
known at what stage of metabolism the ring-methyl group is oxidized, but
ring-methyl oxidation products (alcohol, aldehyde, or acid) with an intact
formamidine group have not been reported.
CDM is not a substrate for mammalian hepatic glutathione transferases
(Ghali, G. and Hollingworth, R. M., unpublished). No in vitro studies of the
conjugation of CDM or its metabolites by glucuronyl transferases,
sulfotransferases, or other conjugating enzymes have been reported, although
from the results described on the aglycones present after urinary
deconjugation, direct conjugation is a possible reaction with formamidines
(Sen Gupta and Knowles, 1970).
MATERIALS AND METHODS
Synthesis of chlordimeform and its major metabolites
Chlordimeform (CDM) and its ^-demethylated metabolites, DCDM and DDCDM,
were synthesized by coupling 4-chloro-o-toluidine with N^-dimethylformamide,
N-methylformamide, or formamide in the presence of phosphorous oxychloride
(Hollingworth, 1976).
In a typical synthesis of chlordimeform, (J.JY-dimethylformamide (19 g,
0.260 moles) in 25 ml of dry benzene was added to phosphorous oxychloride
(12 g, 0.078 moles) in 20 ml of dry benzene in a 250 ml three-neck flask
attached to a water condenser. The mixture was stirred at room temperature
for 30 min, then 4-chloro-o-toluidine (7.4 g, 0.052 moles) in 30 ml of dry
benzene was added at room temperature with stirring for a further 3 hr.
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In the case of DCDM and DDCDM, the order of addition of reactants was
changed in order to avoid the accumulation of a sticky intermediate complex
i.e., the formamide and the toluidine were added simultaneously to the
phosphorous oxychloride in the flask. Furthermore, the formamide was
dissolved in acetonitrile and the stirring continued for only one hr at room
temperature.
In the case of CDM and DCDM, the precipitate from this reaction (crude
hydrochloride salt) was collected by filtration and treated with 250 ml of
ice-cold 1 N sodium hydroxide under cooling and continuous stirring for about
10 min. The oily layer was then extracted with 100 ml of benzene. The
benzene layer was washed with water and dried over anhydrousj.sodium sulfate.
The compound was either redistilled (CDM; bQ _ 103-108°, nD 1.5918), or
recrystallized (CDM, DCDM, and DDCDM) several times until purity was evident
by thin layer chromatography (Whatman K5F silica gel, developed with
benzene:diethylamine, 95:5) and melting point (Fisher-Johns hot plate). CDM
and DCDM were recrystallized from benzene while DDCDM was recrystallized from
ether by the gradual addition of hexane.
In the case of DDCDM, which is more easily hydrolyzed under basic
conditions, the precipitate was taken into a separatory funnel which
contained prechilled ether and an equal volume of ice-cold 0.5 N sodium
hydroxide, and shaken continuously for 2-3 min. The ether phase was then
separated, washed several times with water, and dried over anhydrous sodium
sulfate. The compound was recrystallized several times until purity was
evident as before. All three of these formamidines as the free base gave
colorless cystals with melting points of 30-31°, 91-92°. and 88°
respectively. The hydrochloride salts of these formamidines were made by
passing HC1 gas into their ethereal solutions. The salts were recrystallized
from ethanol-acetone (1:1) giving water soluble white crystals with melting
points of 221-223°, 192-194° and 220-221° for CDM, DCDM and DDCDM
respectively.
The formamidine metabolite, Nkformyl-4-chloro-<>-toluidine (NFT), was
synthesized by refluxing 4-chloro-o-toluidine with an excess of formic acid
in benzene with concomitant removal of the water produced (Fieser and Jones,
1955). After removing the solvent, the product was recrystallized from 1:1
ethanol-acetone mixture producing colorless crystals with a sharp melting
point at 120°C.
The urea metabolite, 4-chloro-o-tolylurea (DDCDU). was synthesized by
the reaction of equimolar potassium cyanate and 4-chloro-o-toluidine in
acetonitrile (Geissbuhler jt _al. 1975) at room temperature. The urea
produced in this reaction precipitated as a highly insoluble product in good
yield. The product was purified by washing the precipitate several times
with acetonitrile and recrystallization from dimethylsulfoxide. The material
so obtained was a white powder (m. 204 ).
1-Methyl-3-(4-chloro-o-tolyl)urea (DCDU) was synthesized by the reaction
of equimolar amounts of 4-cihloro-o-toluidine and methyl isocyanate in
benzene. The isocyanate was added to the toluidine with stirring at 40°.
71
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The precipitate was filtered off and washed with water and then with
acetonitrile and recrystallized from dimethylsulfoxide (m. 213°).
The structures of these compounds were confirmed by elemental analysis,
and infrared, nuclear magnetic resonance, and g.c.- mass spectroscopy.
4-Chloro-£-toluidine was purified from the commercial material (Aldrich
ChCiflical Co.) by vacuum distillation. A further metabolite,
j-chloroanthranilic acid, was obtained from Ciba-Geigy Agrochemicals,
Greensboro, NC.
14
Chlordimeform labeled with C in the ring methyl group with specific
activity of 20 UCi per mg (4 mCi per mmole) and a radiochemical purity of
96-99/1 (major impurity^T, formed on storage) was obtained from Ciba-Geigy
Agrochemicals. Other^ C-labeled metabolites were biosynthesized by
incubating 20 yCi of C-chlordimeform for 20 min with the microsomal
fraction of mouse liver and NADPH (1.7 mM) in Tris buffer (pH 7.6) at 37°,
and subsequent extraction of the metabolites with chloroform, and
purification by preparative two-dimensional TLC (Knowles and Benezet, 1977).
Compounds were eluted from the TLC plates with acetone.
Effect of mixed function oxidase inducers and inhibitors on the acute
toxicity of CDM and its N-demethylated metabolites
The mice were pretreated with either of two hepatic mixed function
oxidase (MFO) inhibitors or with one of three MFC inducers. The inhibitors
SKF 525-A (in 0.9% NaCl) and piperonyl butoxide (in corn oil) were
administered ip at the rate of 50 and 400 mg/kg respectively two hours prior
to subsequent treatment with graded oral doses of CDM, DCDM, or DDCDM as
described before. In the case of the inducers, phenobarbital (in saline
solution) was administered ip at four successive daily doses of 50 mg/kg
prior to assay on day five. Aroclor 1254 (in corn oil), a polychlorinated
biphenyl mixture, was given once at 500 mg/kg ip five days prior to
treatment, and 3-methylcholanthrene (in corn oil) was administered ip in two
successive daily doses of 40 mg/kg with the second dose two days prior to the
toxicity tests. Control animals were pretreated with the vehicle alone.
LD,. 's were determined as described in Section 1.
