United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600/1-78-065
November 1978
Research and Development
Oft
Mechanisms of
Pesticide
Degradation
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5 Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-6QO/1-78-065
November 1978
MECHANISMS OF PESTICIDE DEGRADATION
by
Fumio Matsumura
Department of Entomology
University of Wisconsin
Madison, Wisconsin 53706
Grant No. R-801060
Project Officer
Robert F. Moseman
Analytical Chemistry Branch
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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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.
11
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FOREWORD
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, environmental 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 preparing 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 represents a research effort to enhance our
knowledge of the mechanisms by which pesticides are degraded
in living organisms. The results of several studies dealing
with biochemical defense mechanisms and transformation of
pesticides are addressed in this report.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
111
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PREFACE
As a group of chemicals pesticides certainly represent one of
the most toxic materials that are intentionally broadcast in the
environment by man. Many of these chemicals are biologically
active, persistent enough, and accumulate in biological systems;
and therefore must be regarded as environmental contaminants. Yet,
these chemicals are often very useful in maintaining quality of
agricultural products, our environment, and protecting man from
diseases and famines. Assessment of risk/benefit; ratio for a
pesticide or any compounds for that matter, is not easy, however.
Ideally such judgements must be based upon sound scientific facts
and economic or aesthetic values that are produced by the chemical.
And yet, few environmental events are definitive, and clear cut.
As a result, we are often forced to come to a decision before all
facts become available. If the past decade can be viewed as a
typical period, environmental issues of pesticidal toxicology are
crisis filled. The cases such as DDT, dieldrin, 2,4,5-T (TCi)D)
attest to this view. Urgent issues may suddenly crop up and
disturb daily routines or basic work schedules. My personal feel-
ing is that the U.S. scientific community has really reacted well,
and time and time again have proven that many able scientists are
willing to spend many hours to face the issue.
In this report we have covered three important issues: they
are questions on a) toxaphene, now number 1 chlorinated insecti-
cide, b) chlordimeform, a controversial, behavior modifying
pesticide, and c) anaerobic degradation systems of various pesti-
cides, a new area of research design to probe into unanswered
degradative force in animals. All these topics have been chosen
as a result of deliverate considerations on acute needs for toxi-
cological information. Toxaphene has been on the RPAR (rebuttable
presumption against registration) list, and yet at the time this
study was initiated nobody even knew what the molecular structure
of the toxic ingredient of toxaphene was. Chlordimeform has been
suspended for use by the request of the manufacturer because of
its health implications. It is now reinstalled with stringent
precautionary measures. Our own viewpoint is that scientific
facts are the best basis for any regulatory decisions. On the
other hand, since the resources and time are limited all scientists
make very conscious efforts to concentrate on manters of utmost
significance. We are confident that the data generated by this
project are of good use to that end. and that our collaboration
between university communities and the government agencies such
as EPA will continue in r.he future.
IV
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ABSTRACT
This research project was initiated with the overall objective
of determining (1) the chemical structures of toxic components of
toxaphene, (2) to study anaerobic metabolism to degrade toxaphene
and other pesticides, and (3) to understand toxic action mechanism
of chlordimeform.
As a result of intensive efforts the molecular structures of
three of the most toxic principles of toxaphene were identified.
Together these components comprise at least 70% of toxaphene's
toxicity toward mice. This is the first time that the structure
of toxic components of toxaphene became apparent despite the wide-
spread use (over 1 billion pounds, which is comparable to DDT) of
toxaphene in the last 3 decades. Toxaphene on the other hand
degrades relatively faster than other chlorinated pesticides such
as DDT and dieldrin. The reason for it is that toxaphene is sus-
ceptible to reductive degradative forces.
Chlordimeform was found to affect amine regulatory mechanisms
in animals. Such actions explain some of the subtle effects of
this pesticide on animals. Inasmuch as that biogenic amines are
known to play many important biological roles such as controlling
emotion, behavior and circulatory functions of the body.
This report was submitted in fulfillment of Research grant,
R801060 from the U.S. Environmental Protection Agency. This
report covers the period of May 1, 1975 through April 30, 1978,
the date that the project was completed.
v
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TABLE OF CONTENTS
page
CHAPTER I Chemical Studies on Toxaphene 1
Abstract 1
Introduction 1
Conclusions 2
Recommendations 2
Materials and Methods 3
Results and Discussion '. 3
Identification of toxic components 3
Degradation of toxic components in lake
sediments by microorganisms 4
Degradation in animals (rats) 5
CHAPTER II Reductive Degradation of Pesticidal Chemicals.. 6
Abstract 6
Introduction 6
Conclusions 7
Recommendations 7
Materials and Methods 7
Anaerobic degradation in vitro of l "*C-mexacarbate
by the 20,000 g supernatant 8
Sephadex gel-filtration 8
Anaerobic degradation in vitro of DDT and
1 "* C-mexacarbate by the flavoprotein preparation. 8
Results and Discussion 9
CHAPTER III Health Effects of Chlordimeform 16
Abstract 16
Introduction 1.6
Conclusions 16
Recommendations 17
Materials and Methods 17
General Procedures 17
Brain amine levels and amine oxidase (MAO) 17
Fluorescence Histochemistry 17
Results and Discussion 18
Studies in higher animals 18
Visualization of brain amine in cockroaches 21
REFERENCES 2.4
APPENDIX 27
GLOSSARY 30
VI
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LIST OF ILLUSTRATION
Figure Number page
Figure 1 UV absorption and degradation activity (DPM)
for the column fractions 13
Figure 2 UV spectra of flavoprotein preparations and
FAD in phosphate buffer 14
LIST OF TABLES
Table Number
1 Toxicities of toxaphene and toxicant Ac to
fathead minnow and mosquito larvae 4
2 Degradation of C-mexacarbate by the 20,000 g
supernatant from rat intestines 11
3 Anaerobic degradation of DDT and C-mexacarbate
by the flavoprotein preparation from rat
intestine 12
4 Serotonin and norepinephrine levels in
whole rat brain 18
5 Potentiation of chlordimefonn toxicity by
tryp tamine 20
3
6 In vivo metabolism of H-L-DOPA and accumulation
of amines as affected by chlordimeform in the
American cockroach 21
7 Effect of chlordimeform on adenylate cyclase and
octopamine-induced stimulation of adenylate
cyclase in roach thoracic ganglia in si-tu 22
V1JL
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CHAPTER I
CHEMICAL STUDIES ON TOXAPHENE
ABSTRACT
Toxaphene is the most widely used chlorinated insecticide in
the U.S.A. today with an annual production of about 50 million
pounds, and a total usage of one billion pounds in the last 25
years. Because of its extreme complexity, it would be both dif-
ficult and impractical to thoroughly chemically characterize all
the components of toxaphene, thus it is very important to limit
study only to the major toxic components. At the time this pro-
ject was initiated nobody had knowledge on molecular structure of
toxaphene. Our isolation and identification efforts led us to
elucidation of three major toxic components of toxaphene. Together
they constitute at least 1/2 to 2/3 of the toxicity of toxaphene
to mice.
