TABLE 2. Effects of p-chlorophenylalanine, DL-a-methyl-p-tyrosine or

Phenylephrine on the Acute Lethality of Chlordimeform in Male Rats a
Chlordimeform LD5q? 95% Confidence Limits
Drug Treatment ng/kg of the LDsg
Control 238 202-271
p-chlorophenylalanine® 238 213-263
DL-a-methy1-p-tyrosine4 233 204-275
Phenylephrine® 241 220-271

a. Charles River Breeding Laboratories, Wilmington, MA.

b. at 24 hours after chlordimeform by the method of Finney (1971).
c. ip 100 mg/kg for 3 days prior to chlordimeform.

d. ip 500 mg/kg 20 hours prior to chlordimeform.

e. ip 1 mg/kg immediately after chlordimeform.

From Robinson and Smith, 1977.

The lethality of chlordimeform varied greatly in the two experiments,
in which rats from two different sources were employed. Such a strain
difference is not unusual. Rats from these two suppliers have been shown by
Stavinoha and coworkers (1969) to react differently to chronic poisoning by
the organophosphate insecticide Di-Syston (disulfoton).

IV. Discussion.

The metabolism and excretion of chlordimeform by rats is quite rapid.
Within 12 hours after the oral administration of 14c_1abeled chlordimeform,
over 70 percent of the administered radioactivity has appeared in the urine,
and approximately 4 percent has appeared in the feces, according to Knowles
and Sen Gupta (1970). Knowles and Roulston (1972) found that less than
17 percent of this labeled material in the urine is unmetabolized or only
demethylated. These are the only forms capable of exerting appreciable
activity. Thus chlordimeform should not be expected to exert toxicity over
an extended period of time, unless it causes initial, irreversible effects.
In our experiments it was found that the rats that died did so within a
fairly short time, as would be expected with a toxicant which is rapidly
inactivated and excreted.

The observations by other investigators that the toxic signs of acute
poisoning resembled those of acute serotonergic or adrenergic stimulation
were reinvestigated in these two experiments. The compounds used affect
selectively the adrenergic or serotonergic neuroeffector systems. Those
that modify adrenergic nerve function will be discussed first (Fig. 1).

5



25.

26.

27.

28.

29,

30.

Robinson, C. P., P. W. Smith, J. D. Zelenski, and B. R. Endecott;
Lack of an Effect of Interference with Amine Mechanisms on the
Lethality of Chlordimeform in the Rat. TOXICOL. APPL. PHARMACOL.,
33:380-383, 1975.

Robinson, C. P., and P. W. Smith: Lack of Involvement of Monoamine
Oxidase Inhibition in the Lethality of Acute Poisoning by Chlordimeform.
J. TOXICOL. ENVIRON. HEALTH. In press, 1977.

Sen Gupta, A. K., and C. 0. Knowles: Metabolism of N'-(4-chloro-o-tolyl)
-N, N-dimethylformamidine by Apple Seedlings. J. AGR. FOOD CHEM., 17:
595-600, 1969.

Sen Gupta, A. K., and C. 0. Knowles: Galecron—!4¢(y'-chloro-o-toly1)-N,
N-dimethylformamidine) Metabolism in the Goat. J. ECON. ENTOMOL., 63:
951-956, 1970.

Stavinoha, W. B., L. C. Ryan, and P. W. Smith: Biochemical Effects
of an Organophosphorous Cholinesterase Inhibitor in the Rat Brain.
ANN. N.Y. ACAD. SCI., 160:378-382, 1969.

Yamori, Y., W. DeJong, H. Yamabe, W. Lovenberg, and A. Sjoerdsma.
Effects of L-Dopa and Inhibitors of Decarboxylase and Monoamine Oxidase
on Brain Noradrenaline Levels and Blood Pressure in Spontaneously
Hypertensive Rats. J. PHARM. PHARMACOL., 24:690-695, 1972.

10


SEROTONERGIC NERVE

MAO INHIBITED BY MITOCHONDRIA (CONTAIN MAO)
CHLORDIMEFORM +
“N

INHIBITED BY
P-CHLOROPHENYLALANINE
|

| sToRES 0

v OF
TRYPTOPHAN > —®SEROTONIN
Zz

7
7
DEPLETED” PRE-SYNAPTIC

BY MEMBRANE POST- SYNAPTIC

MEMBRANE

RECEPTOR FOR SEROTONIN

“BLOCKED BY METHYSERGIDE

RESERPINE
FIGURE 2.