Subcellular fractionations
Animals were killed by a blow to the head. The desired tissues such as
livers, brains, and lungs were rapidly removed and washed with cold 0.25 M
sucrose solution. The tissues were then weighed, minced, and homogenized in
fresh prechilled sucrose at the rate of 5 ml/g tissue using a Potter-Elvehjem
type glass homogenizer fitted with a teflon pestle. The system was kept ice
cold throughout. The microsomal and mitochondrial fractions were isolated by
differential centrifugation, initially 600 x g for 10 min, then the
supernatant was spun down at 12,000 x g for another 10 min to isolate the
mitochondrial fraction, and then by further centrifugation of the supernatant
at 105,000 x g for one hr using a Beckman L2-65 ultracentrifuge to separate
theomicrosomal fraction. The resulting microsomal pellet was kept frozen at
-20 for a maximum of four days before determination of the MFO activities.
72
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The supernatant from the 105,000 g spin was used as the source of
soluble enzymes. It and the mitochondrial fraction was used within two hours
of preparation. Hepatic microsomes were also prepared from mice pretreated
with MFO inducers or inhibitors as already described.
Metabolism of chlordimeform in vitro
Microsomal pellets (or mitochondrial fractions) of hepatic, brain, orQ
lung tissues were resuspended in ice-cold 0.05 M Tris buffer (pH 7.6 at 37 )
to a final concentration corresponding to 5 g of liver and 20 g of brain or
lung tissues per 100 ml. Soluble fraction of liver was diluted 5 times with
buffer. Rates of metabolism were determinedj.at.j37 in an incubating shaker
in open 50 x 5 mm tubes containing 4.2 x 10~ M C-labeled substrate (CDM,
DCDM, or DDCDM), 10 yl 10"^M NADPH in the Tris buffer, and 50 yl of
microsomal (or raitochondrial) suspension or liver soluble fraction. The
final incubation volume was 60 yl. The substrate was added as a solution in
hexane and the hexane was carefully evaporated before addition of the other
components. Five yl samples were taken from the incubating mixture at
several different times over 10 min after the addition of enzyme, spotted on
silica gel TLC plates (Whatman K5F, 250 y) along with the chromatographic
standards in some runs, and developed in a two dimensional system modified
from Knowles and Benezet (1977). The first system consisted of
benzene:acetonerdiethylamine (95:5:5). while the second system consisted of
benzene:dioxane:acetic acid (90:10:1). Individual metabolite zones were
located by autoradiography on X-ray film and scraped for scintillation
counting in a toluene base scintillation cocktail containing 0.4J
2,5-diphenyloxazole and 4$ Cab-0-Sil powder. The nature of the metabolites
was confirmed by cochromatography in the system above and by mass
spectrometry in some cases.
To study the effect of the absence of molecular oxygen on the microsomal
metabolism of CDM, a modified Warburg vessel with two gas valves was used.
The enzyme and NADPH were placed in one compartment while the substrate was
placed in the other. A source of nitrogen was connected to the gas inlet
while both valves were open. The nitrogen was allowed to pass for 10 min
before the valves were closed. This method gave equivalent results to a
second one in which the flask was alternately evacuated and then flushed with
N_. However, the latter method resulted in foaming and possible enzyme
denaturation. The vessel was warmed to 37° and the enzyme and NADPH were
admitted to the substrate compartment and mixed well before incubation for 10
min in a shaking water bath at 37°. The reaction was stopped by dropping the
vessel in a cooling mixture (-20°).
The effect of carbon monoxide on the microsomal metabolism of CDM was
also studied by bubbling CO gas for 5 to 10 min at a rate which did not cause
foaming into a mixture of the enzyme and NADPH before adding them to the
substrate in the usual incubation tubes.
Metabolism of chlordimeform in vivo
14
Male Swiss white mice (23-25 g) were orally dosed with C-chlordimeform
after pretreatment with piperonyl butoxide, phenobarbital,
73
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3-methylcholanthrene, or vehicle as previously described. Four mice were
used for each treatment and each mouse was given 10 yCi of the toxicant (1:1
mixture of labelled and unabelled CDM) in 0.25 ml corn oil. This dose of COM
amounted to 40 mg/kg. After 40 min, the animals were sacrificed. Blood was
collected in heparinized vials. Livers and brains were rapidly removed,
placed in preweighed scintillation vials, and immediately frozen in an
acetone/dry ice bath and stored at -20° for analysis. Total tissue
•adioactivity was assayed using a 50 mg sample of well-macerated tissue in a
scintillation vial. The tissue was digested overnight with 0.5 ml of
Soluene-100 (Packard Instrument Co., Downers Grove, ID at room temperature,
bleached with 0.5 ml 30% hydrogen peroxide, and then treated with 0.2 ml
glacial acetic acid to overcome chemiluminescence. Scintillation cocktail
(10 ml, Aquascint 1, ICN) was added to the vials which were counted in a
scintillation spectrometer (Packard Tri-Carb 3310). Efficiency of counting
was determined with C-toluene internal standard.
To study the nature and magnitude of the individual metabolites, about
200-400 mg of each tissue was weighed and homogenized in 2 ml 2% HC1, 0.5 ml
10% trichloroacetic acid (TCA) was added, and the mixture was centrifuged for
30 min at 3,000 rpm. The supernatant was transferred to another tube and the
pellet re-extracted with another 2 ml of 2% HC1 followed by TCA and
centrifugation as before. The two supernatants were combined, treated with
another 0.5 ml of 10% TCA and spun down for a further 30 min. The clear
acidic extract was then transferred to a larger tube, the pH was carefully
adjusted to 9.0 using 1 N NaOH with rapid mixing, and the solution was
extracted three times with prechilled ether- The combined ether
extractswere dried over anhydrous sodium sulfate, and evaporated to near
dryness using a gentle stream of nitrogen under cooling conditions to
minimize evaporative losses of volatile components. The residual tissue
material from the acidic extractions was further extracted with two portions
of ether to remove any residual NFT. These two ether extracts were combined,
dried over anhydrous sodium sulfate, and evaporated to near dryness as
before. The ethereal extracts were analyzed by two-dimensional
TLC/scintillation counting as already described. The nature of the
metabolites was determined initially by cochromatography on TLC.
Rates of hydrolysis of chlordimeform and its N-demethylated metabolites
The rates of hydrolysis of CDM, DCDM, and DDCDM were investigated at pH
7.4 and pH 8.7. A 10 mM stock solution of each compound was made in
methanol. One ml of each of these solutions was placed in a 12 ml graduated
tube with glass stopper and made to 10 ml using 0.01 M Tris buffer which was
prewarmed to 37°. A 5 yl sample (zero time) was taken right after mixing and
before placing the tube in a shaking water bath at 37°. Samples (5 ul) were
taken at 5 min intervals over the first 30 min then less frequently for 3 hr
and every 24 hr until the reaction was completed.