INTRODUCTION
Toxaphene is a widely used insecticide. Two-thirds of its
production is used for cotton insect control while other uses
include vegetables, small grains, soybeans, and control of exter-
nal insects on livestock. It has also been employed extensively
in fish eradication programs. Its annual production is about 50
million pounds (1971 estimate, Environmental Protection Agency,
1972) with a total usage of one billion pounds in the past 25 years
Despite this wide usage, little has been known about the chemistry,
toxicity, metabolism, or environmental fate of its components.
Only recently a major effort toward answering these questions
has been made by Casida et al. (1974), who were successful in
isolating and identifying a toxic component of toxaphene,
2,5-endo ,6-exo-S,9,10-heptachlorobornane. According to them, at
least 175 polychlorinated 10-carbon compounds were recognized by
their methods (Holmstead et al., 1974). The components were
described as polychlorobornanes, polychlorbornenes, and poly-
chlorotricyclenes with 6 to 10 chlorine atoms per component. Also
isolated was a CiQH]_QClg component which was more toxic to mice
and houseflies than the above component; however, no structure
was proposed for the latter component.
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Isolation and identification of all the components of toxaphene
would be a monumental task. Therefore, at this stage of our under-
standing of toxaphene, it was necessary to limit study to the
chemical elucidation of major coxic components. Also from an
environmental standpoint, it is important to assess the hazards
in terms of its toxicity to nontarget organisms, particularly
aquatic species. This is of obvious importance since results from
several studies on the toxicity of toxaphene to nontarget organisms
(Piinentel, 1971) indicate, toxaphene to be quite toxic to fish, many
aquatic invertebrates, and most insects.
CONCLUSIONS
Toxaphene is a very unusual chlorinated insecticide in that its
residues are not generally found in the human tissues, foods and
drinking water despite its extensive use which is comparable to
DDT. The reason for this lack of residue data can be multifold:
e.g. (a) the difficulty in establishing analytical procedures
because of its chemical complexity, (b) the lack of knowledge as
to which component to study, arid (c) general lability of the
major components via metabolic and photochemical processes etc.
To answer the question whether toxaphene is an environmentally
damaging chemical or not, one must first clearly identify and
establish its most toxic fraction or fractions so that their
environmental fates and effects can be closely studied. Also
the basic requirement for any toxicological study is the know-
ledge on the structure of the toxicant involved. The data shown
in this work provide the basic means to that end.
At least now we know the chemical structures and some proper--
ties of toxic components of toxaphene. With this knowledge
scientists can start searching their residues in the environment
because they will know what to look for. Also metabolic studies
which were impossible before can be initiated to increase our
knowledge on toxicological behavior of toxaphene.
RECOMMENDATIONS
1. With the knowledge on these toxic components of toxaphene,
residue monitoring programs specifically aimed at finding
them should be initiated.
2. Metabolic studies particularly aimed at finding possible
toxic metabolic products of toxaphene should be encouraged
in animals and in the environment.
3. Determine parameters for environmental toxicological infor-
mation such as bioaccumulation, half--Life in soil, evapor-
ation, ecotoxicity etc. of these toxic components of
toxaphene.
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MATERIALS AND METHODS
Components and fractions of toxaphene have been separated by
a combination of methods including column chromatography, thin-
layer chromatography, reverse phase thin-layer chromatography,
and preparative gas chromatography.
For analysis of purified components, mass spectra were taken
on a Finnigan 1015 qaadropole mass spectrometer. Infrared (IR)
spectra were obtained on a Beckman IR33 spectrophotometer.
Fourier Transform NMR spectra were obtained on a 90 MHz Bruker
FTNMR spectrometer.
A number of test organisms were used for bioassay of toxaphene
fraction toxicity. They were: 1) mosquito larvae, Aedes aegypti;
2) freshwater blue-green algae, Anaeyst-Ls nidulans (TX20); 3) brine
shrimp, Art em-La salina; and 4) fathead minnow, Pimephales promelas
Bioassay procedures involved LC^n determinations for brine shrimp
and mosquito larvae (Nelson, 19/4), while toxicity to algae was
measured as a decreased k value, a growth rate constant (Batterton
et al. , 1971). Chandurkar et al. , (1978) provided toxicity test-
ing on minnows.
RESULTS & DISCUSSION
Identification of toxic components
Fractions separated by the above methods show varying toxici-
ties to four aquatic organisms, a blue-green alga, brine shrimp,
fathead minnow and mosquito larvae. Employing a combination of
preparative TLC and GC methods, a toxic fraction of toxaphene
1.87, 1.75 and 1.35 times more toxic than toxaphene to mosquito
larvae, brine shrimp, and algae, respectively, has been isolated.