In a similar manner, the effects of compounds that affect serotonergic
nerve function were observed (Fig. 2). Inhibition of MAO by chlordimeform
also causes accumulation of serotonin, as MAO also deaminates and thus
inactivates serotonin. Depletion of serotonin to reduce the quantity avail-
able for accumulation was accomplished by using p-chlorophenylalanine, which
selectively interferes with serotonin synthesis. Methysergide was used to
specifically block the serotonin receptor. If serotonin accumulation played
a major role in chlordimeform lethality, both serotonin depletion and sero~
tonergic receptor blockade should have afforded some protection. Because
this did not occur we infer that serotonin accumulation subsequent to MAO
inhibition does not contribute much to chlordimeform lethality in the rat.

In like fashion, neither the depletion of both NE and serotonin by
reserpine, nor the blockade of both serotonergic and adrenergic receptors
with methysergide and phentolamine given at the same time, altered the LDog
of chlordimeform.

We conclude from these experiments that although MAO inhibition may
contribute to the symptomatology of acute chlordimeform poisoning, it does
not play a major role in the relatively sudden deaths that result from
lethal doses.

Thus it would appear that none of the drugs tested shows promise in the
treatment of acute chlordimeform poisoning. Likewise, it is unlikely that
aerial applicators or others would incur extra risk if exposed to chlordime-
form while taking any of those agents tested that are in current use for

therapeutic purposes.
7



NORADRENERGIC NERVE

MAO INHIBITED BY MITOCHONDRIA (CONTAIN MAO)
CHLORDIMEFORM ‘
N

INHIBITED BY

OL- @- METHYL -P-
TYROSINE STIMULATED BY PHENYLEPHRINE
l
| STORES 0 RECEPTOR FOR NE
OF (ALPHA~ ADRENERGIC RECEPTOR)
TYROSINE —> —» > NE a x
BLOCKED BY PHENTOLAMINE
7
7 /
DEPLETED~ PRE~SYNAPTIC
BY MEMBRANE Pi -
nesenPine oor goynarric

FIGURE 1.

After NE is released by nerve impulses from the pre-synaptic membrane,
some of it combines transiently with its receptor to initiate post-junctional
activity. An appreciable amount of NE is actively taken back into the
adrenergic nerve, and part of this reenters the pre-synaptic granules to be
used again.

Some NE diffuses into mitochondria where it may be inactivated by MAO,
an enzyme which oxidatively deaminates it. Chlordimeform inhibits MAO, and
thus allows the buildup of NE in the region in which it is released. The
compound DL-o-methyl-p-tyrosine inhibits the synthesis of NE by inhibiting
the enzyme, tyrosine hydroxylase. This decreases the quantity of NE avail-
able, without appreciably reducing serotonin levels. This should reduce
lethality resulting from the acute buildup of NE. Phentolamine blocks the
alpha-adrenergic receptor and thus should be able to reduce lethality
resulting from acute stimulation of these receptors by the accumulated NE.
Phentolamine also reduces reuptake of NE into adrenergic nerves and thus
would contribute to NE accumulation, but the receptor blocking effect would
predominate. Phenylephrine is a directly acting agonist at the alpha-
adrenergic receptor. If stimulation of alpha~adrenergic receptors is a major
contributor to the lethality of chlordimeform, phenylephrine should increase
chlordimeform lethality. Because no drug affecting the adrenergic neuro-
effector system affected the lethality of chlordimeform, it seems that the
effects of MAO inhibition on adrenergic nerve function contributes little to
chlordimeform lethality.


13.

14,

15.

16.

17.

18.

19.

20.

21.

22.

23.

24,

Ehrhardt, D. A., and C. 0. Knowles: Metabolism and Translocation of
N'-(4-chloro-o-tolyl)-N,N-dimethylformamidine (chlorphenamidine) and

its Hydrochloride Salt in Grapefruit Seedlings. J. ECON. ENTOMOL. 63(4):
1306-1314, 1970.