The amount of the hydrolysis product, N_-fortnyl-4-chloro-o-toluidine
(NFT), was monitored by injecting the samples immediately into a Water
Associates Liquid Chromatograph equipped with a 30 cm x 4 mm i.d. Micron
Bondapak C-18 column, UV absorbance detector (Model 440) of a fixed
wavelength (245 nm), and a Water Associates Pump Model M-6000A. An
74
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attenuation unit full scale of 0.05 to 0.1, a flow rate of 2 ml per minute,
and a pressure of 2000 psi were used. The mobile phase was a mixture of
acetonitrile:water:methanol (45:45:10). All solvents were "L.C. Distilled in
Glass" grade. Retention time for the hydrolysis product under these
conditions was 3.5 min. Retention times for the parent formamidines were
much longer (> 20 min). The experiment was duplicated for each compound at
each pH and the results averaged. The pH of the reactions was monitored and
was steady throughout the experiment. In order to ensure that the reaction
had reached completion, the tubes were boiled for 30 min at the end of the
reaction and the amount of the hydrolysis product was checked before and
after boiling. However, no change in the amount of the hydrolysis product
was observed.
The NFT peak heights were measured at each time interval and a first
order plot relating time to log[(ha - h )/(ha - h )] was made, where ha is
the final peak height of NFT at ta and 80 is the peak height at t , and h is
the peak height at a given time, t. The pseudo first order rate constant (k)
was calculated from the equation, k = slope x 2.303, and the half life (
of each compound was calculated from the equation, t1/2 = 0.693/k.
RESULTS
Toxicity and symptoms in mice after oral exposure to CDH and its metabolites
Data regarding the acute toxicity of COM and its major metabolites have
already been shown in Table 1. Additional studies with two of the more minor
urea metabolites (DCDU and DDCDU) indicated LD,.. values over 500 mg/kg and an
absence of the rapid excitatory symptoms typical of the three formamidines.
The symptoms elicited by lethal doses of CDM and the two jf-demethylated
metabolities, although not identical, are very similar. These compounds are
rapidly acting and symptoms are manifested as restlessness, hyperreflexia,
tremors, particularly of the head and forelimbs, developing to one or more
episodes of clonic convulsions. Death occurs generally within the first hour
during one of these convulsive episodes and is marked by labored breathing
and gasping. The tremors are not seen in the case of animals poisoned with
the NUdemethylated derivatives and the convulsive stage is significantly
longer. The CDM-treated animals usually show signs of locomotor difficulty
partly due to frequent hyperextension of the hind legs a later stages of
poisoning, but this was not seen with DCDM and DDCDM.
Since N_-demethylations appear to be the only metabolic reactions leading
to more acutely toxic products, and these are most likely catalyzed by
microsomal mixed function oxidases (MFO), the effect of ip pretreatment with
inhibitors or inducers of hepatic MFO systems on the toxicity of CDM was
studied. The results are shown in Table 14. Surprisingly, the MFO
inhibitors (SKF 525-A and piperonyl butoxide) had no effect on the toxicity
of CDM. Neither did the MFO inducer, phenobarbital. The other two inducers,
3-methylcholanthrene and Aroclor 125M had the even more paradoxical effect of
decreasing the toxicity of CDM about 2-fold. The same trends were also seen
in the effect of some of these pretreatments on the toxicity of DCDM and
DDCDM. 3-Methylcholanthrene had a clear protective effect, approximately
75
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doubling the LD_Q for both DCDM and DDCDM. Aroclor 1254 was tested only with
DCDM but had a comparable protective action. Piperonyl butoxide pretreatment
again did not have any effect on the toxicity of DCDM or DDCDM.
TABLE 14. THE EFFECT OF SEVERAL PRETREATMENTS AFFECTING MICROSOMAL OXIDATIONS
ON THE TOXICITY OF FORMAMIDINES TO MICE
LD5 (±95? Conf. Intervals), mg/kg
Pretreatment Formamidine Control Pretreated
Piperonyl butoxide
SKF 525-A
Phenobarbital
Aroclor 1254
3-Methylcholanthrene
Piperonyl butoxide
Aroclor 1254
3-Methylcholanthrene
Piperonyl butoxide
3-Methylcholanthrene
COM
COM
COM
COM
COM
DCDM
DCDM
DCDM
DDCDM
DDCDM
233(183-295)
290(223-377)
250(225-279)
295(268-324)
250(223-280)
185(162-209)
167(145-199)
167(145-199)
76(61-95)
100(81-123)
260(206-327)
280(215-364)
266(229-309)
510(425-612)
410(360-462)
190(168-214)
350(273-447)
310(258-371)
84(67-105)
170(132-210)
Non-enzymatic hydrolysis of chlordimeform and its N—demethylated metabolites.
Since it has been suggested that much of the NFT produced from these
formamidires arises by non-enzymatic hydrolysis, and because this hydrolysis
is a clear detoxication reaction (although potentially leading to
mutagenic/carcinogenic anilines), we measured the rates of hydrolysis of CDM,
DCDM, and DDCDM. As shown in Fig. 28 plots of log C(ha - h )/(ha - h )]
against time were linear, indicating a pseudo first order rate of hydrolysis
for each compound. First order kinetics were observed over at least 60% of
the total reaction. The rate constants (k) and half lives (t^) calculated
from these plots at 37° and either pH 7.4 or 8.7 are given in Table 15.
Under 'physiological conditions' i.e. pH 7.4 and 37°, the formamidines
are relatively stable with half lives ranging from 161 min for DDCDM to 538
min for CDM. In each case, raising the pH to 8.7 approximately triples the
rate of hydrolysis. The hydrolysis product in each case, NFT, could also be
hydrolyzed further to CT. However, under the reaction conditions and times
employed here, NFT was stable i.e. no hydrolysis was detected in 12 hr at pH
8.7.
Metabolism of formamidines by mouse enzymes in vitro
In order to determine which tissue(s) and subcellular fraction(s) were
most active in degrading CDM, a preliminary study was conducted with mouse
liver, brain, kidney, heart, skeletal muscle, and lung homogenates fortified
76
-------
0.10
.__CDM
DCDM
o>
o
. 0.03.,
0.02.
0.01 ..
0.00
10 20 30 40 50 60 70 80 90 100 110 120 130
Time ( min )
Figure 28. Rates of hydrolysis of chlordimeform and its N-demethylated
metabolites at pH 7.4 and 37 .
77
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TABLE 15. RATE CONSTANTS AND HALF LIVES FOR THE HYDROLYSIS OF CHLORDIMEFORM AND ITS _N-DEMETHYLATED
METABOLITES.