The toxic A fraction, though it behaves as a single component
in various chromatographic systems, was found to consist of two
components on the basis of nuclear magnetic resonance (NMR) spec-
troscopy. They were further characterized by infrared and mass
spectrometry as octachlorobornanes. We have also noticed that
there is a persistent contaminant in the preparations of toxic
fraction A. In view of the implication of recent reports (Turner
et al. , 1971) we have examined this contaminant (hereinafter
referred to as toxicant Ac), and now report the identification
of such a component of toxaphene.
The toxicity of this toxicant Ac was determined using fish,
fathead minnow and mosquito larvae as test organisms. The data
are presented in Table 1. The comparison of LC^Q values of this
toxicant Ac with that of standard toxaphene shows that it is
approximately four times more toxic to fish than standard toxa-
phene, and that it is just as toxic to mosquito larvae as
standard toxaphene.
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The toxicity data against mosquito larvae show that toxicant
Ac is not as toxic as toxic fraction A12, indicating that the
former cannot; account for the toxicity of the latter. On the
other hand, the toxicity of toxicant Ac to fish raises the pos-
sibility that there are components in toxaphene with unfavorable
selective toxicity to nontarget organisms as compared to insects.
TABLE 1
Toxicities of Toxaphene and Toxicant Ac to_
Fathead Minnow and Mosquito Larvae
Compound(s) Fathead Minnow Mosquito Larvae
(48-hr LC50, ppb) (24-hr LC50> ppm)
Toxaphene
Toxicant Ac
77.55
18.77
0.3755
0.3788
Our finding here gives strong support to the idea that
toxic fraction A (= toxicant A) is the most toxic component of
toxaphene. In view of the uncertainties about the real toxicities
of the other two major components, toxicant B and toxicant C, the
identification of the structures and the toxicities of toxicant
Ac and A at this stage certainly helps clarify the question as
to which component of toxaphene to study for its environmental
behavior in order to assess the potential ecotoxicity of toxaphene.
Degradation of toxic components in lake sediments by microorganisms
Degradation of toxicant A (= toxic fraction A) and toxicant
B was found to proceed at a faster rate than that of crude toxa-
phene in lake sediments. Under the experimental condition the
half-life of A and B was about 30 days. In all tests, degradation
of toxicant C, on the other hand, was found to be even faster than
A or B. The rate of degradation did not vary appreciably when
the incubation condition was changed from anaerobic to aerobic.
This is contrary to the generally held belief that toxaphene
degrades faster under anaerobic conditions.
To study microbial degradation Pseudcmonas putida was select-
ed because of its well known ability to degrade camphor-type
molecules. Degradation of toxaphene by the microorganism proceeds
under aerobic conditions. Again the degradability of toxicant C
was much higher than A or B by microorganisms. It was not pos-
sible to isolate and determine any of the metabolic products.
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However, because of the stimulatory action of oxidative cofactors
(e.g. NADPH) it was concluded that the metabolism is mainly
carried out in an oxidative enzyme system. The rates of degrada-
tion of A and B were slightly faster than that of the total crude
toxaphene (faster by 10 to 307o) .
Degradation in animaIs _(rats)
Toxaphene was found to be metabolized in the rat via dechlor-
ination and oxidation reactions. The enzyme systems responsible
for such degradation were studied in detail. In short the animal
systems degrade toxaphene in two step reactions. First chlorine
molecules are stripped off from toxaphene via dechlorination
system which is stimulated by a cofactor, NADPH. Oxidative
attacks occur either directly on the less chlorinated members of
the original toxaphene complex, or on the dechlorination reaction
products. One dechlorination product of toxicant C was isolated
and identified. Also several hydroxylation (= oxidation) products
of toxicant C exist as judged by the result of a specific deriva-
tization reaction. Apparently several nonchlorinated sites on
the bornane ring are susceptible for oxidative attacks by the
animal enzymes.
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CHAPTER II
REDUCTIVE DEGRADATION OF P'fiSTIClDAL CHEMICALS
ABSTRACT
A low molecular weight flavoprotein from rat gut walls is
active in reductive reactions, namely, dechlorination and N-
demethylation. Reductive dechlorination is stimulated by FAD
addition. It; provides a logical explanation as to how mammalian
systems could initiate DDT degradation process which involves the
initial reductive dechlorination step as a rate limiting reaction.
INTRODUCTION
Biotransformation of foreign compounds (Xenobiotics) is of
utmost importance to the survival of living organisms against
environmental pollutants. The three major primary enzymatic
processes involved are oyi.dati.ve, reductive and hydrolytic
systems. The mechanisms of oxidation and hydrolysis are reason-
ably well understood. On the other hand, the reductive reactions
are not fully established, and even among the known reactions
their mechanisms are not fully understood (Gillette, 1971).
Exception to this is the reduction of nitro and azo compounds
where microsomes are capable of forming corresponding anilines
under anaerobic conditions (Rose and Young, 1973; Symins and
Jachau, 1974). Recently several papers have appeared describing
reductive reactions on pesticidal chemicals, indicating the
trend for a renewed interest to the role of reductive reactions
in respect to detoxication of foreign compounds. Under anaerobic
conditions, DDT is converted to TDE by avian and mammalian livers,
microsomes, intestinal microflora, and several bacterial and soil
microorganisms.
Our interest in this subject stems out of the observation of
reductive metabolism of DDT to TDE, the first indispensable step
for DDT degradation. It has been debated for some time whether
the reaction is due to the microbial action in the alimentary
system. To be sure there have been some evidence that avian and
mammalian livers are capable of metabolizing DDT to TDE under
anaerobic conditions in vitro. However, the functional meaning
of such systems in v'vo as well as the enzymatic basis of such
activities have not been fullv clarified.
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CONCLUSIONS
The discovery that flavoproteins-flavin cofactor combinations,
whether they are microbially derived or mammalian in origin, are
capable of degrading various xenobiotic substrates in the presence
of FAD or other flavin cofactors under anaerobic conditions helps
clarify many phenomena hitherto considered and unexplainable. For
instance, it has been shown by French and Hoopingarner (1971) that
cell membrane fractions from Escherichia aoli actively convert DDT
to TDE, and that the reaction is strongly stimulated by FAD.