Finney, D. F.: Probit Analysis, 3rd Edition, Cambridge at the
University Press, London. 333 pp., 1971.

Jesperson, S., and J. Scheel-Kruger: Evidence for a Difference in
Mechanism of Action between Fenfluramine- and Amphetamine-induced
Anorexia. J. PHARM. PHARMACOL., 25:49-54, 1973.

Johnson, B. T., and C. 0. Knowles: Microbial Degradation of the
Acarcide N-(4-chloro-o-tolyl)-N, N-dimethylformamidine. BULL. ENV.
CONTAM. TOX., 5:158-163, 1970.

Kenaga, E. E., and W. E. Allison. Commercial and Experimental Organic
Insecticides. BULL. ENTOMOL. SOC. AMER., 15:85-148, 1969.

Knowles, C. 0., and W. J. Roulston: Antagonism of Chlorphenamidine

Toxicity to the Cattle Tick Boophilus Microplus by Piperonyl Butoxide.
J. AUST. ENT. SOC., 11:349-350, 1972.

Knowles, C. 0., and W. J. Roulston: Toxicity to Boophilus Microplus
of Formamidine Acaricides and Related Compounds, and Modification of
Toxicity by Certain Insecticide Synergists. J. ECON. ENTOMOL., 66:
1245-1251, 1973.

Knowles, C. 0., and A. K. Sen Gupta: WN'-(4-chloro-o-tolyl)-N, N-
dimethylformamidine-14(Galecron) and -4-chloro-o-toluidine 14C
Metabolism in the White Rat. J. ECON. ENTOMOL., 63:856-859, 1970.

Knowles, C. 0., and S. P. Shrivastava: Chlordimeform and Related
Compounds: Toxicological Studies with House Flies. J. ECON. ENTOMOL. ,
66:75-79, 1973.

Lin, T. H., H. H. North, and R. E. Menzer: The Metabolic Fate of
Chlordimeform N-(4-chloro-o-tolyl)-N', N'-dimethyl formamidine in
Human Embryonic Lung Cell Cultures. J. AGR. FOOD CHEM., 23:257-258,
1975.

Lockett, M. F., D. L. Stuart, R. Wadley, A. R. Goss, and H. H. Siddiqui:
Some Effects of Dihydroergocristine and of Phentolamine Mesylate on
Renal Function in Rats. J. PHARM. SCI., 21:648-655, 1969.

Pletscher, A., P. A. Shore, and B. B. Brodie: Serotonin as a Mediator
of Reserpine Action in Brain. J. PHARMACOL. EXPER. THER., 116 :84-89,
1956.



Technical Report Documentation Page

T. Report No. 7. Government Accession No. 3. Recipient's Catalog No.
FAA-AM-77-19
Title and Subtitle 5. Report Date

August 1977

& Performing Organization Code

THE ROLE OF MONAMINE OXIDASE INHIBITION IN
THE ACUTE TOXICITY OF CHLORDIMEFORM [8. Performing Organization Report No.

7 Aether’) Paul W. Smith, * Casey P. Robingon,**
Jane D. Zelenski, and Boyd R. Endecott

9. Performing Organization Nome and Address 10. Work Unit No. (TRAIS)
FAA Civil Aeromedical Institute

P.O. Box 25082 TT. Contract or Grant No.
Oklahoma City, Oklahoma 73125* ae

College of Pharmacy, 0.U., Oklahoma City 73190 13. Type of Report and Period Covered

12. Sponsoring Agency Name and Address

Office of Aviation Medicine
Federal Aviation Administration
800 Independence Avenue, S.W.
Washington, D.C. 20591 FAA

14" Sponsoring Agency Code

15. Supplementary Notes

Research leading to preparation of this report was performed under
Tasks AM-A-75-TOX-28 and AM-A-76-TOX-28.