CDM DCDM DDCDM
pH 7.4 pH 8.7 pH 7.4 pH 8.7 pH 7.4 pH 8.7
Rate constant (k, min~~ )
Half life (t1/2, min)
1.29 x 10~3
538
3.10 x 10~3
. 224
2.78 x 10~3
250
7.80 x 10"3
88.8
4.33 x 10~3
161
1.25 x 10~2
55.2
00
-------
with NADPH (1 mM) and GSH (5 mM). These, and all other data for metabolism .in
vitro were corrected for the slight spontaneous breakdown of formamidines to
NFT, as measured by blanks run concurrently with the appropriate enzyme
preparation denatured by boiling. Significant metabolism (loss of COM greater
than 5% in 20 rain) was seen only with the liver homogenate. Further studies
with subfractions from these tissues were conducted and the results are shown
in Table 16. Of the liver subfractions, CDM-metabolizing activity was
confined almost exclusively to the microsomal fraction. It is significant
that no detectable metabolism of CDM was seen with brain microsomes. The lung
and liver microsomal fraction was virtually inactive in degrading CDM even
when prepared from mice pretreated with the MFO inducers, phenobarbital and
3-methylcholanthrene. Since the liver microsomal system is so predominant in
the biotransformation of CDM, further metabolic studies in vitro were confined
to this fraction.
TABLE 16. RELATIVE ACTIVITIES OF MOUSE TISSUE MICROSOMES AND OTHER CELLULAR
SUBFRACTIONS IN DEGRADING CHLORDIMEFORM.
g
Cellular subfraction % of added CDM recovered unchanged
Liver
Microsomes 62.9
Mitochondria 98.2
Soluble 98.7
Brain
Microsomes 99.1
Lung
Microsomes 98.2
Control
Buffer 99.4
Means of two independent experiments with two replicates each. Incubation
time: 10 min.
The nature of the microsomal enzymes attacking CDM was investigated, with
the results given in Table 17. In the absence of NADPH, the rate of CDM
degradation was reduced virtually to zero. Replacement of 0_ by Np also
greatly decreased the rate of metabolism (80% inhibition). Even in the
presence of atmospheric 0_ pretreatment of the microsomal suspension with CO
gas inhibited the degradation of CDM by about 75%.
Comparative metabolism of formamidines by mouse liver microsomes
The rates and pathways of microsomal metabolism of CDM, DCDM, and DDCDM
were compared at several times over 20 min. The data presented in Table 18
for 10 min incubations are typical. In addition to the parent compound (CDM).
five other types of metabolites were assayed; DCDM, DDCDM, NFT, CT and a
'Polar1 fraction. This last fraction stayed on or near the origin in the
79
-------
1U
TABLE 17. METABOLITES FORMED IN REACTION OF C-CHLORDIMEFORM WITH MOUSE
LIVER MICROSOMES.
Reaction Conditions % of Substrate recovered asa;
Enzyme NADPH Gas CDM DC DM DDCDM NFT
+ Air
+ - Air
+ + N
+ CO
62.3
98.4
91.5
89.3
26.8
0.0
3.2
7.6
1.1
0.0
0.1
0.2
2.4
0.4
0.2
0.6
Means of two independent experiments with two replicates each. Incubation
Ortime: 10 min.
2-dimensional TLC system employed. The nature of the constituents was not
determined, but rechromatography with a more polar developing system showed at
least 7 components. The nature of the major metabolities (CDM, DCDM, DDCDM and
NFT) was confirmed by GC-MS Analysis. Rather small amounts of the urea
derivatives (CDU, DCDU, and DDCDU) were detected (less than 5%). Since these
compounds are neither highly toxic, nor major metabolites they were not
quantified further.
Liver microsomes degraded the three formamidines in the order
DDCDM>DCDM>CDM. DDCDM was very readily metabolized since only 1556 of the added
DDCDM was recovered intact after 10 min, and 60$ was hydrolyzed to NFT, whereas
63? of the CDM added was recovered unchanged. Only small amounts of DDCDM were
recovered starting from CDM and DCDM, although this may reflect the instability
of DDCDM as much as a slow rate of synthesis. When CDM was the substrate, the
major metabolite (27?) was DCDM with relatively small amounts of the other
metabolites. Thus the first N[-demethylation (CDM to DCDM) occurs readily. The
rate of the second N-demethylation (DCDM to DDCDM) is hard to evaluate because
of rapid further degradation of DDCDM, but it is toxicologically significant
that DDCDM does not accumulate to any high level.
Effect of manipulations of MFO activity on the in vitro metabolism of
chlordimeform.
Intraperitoneal pretreatments of mice with inducers and inhibitors of MFO
considerably affected the rate and type of microsomal metabolism of CDM iji
vitro as shown in Table 19. Despite their lack of effect on toxicity, the MFO
inhibitors, SKF 525-A and piperonyl butoxide, decreased both the rate of
destruction of CDM and the rate of accumulation of the apparently more toxic
metabolites DCDM and DDCDM. Phenobarbital, as expected, showed the reverse
effect, causing a much more rapid loss of CDM and a considerably greater
accumulation of the two N-demethylation products.
3-Methylcholanthrene and Aroclor 1254 were even more effective inducers
as judged by the overall loss of CDM. However, in contrast to the other
compounds, these two inducers also considerably increased the rate of
production of NFT as well as the rate of jY-demethylation. Compared to the
controls, more of both DCDM and DDCDM accumulated after 3-methylcholanthrene
80
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TABLE 18. IN VITRO METABOLISM OF CHLORDIMEFORM AND ITS J4-DEMETHYLATED METABOLITES BY MOUSE LIVER
MICROSOMES.
Percentage of substrate recovered as :
Substrate
CDM
DCDM
DDCDM
CDM DCDM DDCDM
62.72 + 1.58 26.88 +1.10 1.02 + 0.30
49.92 + 2.20 1.83 + 1.50
15.17 + 0.15
NFT
4.22 + 0.70
15.26 -i- 1.36
60.04 + 1.40
CT
1.10 + 0.30
7.43 + 0.40
9.23 + 0.60
Polar
2.33 + 0.70
12.90 + 1.35
10.66 + 0.90
00
Mean (+SD) of two independent experiments with two replicates each. Incubation time: 10 min.
-------
TABLE 19. EFFECT OF SEVERAL PRETREATMENTS AFFECTING MICROSOMAL OXIDATIONS ON THE _IN VIThO METABOLISM OF
CHLORDIMEFORM BY MOUSE LIVER MICROSOMES.
Metabolites, percent as :
00
Pretreatment
Control
Phenobarbital
3-Methylcholanthrene
Aroclor 1254
Piperonyl butoxide
SKF 525-A
62.72
40.04
32.78
19.99
75.03
70.81
CDM
+ 1.53
+ 1.06
+ 2.18
+ 0.46
+ 6.20
+ 2.05
DCDM
26.88
44.86
37.37
54.26
16.86
19.18
+ 1.10
+ 0.12
+ 4.00
+ 0.50
•i- 4.71
-i- 0.16
DDCDM
1.02 + 0.30
4.09 + 0.37
1.40 + 0.17
7.69 + 0.30
0.61 + 0.14
0.67 + 0.03
NFT
4.22 -»-
5.85 +
16.18 +
8.54 +
3.93 +
3.76 +
0.70
0.07
1.33
0.38
0.40
0.01
CT
1.10 +
1.06 +
1.56 +
1.62 +
0.93 +
1.60 +
0.29
0.33
0.04
0.18
0.28
0.01
2.33
3.18
9.59
5.35
1.50
1.54
Polar
+ 0.69
+ 0.33
+ 0.33
+ 0.21
+ 0.57
+ 1.10
a
Means of two independent experiment with two replicates in each. Control is the average of ten
independent experiments with two replicates in each. Incubation time: 10 min.