Similarly Wedemeyer (1966) observed earlier that the reduction
activity on DDT in the cell-free extract from Aerobaoter aevogenes
was stimulated by the addition of flavine mononucleotide (FMN)
with light illumination. Such reductive reaction was strongest
under an anaerobic condition at an acidic pH. As for mammalian
systems, it is interesting to note that basically similar bio-
chemical characteristics are found in the DDT reducing enzyme
in the liver. According to Hassall's (1971) description, the
system is heat-stable, is optimal at an acidic pH, and is stim-
ulated by exogenously added riboflavin. Thus, it is likely that
the underlying basic mechanism common throughout these phemonena
is anaerobic reduction by involving flavoprotein-flavin cofactor
systems.
RECOMMENDATIONS
The meaning and significance of such a flavoprotein involved
system in vivo in elimination of toxic foreign compounds must be
further examined, particularly in relation to other documented
reductive systems such as porphyrin -Fe"^ involved systems (Castro,
1964; Khalifa et al. , 1976; Miskus et at., 1965) and specific
NADPH requiring nitro reductase systems (Gillette, 1971; Hitchcock
and Murphy, 1967; Rose and Young, 1973; Symms and Jachau, 1974).
Nevertheless, this system can be easily distinguished from others
by the stimulated effect of FAD (and other flavins), characteristic
heat stability, and acidic pH requirements, and therefore, we feel
certain that its contribution will be properly assessed in the
near future.
MATERIALS AND METHODS
To study the reductive system in mammals, we have chosen
the 20,000 g supernatant fraction from the homogenate of the
intestinal wall (small intestine) of rats as the source.
Anaerobic degradation activities on DDT and mexacarbate were
investigated. At the end of the reaction, the products were
extracted with diethyl ether, and the degradation activity was
mainly monitored by assaying the radioactivity in the aqueous
phase.
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Anaerobic degradation in vitz>o of C-mexaca.rba_te bv_j;he_J^ j!)_00_jg_
supernatant. ~
The incubation mixture consisted of 1 ml of the 20,000 g
supernatant containing 36 rag ~"resh weight tissues equivalent in
phosphate buffer and C-mexacarbate in 10 ul ethanol. The
cofactors FAD or NADPH were added by using 0.2 ml buffer as the
vehicle. The volume of incubation mixture in each tube was
adjusted to 1.2 ml by using phosphate buffer. Incubation was
carried out in Thunberg tubes. After addition of all consti-
tuents, the tube was evacuated and nitrogen was flushed; this
process was repeated two additional times and finally the niuro-
gen was evacuated and the incubation carried out under reduced
pressure. The system was maintained in a metabolic shaker at
37°C for two hours. At; the end of incubation, the products
were extracted with diethyl ether, and analysis was carried, out
by using thin-layer chromatography (TLC), along with autoradio-
graphy, using benzene-methanol (95:5 v/v) as the mobile phase.
Sephadex gel-filtration
In an attempt to partially purify the protein responsible
for such degradation activities the 20,000 g supernatant was
first prepared by homogenizing the rat intestinal wall in two
parts of buffer, and centrifuging it successively at 8,000 g
for 10 minutes and 20,000 g for one hour. Hydrolysis was
carried out by incubation of the 20,000 g supernatant with
protease at the ratio of 2 mg per ml .supernatant. The resulting
hydrolysate was subjected to a gel-filtration column using
sephadex G-75. Absorption at 260 or 280 nm was used to monitor
the column elution pattern, and the degradation activity was
measured individually for each fraction, using water-soluble
1^C-metabolites of mexacarbate as the parameter. Two major
peaks (I and II) were observed as shown in Fig. 1. Hereafter,
peak II will be referred to as flavoprotein preparation,
Anaerobic degradation in vitro of DDT and *"C-mexacarbateby _the
flavoprotein preparatjLon
5
in
rnl of the
case of
DDT or ^C-mexacarbate in 10 yl ethanol
extraction, and expression of data
flavoprotein
"* C-mexacarbate;
Incu-
Incubation mixtures consisted of
preparation in case of DDT and 2.5 ml
FAD or NADPH; and
bation conditions, extraction, and expression ot data are as
mentioned above. With respect to DDT, analysis was carried out
by gas chromatography using an electron capture detector and two
columns, QF-1 and OV-101 at 185°C. The first column was employed
for qualitative identification of the metabolites, while the lat-
ter was used for both qualitative and quantitative estimations.
As for mexacarbate degradation, ether extracts were analyzed by
TLC (mobile phases were: chloroform-methanol 99:1, benzene-
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methanol 95:5, ether-hexane 4:1, chloroform-acetonitrile 4:1)
along with autoradiography on X-ray films against authentic
reference compounds. The spot corresponding to desmethyl
mexacarbate was isolated and characterized by proton magnetic
resonance spectroscopy.
RESULTS AND DISCUSSION
By using the 20,000 g supernatant from rat intestine, we
could first establish that there was indeed a mexacarbate
degradation system which was strongly stimulated by FAD under
an anaerobic incubation condition (Table 2), In this system
mexacarbate was found to be degraded to relatively polar com-
pounds as judged by the increase in radioactivities in the
aqueous phase. In addition, the major ether-soluble product,
N-desmethyl mexacarbate as judged by TLC analyses and autoradio-
graphy also increased in the presence of FAD.
To study the characteristics of this reductive system, sever-
al tests were conducted using the 20,000 g supernatant. The con-
tinuous treatment of the system with 1 ppm of streptomycin from
the time of dissection to the final incubation {added in the buffer)
had no effect on the activity. Neither did the addition of
cations Fe , Co , Mg cause the change in the degradation
activity. The treatment which caused the activity changes were
10~3M HgCl2 (80% inhibition), CO bubbling (28% inhibition),
lO'^M DFP T50% inhibition), 10 3M N-ethyl maliemide (60% stimula-
tion) , and mersalyl acid (130% stimulation). The pH optimum
was found to be 6.