16. Abstract

This paper presents data from experiments on male rats performed to determine
whether drugs which interfere with central amine mechanisms would decrease the
lethality of the acaricide chlordimeform (and thus be of potential value as
antidotes for accidental poisoning) or increase chlordimeform lethality (and
thus should be avoided by aerial applicators and others in contact with it).
Neither reducing serotonin synthesis with p-chlorophenylalanine, reducing
norepinephrine synthesis with DL-a-methyl-p-tyrosine nor depleting both amines
with reserpine affected the lethality of chlordimeform. Likewise, blocking
a-adrenergic receptors with phentolamine or the serotonergic receptors with
methysergide, or both, did not influence chlordimeform lethality. The
adrenergic agonist drug phenylephrine also did not affect chlordimeform lethality.
Thus, the results indicate that: (1) monamine oxidase inhibition does not play

a major role in acute chlordimeform lethality; (2) none of the drugs tested
shows promise in the treatment of chlordimeform poisoning, and (3) aerial
applicators or others would appear to incur little or no extra risk should

they be taking any of the above drugs during potential exposure to chlordimeform.

V7. Key Words DT —a-methyl-p-tyrosine 18, Distribution Statement

norepinephrine chlordimeform Document is available to the public

serotonin alpha-adrenergic through the National Technical

reserpine block Information Service, Springfield,

methysergide phenylephrine Virginia 22161

phentolamine p-chlorphenylalanine

19. Security Classif. (of this report) 20. Security Clossif. (of this page) ZI. No. of Pages | 22. Price
Unclassified Unclassified 10

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized



10.

11.

12.

References

Abo-Khatwa, N., and R. M. Hollingworth: Chlordimeform: The Relation
of Mitochondrial Uncoupling to Toxicity in the German Cockroach.
LIFE SCI., 11:1181-1190, 1972.

Abo-Khatwa, N., and R. M. Hollingworth: Chlordimeform: Uncoupling
Activity Against Rat Liver Mitochondria. PEST. BIOCHEM. PHYSIOL., 3:
358-369, 1973.

Ahmad, S., and C. 0. Knowles: Metabolism of N'-(4-chloro-o-tolyl)-

N, N-dimethylformamidine (chlorphenamidine) and 4'-chloro-o-formotoluidide
by Rat Hepatic Microsomal and Soluble Enzymes. COMP. GEN. PHARMACOL.,
2:189-197, 1971.

Alpers, H. S., and P. A. Shore: Specific Binding of Reserpine--
Association with- Norepinephrine Depletion. BIOCHEM. PHARMACOL., 18:
1363-1372, 1969.

Aziz, S. A., and C. 0. Knowles: Inhibition of Monamine Oxidase by the
Pesticide Chlordimeform and Related Compounds. NATURE, 242:417-418, 1973.

Beeman, R. W., and F. Matsumura: Chlordimeform: A Pesticide Acting Upon
Amine Regulatory Mechanisms. NATURE, 242:273-274, 1973.

Beeman, R. W., and F. Matsumura: Studies on the Action of Chlordimeform
in Cockroaches. PEST. BIOCHEM. PHYSIOL., 4:325-336, 1974.

Boakes, R. J., P. B. Bradley, and J. M. Candy: A Neuronal Basis for
the Altering Action of (+)-amphetamine. BR. J. PHARMACOL., 45:391-403,
1972.

Bull, D. L.: Metabolism of Chlordimeform in Cotton Plants. ENVIRON.
ENTOMOL., 2:869-871, 1973.

Dittrich, V.: N-(2-methyl-4-chlorophenyl)-N,N' dimethyl formamidine
(C-8514/Schering 36268) Evaluated as an Acaricide. J. ECON. ENTOMOL.,
59:889-893, 1966.

Dittrich, V., and A. Loncarevic: New Insecticides for Asiatic Rice
Borer Control in Paddy Rice. J. ECON. ENTOMOL., 64:1225-1229, 1971.

Dixit, B. N.: Brain 5-hydroxytryptamine and Anterior Pituitary
Activation by Reserpine and its Analogs. ARCH. INT. PHARMACODYN., 189:
100-108, 1971.


The length of time between injection of chlordimeform and death of the
rats that died was dose related. Those receiving large doses (=~ LDgo)
survived only about 15 min, while those dying after small doses ( 7 LDoQ)
survived for approximately an hour. All animals that died during the 24-hour
observation period did so within the first 3 hours for the reserpine-treated
group, and within the first 2 hours for all other groups. Although the exact
time from injection of the poison until death was not recorded for each
individual animal, it was our impression that rats pretreated with the amine
receptor blockers died more quickly than control rats.