-------
and Aroclor pretreatments. However, 3-methylcholanthrene caused only a small
rise in the level of DDCDM compared to phenobarbital and Aroclor 1254.
3-Methylcholanthrene was also the most effective compound stimulating the
pathways to NFT and polar metabolites.
Effects of manipulations of MFO activity on in vivo metabolism of
chlordimeform.
The effect of pretreatment with the inducers phenobarbital and
3-methylcholanthrene and the inhibitor piperonyl butoxide on the levels of CDM
metabolites in the liver, blood and brain was determined 40 min after dosage
with CDM. The 40 min interval was chosen since this was the typical time for
the development of severe toxicity symptoms and death at lethal doses of CDM.
Recoveries of added CDM, DCDM, DDCDM, and NFT by this method of extraction
ranged from 80-8556. The data in Tables 21 and 22 are corrected for this level
of recovery. The recovery of CT was not checked and no recovery correction of
the data for this compound was made.
The percentage of the dose present in the tissues after each treatment is
shown in Table 20. For the purposes of calculation the total blood volume of
the mice was taken as 6.0% of their body weight. Liver weight averaged 1.44 g
and brain weight 0.42 g. Relatively large amounts of radioactivity were
present in the liver with much less in the blood and brain.
TABLE 20. TOTAL RADIOACTIVITY IN TISSUES AFTER AN ORAL DOSE OF CHLORDIMEFORM.
Q
Total radioactivity as percent of the dose
Pretreatment Liver Brain Blood
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
11.15 + 0.26
12.53 + 0.55
11.16 + 0.52
6.36 + 0.34
0.717 + 0.071
0.694 + 0.051
0.693 + 0.037
0.352 + 0.051
1.23 + 0.072
1.33 + 0.081
1.37 + 0.110
0.65 + 0.052
aMean ^ SD (n = 3). Time after dosage, 40 min.
Pretreatment with phenobarbital or 3-methylcholanthrene had no effect on
the levels of radioactivity in the tissues. However, piperonyl butoxide
unexpectedly reduced the amount of radiolabel by about 50% in all the tissues.
This effect proved to be reproducible in a replicate run. The levels of the
individual metabolites in these tissues are presented in terms of the relative
percentage they represent of the total radioactivity recovered (Table 21) and
in terms of their absolute levels (Table 22).
The data for the liver in Table 21 show that pretreatment with
phenobarbital or 3-methylcholanthrene hardly affects the percentage of the
radioactivity present as the parent compound, CDM, which averages about 10$.
However, the MFO inhibitor, piperonyl butoxide radically changes this picture,
reducing metabolism so that 56$ of the radioactivity is present as CDM.
Piperonyl butoxide also increases the proportion of DCDM present in the liver
83
-------
but the other treatments have little effect in this regard. However, the
proportion of DDCDM was much reduced by piperonyl butoxide, and much increased
by the inducer pretreatments. NFT and polar metabolites were greatly reduced
by piperonyl butoxide. The most notable change resulting from inducer
pretreatment was the 140% increase in NFT after 3-methylcholanthrene which was
not seen with the other MFO inducer, phenobarbital.
A rather similar picture is presented in the data for the blood and, to a
lesser extent, for brain also. The few notable differences are the low
percentage of DCDM in the blood and brain after 3-methylcholanthrene
treatment, and the high percentage of NFT in the brain compared to the other
tissues. In fact, 45% of the radioactivity in the brain is NFT in the
3-methylcholanthrene-treated animals compared to 18% in the controls. Clearly
3-methylcholanthrene increases NFT levels in the brain at the expense of DCDM.
The same conclusion holds, to a lesser extent, in the blood also. As in the
liver, so in the blood and brain, piperonyl butoxide greatly increases the
proportion of label present as COM, and, to a lesser extend, DCDM, while
radically decreasing the other metabolites. Polar metabolites are less
important in the brain, perhaps because of the blood-brain barrier.
Since the total levels of radioactivity in these tissues were virtually
identical in the control, phenobarbital, and 3-methylcholanthrene
pretreatments (Table 20), the above conclusions also apply when the data are
presented as the percent of the total dose recovered in each form in Table 22.
However, because piperonyl butoxide pretreatment reduced the total
radioactivity in all the tissues, the data presented in Table 22 are
significantly different for this pretreatment. It is these data which should
be related to the observed toxicity in Table 1. Although COM levels are still
quite elevated after piperonyl butoxide, the DCDM concentration is lower than
the control in the blood and brain, and the amount of DDCDM in all tissues is
extremely low. This is true for the other metabolites (NFT, CT and polar
fraction) also.
On the basis of the data in Tables 1 and 22, an attempt was made to
correlate the effects of the several pretreatments on the toxicity of COM with
the levels of formamidines singly or in combination in liver, blood, or brain.
In this comparison, the levels of COM, DCDM, and DDCDM singly or in the
combinations DCDM plus DDCDM, and COM plus DCDM plus DDCDM (total
formamidines) were examined. The only plausible correlations obtained are
shown in Table 23. The effects on the toxicity of CDM are presented as a
ratio of LD (control)/LD (pretreatment). A ratio of greater than 1.0
indicates tnat the pretreatment increased the toxicity of CDM while a value of
less than 1.0 reveals a decrease in toxicity. Thus a direct correlation of
this toxicity ratio with tissue levels of any 'active' compound(s) would be
expected.
84
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TABLE 21 . EFFECT OF SEVERAL PRETREATMENTS AFFECTING MICROSOMAL OXIDATIONS ON THE
DISTRIBUTION OF CHLORDIMEFORM IN MICE.