Degradation activity on mexacarbate was associated with the
first 3-5 fractions in each peak. However, further examination
of peak I by rechromatography on sephadex G-25 revealed three
peaks, all with very low specific degradation activity on mexa-
carbate. Hence, it was decided that peak II is a better source
for the degradation systems on mexacarbates.
Upon examination of these two peaks, it was found that
fluorescence spectroscopic characteristics (i.e., fluorescence
peak at 520 nm and excitation peak at 450 nm) of peak II and FAD
were practically identical. Moreover, both FAD and peak II
showed an identical UV-absorption peak at 260 to 270 nm (Fig. 2).
Other characteristics such as the resistance to protease, heat
(up to 90°C for 20 min), etc., agree well with our diagnosis that
the system isolated here is a flavoprotein. The molecular weight
of the flavoprotein is likely to be in the order of 6,000 to
10,000 as judged by its relative elution position against stan-
dard components (i.e., cytochrome c:13,000) on the same sephadex
column.
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DDT and mexacarbate were incubated with this flavoprotein
preparation (peak II) in the presence and absence of either
NADPH or FAD (Table 3). It was found that the addition of FAD,
but not NADPH, greatly stimulated the metabolism of both sub-
strates. The most conspicuous increase was observed in the. pro-
duction of TDK in the case of DDT, and desmethylinexacarbate and
water soluble metabolites in the case of rnexacarbate. The
reaction of TDE formation is reductive dechlorination. Moreover,
the formation of desmethylmexacarbate must involve reductive
desmethylation which has never been reported in any biological
systems to our knowledge.
To study the effects of light and oxygen the tubes were
incubated in the dark under the same conditions as described
before. Two flavin cofactors were used, namely, FAD and ribo-
flavin. The overall degradation activities on mexacarbate, in
the presence of FAD, amounted to 36% as of initial substrate
added (Table 3). When the reaction was carried out in the
dark, the overall degradation activities were decreased to 1/3
and was further decreased in the presence of oxygen to 1/10.
When riboflavin was used as the cofactor, degradation activities
amounted to 8070 of mexacarbate degraded in the presence of
light. When the reaction was carried out: in dark, the same
degradation activity on mexacarbate was experienced. Only in
the presence of oxygen in dark did the degradation activities
decrease to 1/10 of the standard value.
The first question one must ask is whether the flavoprotein
system discovered here represents a genuine enzymatic system or
not. Gillette (1971) has previously noted that azo and nitro
compounds may be nonenzymatically reduced to amines by reduced
cofactors such as NADPH, MADH and reduced flavins such as FADH.
Kamm and Gillette (1963) also showed that: FAD can be reduced by
a purified cytochromc c - NADPH system anaerobically, and that
this reduced FAD was capable of nonenzymatically converting
p-nitro-benzoate to p~aminobenzoate.
The system described herein is not stimulated by NADPH or
NADH, and therefore, is different from the above cases. The
degradation activity of mexacarbate was, on the other hand,
reduced in the absence of light indicating that at least some
part of FAD reduction in the standard reaction scheme is carried
out nonenzymatically by some electron donors. The rest of the
FAD reducing reaction which takes place in the dark could be
carried out by enzymatic systems or by flavoprotein itself. It
has been known that flavoproteins are capable of reducing flavin
cofactors. Thus, in view of the essential role of flavoproteins,
it is most logical to assume that the sole function of flavo-
protein here is to convert FAD to FADH which actually reacts
with DDT or mexacarbate to produce respective degradation pro-
ducts. It must be stressed here that FAD, as low as 10 yg per
reaction tube, resulted in maximum stimulation of reductive
10
-------
activities, and furthermore, this FAD concentration is roughly
in the same magnitude of its level in various animal tissues.
Thus, even though the actual reductive reaction itself could be
carried out nonenzymatic, the phenomenon itself should not be
regarded as irrelevant phenomenon in vivo. Also, flavoproteins
are known to be omnipresent in various biological systems. In
animals, they are present in the liver as well as in the alimen-
tary canal. Thus, along with the evidence that favorable
anaerobic conditions exist in the alimentary canal in vivo, the
chance is that such a flavoprotein-flavin cofactor catalyzed
reaction do play significant roles for degradation of certain
xenobiotics.
TABLE 2
Degradation of C-mexacarbate by the 20,000 g supernatant
from rat intestines*
Degradation Products (%)
Incubation Mexacarbate Desmethyl Other Ether Water
conditions Remaining (7o) Mexacarbate Soluble Soluble
20,000 g
Supernatant 88 1 1 0.5
20,000 g
Supernatant
+ FAD 74 7.5 4 5
20,000 g
Supernatant
+ NADPH 85 2 3 0.5
* Averages of 2-3 independent experiments, each experiment
consisting of duplicate sets.
11
-------
TABLE 3
14,
Anaerobic degradation of DDT and C-mexacarbate by the flavo-
protein preparation from rat intestine.1
Substrate
and
Metabolites
DDT
TDK
DDE
6
6
Flavo- Flavoprotein
protein +• FAD
Flavoprotein
+ NADPH
DDT as^Substrate
71 83
21 7
Control2
96
2
2
Mexacarbate
Desmethyl
mexacarbate
Other ether
87
14
C-Mexacarbate as Substrate
54
22
90
91
solubles
Water-
solubles
2
2
10
14
2
2
1
1
1) Figures in this Table are percent of initial substrate added,
average of 2 to 3 separate experiments; each carried out in
duplicates.
2) Control samples refer to flavoprotein incubated without sub-
strate; then after incubation substrate was added and extracted
immediately with ether.
12
-------
E
c
°
co
-------
2.0
Q)
U
c
ro
_Q
L
O
.0
Flavoprotein
FAD
200
300
Wavelength (nm
Figure 2. UV spectra of flavoprotein preparation and FAD in
phosphate buffer.