None of the drugs used in an attempt to modify the lethality of
chlordimeform significantly affected the LDso (Tables 1 and 2).

TABLE 1. Effects of Pretreatment with Reserpine, Methysergide, or
Phentolamine on the Lethality of Chlordimeform in Male Rats*

95%
Confidence 95%

Chlordimeform Limits Confidence
Deo? of the Relative Limits of

Pretreatment (mg? Rg) IDs5 Lethality® Relative Lethality
None 128 (103-149) Lo eee een
Reserpine, © 138 (117-171) 0.89 0.71-1.12
Methysergide,¢ 107 (91-129) 1.15 0.92-1.44
Phentolamine,® 116 (99-142) 1.06 0,.84-1.32
Phentolamine, 107 (89-152) 1.18 0.94-1.46

and methysergide!

a. Holtzman, Madison, WI.

b. at 24 hours after chlordimeform by the method of Finney (1971).

c. im 2.5 mg/kg (48 hr) and 1 mg/kg (24 hr) prior to chlordimeform.

d. ip 3 mg/kg 25 min prior to chlordimeform.

e. ip 10 mg/kg 25 min prior to chlordimeform.

£. ip, phentolamine, 10 mg/kg; methysergide, 3 mg/kg, 25 min prior to
chlordimeform.

From Robinson et al., 1975.


In one experiment the effects of reserpine, methysergide and phentola-
mine on chlordimeform lethality in rats were determined. Reserpine in the
dose and dosage schedule used depletes both norepinephrine and serotonin from
their respective storage sites (Pletscher et al., 1956; Alpers and Shore,
1969; Dixit, 1971). The dose of phentolamine used selectively blocks alpha-
adrenergic receptors (Lockett et al., 1969), while that of methysergide
selectively blocks serotonergic receptors (Jespersen and Scheel-Krug, 1973).
In this experiment 200 white male Holtzman rats were randomly divided into
five equal groups. One group received reserpine (Serpasil for injection,
Ciba) 48 hr (2.5 mg/kg) and 24 hr (1 mg/kg) prior to the administration of
chlordimeform. Three other groups received either methysergide (3 mg/kg in
normal saline), phentolamine (10 mg/kg in normal saline), or both drugs
25 min prior to the injection of chlordimeform. The control group received
no drug prior to the injection of chlordimeform.

In the other experiment the effects of p-chlorophenylalanine, DL-a-
methyl-p-tyrosine and phenylephrine on chlordimeform lethality were examined.
The dose and dosage schedule of p-chlorophenylalanine used was reported by
Yamori and coworkers (1972) to lower brain-stem serotonin to undetectable
values while causing only a slight decrease in brain-stem NE. Boakes and
coworkers (1972) reported that DL-o-methyl-p-tyrosine causes a selective
depletion of NE from rat brain. Phenylephrine causes a direct stimulation
of alpha-adrenergic receptors.

In this experiment, white male rats from Charles River Breeding
Laboratories were divided into three treatment groups of 54 rats each and a
control group of 48 rats. Rats in one treatment group were given 100 mg/kg
p-chlorophenylalanine daily for 3 days prior to the administration of
chlordimeform. Rats in the second group were given 500 mg/kg DL-o-methyl-p-
tyrosine 20 hours prior to chlordimeform. Rats in the third group received
1 mg/kg phenylephrine immediately after chlordimeform. Rats in the control
group received chlordimeform only. Six rats from each of the three treatment
groups were given no chlordimeform, and were observed throughout the
experiment.

All drugs in both experiments were injected intraperitoneally except
reserpine, which was given intramuscularly. Chlordimeform was injected
undiluted at appropriate dosage levels, and the resulting lethalities
observed 1, 3, and 24 hours later. The LD,.'s were determined from curves
plotted from mortality at each chlordimeform dosage level, using the maximum
likelihood method of Finney (1971).

III. Results.

Rats that received only chlordimeform showed signs of central stimula-
tion. Signs of central stimulation were more marked in those that had also
received phenylephrine. No rats that received the pretreatment drugs alone
died.