VIVO METABOLISM AND
—Metabolites as percent of total radioactivity in the liver
Pretreatments
COM
DC DM
DDCDM
NFT
CT
Polar
00
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
10.11+2.79
9.39+1.03
10.79+0.50
56.47+1.47
CDM
7.27+1.03
10.06+0.91
10.25+2.79
61.59+1.98
CDM
6.78+2.03
4.89+0.26
6.92+1.46
33.05+2.70
14.30+1.86
17.88+0.62
14.85+4.11
26.11+1.55
Metabolites as
DCDM
11.34+2.90
8.16+1.69
4.93+0.13
13.15+0.88
Metabolites as
DCDM
27.99+4.65
30.18+0.37
8.84+1.03
32.80+1.95
6.92+0.99
15.66+1.34
13.47+0.47
3.88+0.25
percent of total
DDCDM
4.58+0.91
5.71+0.79
12.36+3.30
1.01+0.07
percent of total
DDCDM
4.37+1.17
5.28+0.44
5. 36+0. 34
1.16+0.11
7.70+1.20
8.58+1.44
18.72+1.58
3.49+1.33
radioactivity
NFT
11.13+0.39
10.82+3.16
19.78+0.47
3.74+1.20
radioactivity
NFT
18.23+0.89
30.33+1.76
45.06+6.32
4.27+1.20
3.26+0.19 30.67+3.97
6.32+0.87 22.17+1.89
2.89+0.51 26.72+2.24
3.09+0.84 4.52+1.34
in the blood3
CT
3.73+0.88
9.92+1.32
3.07+0.81
2.54+0.37
in the brain
CT
6.74+1.19
3.05+1.10
1.72+0.64
3.90+0.79
Polar
36.11+4.50
21.84+2.38
27.27+3.55
4.04+0.35
Polar
13.86+4.20"
12.06+2.91
15.78+5.26
4.62+1.29
aMean (+ SD). Three animals, duplicate assays on each extract.
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TABLE 22. EFFECT OF SEVERAL PRETREATMENTS AFFECTING MICROSOMAL OXIDATIONS IN THE MOUSE ON THE IN VIVO
METABOLISM OF CHLORDIMEFORM.
Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
Pretreatements
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
Pretreatments
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
CDM
1.12+0.28
1.16+0.14
1.20+0.11
3.59+0.11
CDM
0.089+0.015
0.134+0.019
0.141+0.045
0.398+0.022
CDM
0.049+0.
0.034+0.
0.048+0.
0.118+0.
Metabolites found
DCDM
1.59+0.17
2.21+0.11
1.64+0.04
1.66+0.17
Metabolites found
DCDM
0.137+0.030
0.109+0.027
0.066+0.006
0.086+0.011
Metabolites found
DCDM
016 0.202+0.046
002 0.210+0.016
009 0.062+0.003
022 0.116+0.024
in the liver
DDCDM
0.77+0.10
1.93+0.83
1.50+0.04
0.25+0.22
in the blood
DDCDM
0.056+0.008
0.075+0.012
0.167+0.032
0.006+0.001
in the brain
DDCDM
0.031+0.011
0.037+0.005
0.037+0.003
0.004+0.001
as percent of
NFT
0.86+0.12
1.06+0.19
1.86+0.02
0.23+0.10
as percent of
NFT
0.136+0.010
0.140+0.034
0.269+0.026
0.024+0.008
as percent of
NFT
0.130+0.016
0.210+0.004
0.314+0.060
0.015+0.006
total dose
CT
0.36+0.02
0.78+0.11
0.32+0.07
0.20+0.05
total dose
CT
0.046+0.012
0.132+0.020
0.045+0.009
0.016+0.003
total dose
CT
0.048+0.008
0.021+0.009
0.012+0.004
0.014+0.005
Polar
3.42+0.52
2.74+0.24
2.99+0.04
0.29+0.10
Polar
0.410+0.036
0.228+0.220
0.406+0.037
0.025+0.003
Polar
0.098+0.028
0.086+0.019
0.108+0.030
0.016+0.004
Mean (+_SD). Three animals, duplicate assays on each extract.
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TABLE 23. COMPARISON OF THE EFFECT OF PRETREATMENTS ON THE TOXICITY OF CDM TO
MICE AND ON THE LEVELS OF SELECTED FORMAMIDINES IN THEIR TISSUE.
% of dose as :
DCDM in:
Pretreatraent
Toxicity Ratio"
Brain
Blood
Total Formamidines
in Brain
Control
Phenobarbital
3-Methylcholanthrene
Piperonyl butoxide
1.00
0.94
0.61
0.89
0.202
0.210
0.062
0.116
0.137
0.109
0.066
0.086
0.282
0.281
0.147
0.238
Calculated as LD,-0(control )/LD (pretreatment). Data from Table 1.
Table 23 shows that only three correlations relate toxicity to metabolite
levels in all the treatments with any degree of fidelity. The levels of DCDM
in brain and blood (which might reasonably be expected to be correlated with
each other) show the same general trend as the toxicity ratios but are not
well correlated quantitatively with toxicity. However, by combining the
levels of all formamidines present in the brain, a correlation is achieved
which matches the pattern of toxicity changes quite well quantitatively.
DISCUSSION
Toxicity tests with CDM and its metabolites establish that only those
compounds which retain the formamidine nucleus (CDM, DCDM, DDCDM) have a high
acute toxicity and induce strong excitatory symptoms. Only after the initial
excitatory phase (and most mortality) is complete do the mice show the
prolonged depressed behavior typical of the non-formamidine metabolites.
Because of the clear increase in toxicity and more rapid toxic action in the
order CDM
-------
3-methylcholanthrene actually decreased the toxicity of COM to about half its
normal value.
Despite the unexpected results obtained in the toxicity studies, the MFC
inducers and inhibitors do have their predicted effects on hepatic microsomal
metabolism when administered in vivo. The data in Table 19 show that
i> nibitor pretreatments of the mice reduce the amount of COM destroyed by the
isolated microsomes. Even in the control microsomes the degradation of CDM
was quite rapid (39% in 10 min), but this was greatly increased in microsomes
from inducer-pretreated mice, with 60%, 67%, and 80% of the CDM destroyed
after the phenobarbital, 3-methylcholanthrene, and Aroclor treatments
respectively. The two MFO inhibitors had similar effects on the pattern of
metabolites produced from CDM. All metabolites were reduced, although the
effect was rather small with NFT compared to the decrease in DCDM, DDCDM, and
polar metabolites. The three inducers, however, produced rather different
changes in the spectrum of metabolites. Phenobarbital and Aroclor
considerably increased the amounts of DCDM and DDCDM, with less effect on the
amount of NFT. 3-Methylcholanthrene on the other hand increased the level of
NFT by 260% but increased DCDM only by 35% and there was np increase at all
in the level of DDCDM.
Two further conclusions can be drawn from these results. First,
phenobarbital in known to increase the activity of those MFO reactions
catalyzed by cytochrome P-450 while 3-methylcholanthrene induces
predominantly the related cytochrome P-448 (Conney, 1967; Alvares et al.,
1973). Aroclor 1254, a mixture of polychlorinated biphenyls, induces both
types of cytochromes (Alvares et ^., 1973). All three inducers increase the
rate of N[-demethylation of CDM, though the P-450 inducers are more effective,
but the hydrolysis of the formamidines to NFT appears to be rather
selectively increased by the P-448 inducers, particularly
3-methylcholanthrene. The same relationship is true for the 'polar1
fraction. Thus much of the cleavage of the formamidines to NFT is probably
catalyzed by cytochrome P-448.