14
-------
Cl C
a ci
DDT
r~^ \
IDE
g
OCNHC
H.
o
3
v_
3
Mexacarbate
o
OCN
^3
3
^
3
Desm ethyl
Mexacarbate
15
-------
CHAPTER III
HEALTH EFFECTS OF CHLORDIMEFORM
ABSTRACT
Chlordimeforrn, a novel type of insecticide-acaricide was
found to cause changes in the level of biogenic amines in rats
and cockroaches. It also causes a marked decrease in blood
pressure in the carotid arteries of rabbits: an observation
consistent with the changes in amine levels.
INTRODUCTION
Chlordimeform, also known as Galecon^ or Fundal®, is an
important insecticide and miticide. Despite its importance and
usefulness, exactly how it poisons has been a mystery until
quite recently. Abo-Khatwa and Hollingworth (1972) reported
that chlordimeform was a respiratory poison in cockroaches.
Although such a property of chlordimeform might explain its
effects upon respiratory function (oxyger. utilization) in
insects, it does not explain the effects on the nervous system.
Beeman and Matsumura (1973) found that chlordimef:orm is a potent
inhibitor of monoamine oxidase (MAO) in the rat. Furthermore,
they could demonstrate the actual accumulation of amines in the
rat brain, the expected result of ftAO inhibition. The impor-
tance of these findings in the mechanism of toxic action of
chlordimeform is the subject of this research.
CONCLUSIONS
In conclusion, we have shown that chlordimeform interferes
with amine function in the nervous system in a variety of ways.
Specifically, chlordimeform causes a build-up of the amines
5-hydroxytryptamine and to a lesser extent norepinephine in the
rat brain, prevents the behavioral effects of reserpine in the
rat (reserpine depletes amine stores in the brain), inhibits
monoamine oxidase from rat liver, and causes low blood pressure
in rabbits.
In the American cockroach it directly stimulates the heart,
enhances the toxicity of the. amine, tryptamine, inhibits amine
N-acetyltransferase from cockroach head, causes accumulation of
indoleamines in living cockroaches, and blocks the action of
octopamine in the cockroach nervous system. It also inhibits
tryptantine metabolism in mites.
16
-------
RECOMMENDATIONS
On the basis of the findings summarized here, we propose that
a major biological effect of chlordimeform is its action on amine-
related systems. In the future, chlordimeform and its metabolites
should be tested in more detail for their effects on amine function,
specifically re-uptake, leak-out from presynaptic storage, meta-
bolism (not just MAO but many other enzyme systems) and the range
of its action on amine receptors either as agonist or antagonist.
MATERIALS AND METHODS
General Procedures
The effects of chlordimeform on a variety of amine functions
in insects, mites and mammals were assessed using established
procedures.
Brain Amine Levels and Amine Oxidase (MAO)
To study the change in amine levels male rats were first
treated with 200 mg/kg of chlordimeform, were killed after 1 hr,
and their brains quickly removed. The serotonin and norepinephrine
levels in the whole brain were then measured by fluorometer
(Maickel et al. , 1968). To study the relationship between this
increase in amine levels, and a decrease in amine breakdown by
MAO, we have examined the effect of chlordimeform on the MAO of
the rat liver.
Fluorescence Histochemistry
Histochemical investigation in the roach brain: To ascertain
that the basic amine regulatory mechanisms in the central nervous
system of the American cockroach are similar to those found in
mammalian brain, the histochemical experiment of Frontali (1968)
was repeated. In this experiment biogenic amines were made visible
by treating the freeze-dried roach brain with formaldehyde vapor.
The brains were then embedded and sectioned, and the sections
viewed through a fluorescence microscope.
Measurement of Effects of Chlordimeform on Amine Receptors
in the Cockroach CNS: To study the effects of chlordimeform on
the CNS receptors for biogenic amines to the cockroach we adopted
the method of Nathanson and Greengard (1973). This method
measures stimulation of adenylate cyclase in the cockroach central
nervous system as a result of the addition of exogenous biogenic
amines.
17
-------
RESULTS AND DISCUSSION
Studies in Higher^Animals
Acute toxicity and poisoning symptoms: The acute toxicity of
chlordimeforrn to mammals is relatively low. By our estimation the
acute, intraperitoneal LD5Q values for rats is 200 mg/kg. Acute
oral and intraperitoneal LDc~ values for mice and rabbits are also
estimated to be of the same order of magnitude (CUBA information
sheet).
Initially, chlordimeform causes nervous excitation in rats and
mice. They exhibit tremors and become extremely hypersensitive.
In rats, gradual dilation of pupils took place over a 1-hr period.
Throughout the entire duration of this early excitation period the
animals have not been observed to show cholinomimemtic symptoms as
slowing of the heart beat, salivation, urination or muscle sp>asms.
Following these initial periods of hyperexcitation the animals
gradually fall into a state of sedation. The transition can be
clearly recognized, since they no longer make attempts to run
around. Instead, they stay motionless in a characteristic low
posture unless they are disturbed. The state of sedation induced
by chlordimeform differs from the one induced by general sedatives
such as phenobarbital, in that in the former case the animal
remains alert to disturbances such as clapping of hands, showing
quick jumping and running responses. The recovery occurs gradually
and in most cases, the animals behave seemingly normal within
24 hours.
Physiological and biochemical effects in vivo -. An intraperi-
toneal injection of 200 mg/kg chlordimeform into rabbits caused a
marked decrease in blood pressure of almost 50% within 30 min
of injection. The brain itself also gives evidence of amine
dysfunction. Whole brain level of amines were measured after
chlordimeform treatment.
The result, shown in Table 4, indicates that the amine levels,
particularly that of serotonin, were noticeably high in the brains
of chlordimeform-treated rats.