It has been suggested (Ahmad and Knowles, 1971a), that the hydrolytic
production of NFT is largely non-enzymatic, although Morikawa est jil. (1975)
concluded that the MFO system also might be involved. As pointed out
earlier, this does not prove that the MFO system produces NFT directly.
However, the data in Table 19 cannot be interpreted except in terms of a
direct MFO-catalyzed hydrolysis of one or more of the formamidines. The
rates of spontaneous hydrolysis of CDM, DCDM, and DDCDM are shown in Table
15. Although hydrolysis to NFT does occur, it is relatively slow e.g. at pH
7.4 and 37°, as used in the MFO studies above, in the 10 min incubation
period employed, the percentage hydrolysis of CDM, DCDM, and DDCDM to NFT
would be 1.3, 2.7, and 4.2 respectively. This compares to the presence of
16.2% of the added CDM as NFT in the MFO assay after the 3-methylcholanthrene
pretreatment, and this value has already been corrected for spontaneous
production of NFT (boiled enzyme blank). The mechanism of this formamidine
cleavage reaction is unknown. One possibility is that the primary MFO attack
in this case is by hydroxylation of the amidine carbon which may lead either
to formamidine cleavage and release of NFT or, by tautomerization, to the
corresponding urea derivatives.
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The second conclusion in comparing Tables 1 and 19 is that it is
difficult to relate these i£ vitro metabolic results to the effects of the
same pretreatments on toxicity. The two pretreatments which decrease the
toxicity of COM i.e. Aroclor and 3-methylcholanthrene do not reduce the level
of any metabolite, and Aroclor greatly increases the level of the more toxic
metabolites, DCDM and DDCDM. These pretreatments do reduce the amount of CDM
remaining, but the LD 's of the other four treatment were not greatly
different, yet the amount of CDM unmetabolized ranges from 40J to 75%.
A partial answer to this lack of evident correlation of toxicity and
metabolism probably lies in Table 18 where the metabolism of CDM, DCDM, and
DDCDM is compared in hepatic microsomes from untreated mice. Clearly the
JT-demethylated products are more rapidly degraded than CDM. DDCDM, the most
toxic of these formamidines is also the least stable with only 15% of the
parent compound recoverable after 10 min and 60% conversion to NFT. Toxicity
and metabolic stability are therefore negatively correlated. Thus the more
potent metabolites (DCDM, DDCDM) are also the ones likely to have the
shortest survival times in the body.
Because of the confusing metabolism-toxicity relations so far developed,
a study was performed to analyze the levels of each of the critical
metabolites in liver, blood, and brain at a time chosen to coincide with the
peak of symptoms in mice given lethal doses of CDM. Because of time and
radioisotope limitations this study was confined to phenobarbital,
3-methylcholanthrene, and piperonyl butoxide as the pretreatments. The data
for recovery of total radioactivity in the three tissues again gave an
unexpected, but important, result. Phenobarbital and 3rmethylcholanthrene
pretreatments have no significant effect on the total C present in any
tissue compared to the control. Piperonyl butoxide reduces this to half the
control value in each case. Thus the piperonyl butoxide must reduce uptake
of the oral dose of CDM. Why this decreased uptake should occur is not
obvious, especially since the piperonyl butoxide was given ip and the CDM
orally. One possibility is that the uptake of CDM from the gastrointestinal
tract is speeded by rapid metabolic destruction of CDM in the liver. When
this metabolism is decreased by piperonyl butoxide, the rate of uptake is
slowed. This interesting effect deserves to be studied further.
Examination of the distribution of CDM and its metabolites in the three
tissues when expressed as the % of the total metabolites recovered (Table 21)
reveals a picture qualitatively similar to that seen in Table 19 with
metabolism by the hepatic microsomes in vitro, but there are some notable
quantitative differences. With the control and inducer treatments in vivo
relatively little unchanged CDM was present in the liver (about 10J of the
quantitative differences. With the control and inducer treatments jji vivo
relatively little unchanged CDM was present in the liver (about 10% of the
recovered radioactivity) indicating a very rapid biodegradation for this
compound. DDCDM was found to be a much more prominent metabolite in vivo
than JUi vitro.
Further, major differences in the tissue levels of CDM between the
inducer pretreatments and control are not evident in most cases jin vivo. A
further difference between the in vivo and J.n vitro results is the high
89
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effectiveness of piperonyl butoxide in preventing the degradation of COM
(e.g. in the brain; 56% as COM versus 10% as COM in the control). In
particular the proportion of metabolites present as DDCDM was greatly reduced
with this pretreatment. Thus it is again evident that in vitro metabolic
studies have only a limited ability to predict rates of metabolism j.n vivo
although pathways of metabolism can often be deduced in vitro.
The crucial comparison in relating the toxicity of COM to its metabolism
lies with the data in Table 22, and the further correlations in Table 23.
The concentration of neither CDM nor DDCDM in any tissue correlates well with
toxicity after the different pretreatments e.g. if CDM itself were the major
toxicant, the data in Table 22 suggest that the piperonyl butoxide
pretreatment should clearly increase the toxicity of CDM since the levels of
this compound are elevated 2-3 fold in all' tissues compared to the control.
On the other hand, if DDCDM were the major toxicant, piperonyl butoxide
should greatly decrease the toxicity of CDM because the tissue levels of
DDCDM are considerably lowered. In fact, neither of these results is seen;
piperonyl butoxide has only a minor effect on the toxicity of CDM. Similar
comparisons with the other pretreatments lead to the same general conclusion.
Of the individual formamidines, the trend in DCDM levels can be related to
the toxicity quite closely (Table 23) and a reasonable quantitative
correlation is obtained between the toxicity ratio and DCDM in brain
(correlation coefficient, r = 0.897) and blood (r = 0.884) for the four
treatments. However, in most cases all three of the formamidines are present
in the tissues at roughly comparable concentrations. The most reasonable
assumption, based on our knowledge of their pharmacological actions, is that
all three have some innate toxicity and the overall effect on the animal is
the sum of the effects of the three compounds acting in concert rather than
the action of only one compound such as DCDM. This hypothesis receives
support from the data in Table 23 where an excellent quantitative correlation
is obtained between the toxicity ratio after the various pretreatments and
the total level of formamidines in the brain. The correlation coefficient
for this relationship is 0.985. Further support for this view comes from
preliminary experiments where we have observed that the toxicities of CDM,
DCDM and DDCDM are additive in mice - each can replace an equivalent toxic
fraction of another without a change in lethality or symptoms.
In this relationship, the major factor underlying the lowered toxicity
of CDM after 3-methycholanthrene pretreatment is the strongly increased
activity in cleaving the toxic formamidines to the poorly toxic metabolite,
NFT, probably through the induction of cytochrome P-448. The same reason may
underly the lower toxicity after Aroclor treatment which also induces P-448
and increases NFT production. Piperonyl butoxide, as one might expect, tends
to preserve the formamidines from further degradation and should, therefore,
increase the toxicity of CDM by our hypothesis. However, this effect is
balanced by the reduced rate of penetration of CDM after piperonyl butoxide
pretreatment so that, overall, the tissue levels of total formamidines differ
little from those of the control, and toxicity of CDM is not changed greatly.
Phenobarbital pretreatment does not alter the pattern of metabolites found in
the brain very significantly, and thus has no effect on the toxicity of CDM.
Our results offer no support for the idea that ^-demethylation is an
90
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obligatory activation reaction for CDM, or that the most toxic compound,
DDCDM, is the sole or even major toxic species iin vivo.
These conclusions must be tentative since only the determination of a
time-concentration profile for each of the major metabolites in each tissue
containing important sites of action would be definitive in determining the
role of the individual formamidines in such a complex toxicological
relationship.
The reason why DCDM and DDCDM are more toxic than CDM when given orally
(Table 1) is unclear. Comparisons of the relative potencies of CDM and DCDM
on most biochemical and physiological systems which plausibly might be
involved in their toxicity show no striking differences between the
compounds. DDCDM, as a relatively newly discovered toxic metabolite, has,
unfortunately, been less widely studied. As shown in Section 1, DCDM is only
slightly better than CDM as an inhibitor of MAO. In Section 4, DCDM was
found to be somewhat less effective than CDM as a local anesthetic, at least
on the frog sciatic nerve. Although only preliminary experiments were
performed to assess the effects of DCDM on the cardiovascular system of the
dog, using the methods described for CDM in Section 2, DCDM was
quantitatively and qualitatively rather similar to CDM although some
differences in action were noted. Further, the two compounds acted very
rapidly in this system (Table 5), suggesting that J^-demethylation of CDM (or
DCDM) is not obligatory for the cardiovascular effects which arise through
peripheral actions. For this reason we did not include further work with
DCDM in our cardiovascular studies.
The only major difference in pharmacological potency between CDM and its
N^demethylation products so far known is in their a-adrenergic and octopa-
minergic actions. DCDM has a much stronger effect than CDM as a partial
agonist at a-adrenergic sites (rabbit ear artery) as shown by Robinson and
Bittle (1979). The same distinction is also found in their action as
octopaminergic agonists in invertebrates, a type of system which is analogous
to the a-adrenergic system of vertebrates. In this case DDCDM is also
active, but somewhat less so than DCDM, while CDM itself is relatively
ineffective (Hollingworth and Murdock, 1980).
However, although it is attractive to try to relate the higher toxicity
of DCDM and DDCDM to their greater potency in stimulating a-adrenergic neuro-
transmission, there are problems with this hypothesis. a-Adrenergic agonists
(e.g. NE itself) are central depressants not central stimulants, as seen with
formamidines at lethal levels (Feldberg, 1963). Further, we found that phen-
tolamine, an a-adrenergic blocker, did not prevent the peripheral cardiovas-
cular depression caused by CDM (Section 2). Robinson et jd. (1975) reported
that phentolamine given ip to rats did not reduce the lethality of CDM, and
concluded that "it seems improbable that death following chlordimeform
poisoning in rats results from stimulation of a-adrenergic . . .receptors."
This view was strengthened by the subsequent observation (Robinson and Smith,
1977) that phenylephrine, itself an o-agonist, did not increase the toxicity
of CDM as might be expected if CDM, or a metabolite, had important actions at
the same site.
91
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The exact a-adrenergic effects of DCDM (and probably DDCDM) are
difficult to predict since partial agonists are, by definition, partial
antagonists also and thus may stimulate a-adrenergic transmission at one dose
and partially block it at a higher dose. This action of the formamidines
deserves further study, particularly with regard to the central effects of
CDM, but as pointed out above, the evidence presently available does not
strongly support this type of action as a major factor in the acute toxicity
cf CDM, whereas local anesthetic-like effects can explain most of the
symptoms observed. The greater toxicity of DCDM and DDCDM may therefore lie
as much in the area of pharmacokinetics (more rapid accumulation at the
site(s) of action) as in the area of pharmacodynamics (greater potency at, or
a different, site of action).
Overall, the toxicological actions of CDM, and probably related
formamidines, is rather complex, with several active compounds present
simultaneously, each of which may have several significant actions on the
nervous system. Much is now known concerning the neurotoxicology of
formamidines and its relationship to their toxicity, but numerous questions
remain to be answered before a satisfactory and complete picture of their
pharmacological and toxicological effects can emerge.
92
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1Q1
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
i. REPORT NO.
EPA-6QQ/i-an~n?ft
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Toxicity, Interactions, and Metabolism of
Formamidine Pesticides in Mammals
6. REPORT DATE
Mav 198Q
8. PERFORMING ORGANIZATION COOK
7. AUTHCRIS)
R. M. Hollingworth and G.K.W. Yim
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Purdue University
Department of Entomology
West LaFayette, Indiana 47907
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
Grant No. R-803965
12. SPONSORING AGENCY NAME AND ADDRESS
US Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The overall goal of this research project was to investigate the mechanism(s) of
acute toxicity of formamidine pesticides in mammals using chlordimeform (N/-(4-chloro-o-
tolylJ-N^-dimethylformamidine) and its metabolites as the primary model compounds. The
role of biotransformations, particularly N-demethylation reactions,, in generating poten-
tially toxic metabolites was also studied. !
By comparing the effects of hepatic microsomal mixed function oxidase inducers and
inhibitors administered in vivo on the toxicity, metabolism, and distribution of metabo-
lites in mouse tissues, it was concluded that although N-demethylation products are
innately more toxic than chlordimeform, they are also less stable, and the best correla-
tion of toxicity was obtained with the total level of formamidines in the brain, rather
than with the level of any individual metabolite.
In a series of studies with dogs, rabbits, and cats, the cause of death was found to
be cardiovascular collapse accompanied by respiratory arrest. Cardiovascular collapse
resulted primarily from a peripheral local anesthetic-like effect of chlordimeform.
Monoamine oxidase inhibition was not a major factor in lethality. Respiratory arrest
was central in origin. Several other central effects of the formamidines were described,
some of which may be local anesthetic actions, and a behavioral profile for chlordimeform
poisoning in the rat was developed. The effectiveness of various drug treatments as
potential therapeutic aids for formamidine intoxication were studied. Formamidines also
nave asoirin-like actions due^tD_flJuLnahi 1 itv t.n inhibit. nrnst.aolaruHn synthesis
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Formamidine pesticides
Toxicity in mammals
Administered In Vivo
06,F,T,
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (THIS Report)
UNCLASSIFIED
21. NO. Or PAUC9
114
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
£PA Form 2220-1 'Rev. 4-77) PREVIOUS EDITION IS OBSOLETE
102
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