TABLE 4
Serotonin and norepinephrine levels in whole rat brain
Chlordimeform Control
Serotonin, ug/g wet weight 0.75 ± 0.07 0.44 ± 0.06
Norepinephrine, ug/g wet weight 0.22 ± 0.01 0.18 ± 0.03
18
-------
Biochemical effects in vitro: The results of MAO assay in
rat liver homogenates indicate that chlordimeform is an inhibitor
of monoamine oxidase (MAO). Also, the degrees of inhibitory
potency of the chlordimeform analogs correlate roughly with those
of the toxicity of these compound to mites.
_3
Acetylcholine (ACh) receptor: It was found that 10 M
chlordimeform had no effect on a muscle which was sensitive to
7 x 10 7M acetylcholine. Thus, chlordimeform poisoning is not
mediated by acetylcholine-related systems as far as its excitatory
aspects are concerned.
In vivo antagonism by reserpine: We have observed that chlor-
dimeform acts as a reserpine antagonist in the rat. Reserpine at
a dose of 10 mg/kg, causes immobility, tremor, and muscle rigidity
in the animal. These symptoms appear within 45 min of injection,
and last at least 30 hr. Rats which were pretreated with chlor-
dimeform (50 mg/kg, IP) 10 min prior to reserpine administration
did not develop tremor, and the muscle rigidity was greatly
reduced. These symptoms did not develop in the chlordimeform-
pretreated rats, even after 10 hr of reserpinization. To demon-
strate the antagonistic action of chlordimeform (50 mg/kg) we
injected chlordimeform into rats 2 hr or 30 hr after reserpinization
(10 mg/kg). In both cases, chlordimeform treatment was followed
within 10 min by the complete disappearance of tremor and rigidity
of the muscles. Such an antagonistic action of chlordimeform
could be explained by a possible action of chlordimeform on brain
amines since tremor and muscle rigidity in reserpine-treated rats
have been associated with low levels of dopamine in the brain.
We have decided that cockroaches are the best initial study
material, despite their general insensitivity to chlordimeform.
Cockroaches, particularly American cockroaches, have been exten-
sively used by physiologists and biochemists. They are most
suited for electrophysiological studies. Not only that, but the
American cockroach is one of two species that has been shown to
have a biogenic amine (serotonin) in the central nervous system.
A basic similarity in insect and mammalian amine regulatory
mechanisms can be inferred from the effects of reserpine or a
MAO inhibitor on the levels of biogenic amine in the cockroach
brain.
General observations in the American cockroach: Injection of
chlordimeform is followed within 5 min by typical symptoms of
intoxication. Symptoms include uncoordination, hyperactivity,
arching, and wing flapping. Prostration becomes irreversible
over a period of several hours, and paralysis begins in 10-20 hr.
Chlordimeform did not cause tremor or twitching, and seldom
induced convulsions, even at a high dose (670 ug/g). A sublethal
dose of chlordimeform (420 ug/g) was followed by symptoms which
lasted at least 6 hr before the insects recovered.
19
-------
Preliminary experiments have established tha: chlordimeform
does riot inhibit housefly head cholinesterase even at 10~3M, nor
did it affect the (Na-K) ATPase of the roach head at this concen-
tration .
From our symptomatological observations we suspected central
nervous system (CNS) involvement in chlordimeform poisoning in
American cockroaches.
Electrophysiological studies in the American cockroach: The
effects of chlordimeform on the electrical activity of the exposed
cockroach ventral nerve cord were studied. Within 10-20 min after
flooding the exposed nerve with 10~3M chlordimeform hydrochloride
solution, the first electrophysiological evidence of damage to the
CNS becomes apparent. This always consists of short volleys of
nerve discharges.
Prolonged exposure (up to 2 hr) to this dose of chlordimeform
results in severe hypersensitivity of the CNS, as evidenced by
long trains of repetitive nerve discharge, both spontaneous and
in response to mechanical stimulation of sensory appendages.
Nerve blockade did not: occur, even after 2 hr of exposure to 10~3M
chlordimeform (Beeman and Matsumura, 1974).
Inhibition of tryptamine, DOPA, and serotonin metabolism:
Chlordimeform inhibited metabolism of 1I+C tryptamine in. the cock-
roach head. To show that such an inhibition of tryptamine meta-
bolism by chlordimeform has some physiological consequences
in vivo the joint actions of chlordimeform and tryptamine were
investigated (Table 5). The results clearly indicate that these
two chemicals act synergistically.
TABLE 5
a/
Potentiation of chlordimeform toxicity by tryptamine—
Mortality, %
Chlordimeform Acetone Chlordimeform
water tryptamine tryptamine
Trial 1 10 0 70
Trial 2 20 0 80
— Data are expressed as mortality 30 hr after injection of
tryptamine (500 yg) or t^O. Ten roaches were used for each
combination (total, 60 roaches). Chlordimeform (100 yg/roach)
was given topically with 5 yl of acetone.
20
-------
In addition, the effect of chlordimeform on the in vivo meta-
bolism of externally applied 3H-L-DOPA (a catecholamine precursor)
was studied in male cockroaches. The results (Table 6) show that
norepinephrine accumulates in poisoned insects to a greater extent
than in unpoisoned ones, in agreement with the observation of
Rutschmann et al, (1965) with established MAO inhibitors in the
rat brain.
Visualization _of _brain amine in cockroaches
By the fluorescence histochemical approach it was possible to
show that reserpine had the expected effects of depleting the amine
storage in the roach brain. On the other hand, any changes in
amine levels brought about by either chlordimeform or tranylcypro-
mine (a typical MAO inhibitor) were subtle and were not detectable
by such a crude qualitative assay method.
TABLE 6
3
In vivo metabolism of H-L-DOPA and accumulation of amines as
affected by chlordimeform in the American cockroach— —
Amounts, % of recovered
radioactivity—
Control
L-DOPA
Norepinephrine
Dopamine
Other metabolites
5,
11.
9.
74,
,85
.05
.60
.17
Total
11.
18.
8,
61.
.36
.36
.65
.75
a/
— After 24 hr metabolism by male roaches. Roaches were extracted
with 10 volumes of acidified n-butanol and the debris removed
by brief centrifugation. The supernatant solution was extracted
with 1 ml of 0.IN HC1, with 15 ml of n-hexane added to aid
separation. Solvent and aqueous phases were concentrated an
spotted on cellulose MN 300 TLC plates along with nonradioactive
reference compounds, and the plates were developed in methanol-
benzene-n-butanol-water (4:4:4:1).
— Chlordimeform given topically at a dose of 100 yg/roach 3 hr
prior to the oral administration of 3H-L-DOPA. The total
poisoning time for chlordimeform was 27 hr.
C /
—' Results expressed in percentages of applied radioactivity
(0.4 nmole of 3H-L-DOPA, specific activity 15 Cl/nmole)
recovered in each fraction. Average of two determinations.
-------
Measurement of the effects of chlordimeform on amine
receptors in the cockroach CNS: It immediately became apparent
that the in vitro method was not sensitive enough to measure the
increcise in the biogenic amine level in vivo as a result of either
MAO I or chlordimeform treatment. However, when their effects were
tested in situ (by using isolated half ganglia) two important
phenomena became known (Table 7). First chlordimeform itself
does not stimulate the adenyl cyclase activity (i.e., it does not
act as a false transmitter in this preparation) and second (at
10"3M) it instead prevented exogenously added octopamine from
achieving the maximum stimulation of adenylate cyclase activity.
On the other hand, chlordimeform at 1.0"5M was found to increase
the rate of cockroach heart beat.
TABLE 7
Effect of chlordimeform on adenylate cyclase and octopamine-
induced stimulation of adenylate cyclase in roach thoracic
ganglia in situ—
_
3
H-c-AMP bound ± S.D., %-•
Control 12.9 ± 0.3
Chlordimeform, 1 x 10~3M 13.3 ± 0.7
Control 15.0 ± 2.6
Octopamine, 2.5 x 10~4M 6.4 ± 1.3
Octopamine, 2.5 x 10"4M + 11.6 ± 0.7
_3
chlordimeform 1 x 10 M
a/
—' Intact hemiganglia (containing adenylate cyclase) were incubated
with no treatment, chlordimeform alone, octopamine alone, or
octopamine and chlordimeform together, and accumulated c-AMP
was measured by competive binding to a c-AMP binding protein.
Procedural details are given in Nathanson and Greengard.
i / O
— Data are expressed as 70 H-c-AMP bound ± standard deviation.
All values are means of three to six determinations. Decreased
binding indicates increased cyclase activity.
So far the direct pieces of evidence supporting the "amine-
theory" in cockroaches are: (a) chlordimeform inhibits metabolism
of l4C-tryptamine in the roach head homogenate in vitro at I50 of
4.4 x 10~1*M; (b) the killing action of chlordimeform is potentiated
by tryptamine, which by itself is noritoxic to the roaches; (c)
increase in indolamines levels are observed in the whole body after
22
-------
application of chlordimeform in vivo, and changes occur in meta-
bolic patterns of serotonin in vioo; (d) chlordimeform at 10"3M
(we have not tested lower concentrations) blocks the stimulatory
action of octopamine on adenyl cyclase in the roach thoracic
ganglion; (e) chlordimeform increases the heartbeat rate in
isolated roach heart preparations, the phenomenon being compatible
with the report that the cockroach heart is innervated by mono-
amine-containirig axons (Miller, 1975),
Indirect evidences are that tranylcypromine, a typical MAO
inhibitor, produces very similar symptoms in the American cockroach.
Also, several MAO inhibitors are good acaricides against the cheese
mite, Trycphagus putrescens.
On the other hand, there are several unanswered problems.
For example; we have so far been unable to detect an increase in
amine levels in vivo in the central nervous system of the cockroach.
Also, it has recently been shown that tryptamine is not metabolized
by oxidative deamination in the roach brain, but rather by
N-acetylation (Nishimura et ai. , 1975). In view of the antagonistic
action of mixed-function oxidase inhibitors (such as sesamex and
piperonyl butoxide), there is a possibility that one of the meta-
bolic products, rather than chlordimeform itself, is an active
agent, at least: in certain species.
These questions, however, do not directly challenge the work-
ing hypothesis that the toxicity of chlordimeform in vivo is
related to the changes in biogenic amine levels in quantity and/or
In quality.
In conclusion, we have shown that chlordimeform can indeed
affect amine regulatory mechanisms and in some instances, can
react with certain amine receptors. On that basis we have proposed
a working hypothesis that chlordimeform acts upon amine-related
systems. Certainly much more information is needed to confirm or
deny such a hypothesis. In the future, chlordimeform and its
metabolic products should be tested for their effects on amine
re-uptake, leak-out from the presynaptic storage, metabolism
(not just MAO but many other enzyme systems) and the range of its
action on amine receptors either as agonist or antagonist.
The key to the safe use of any new pesticide is to provide
the basic toxicological data based upon logical explanation of
its action mechanism and its side effects. Chlordimeform and its
analogs and metabolites possess very peculiar and unfamiliar
properties, particularly as pesticides. It appears very important
to make efforts at this stage to understand the basic mechanisms
of their actions. Hopefully our initial efforts are providing
the means to meet the challenge.
23
-------
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Hitchcock, M. and S. D. Murphy. 1967. Enzymatic reduction of
0,0-diethyl 0-(4-nitrophenyl) phosphorothioate, 0,0-diethyl
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24
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Matsumura, F. and R. W. Beeman. Biochemical and physiological
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Turner, W. Y. 1977. Paper presented to the Division of Pesticide
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Science 152: 647.
26
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APPENDIX
List of technical publications
resulting from this research project (1973-1978)
Matsumura, F. and H. J. Benezet. 1973. Studies on the bioaccumu-
lation and microbial degradation of 2,3,7,8-tetrachlorodi-
benzo-p-dioxin. Environ. Health Perspectives 5: 253.
Benezet, H. J., and F. Matsumura. 1973. Isomerization of y-BHC to
a-BHC in the environment. Nature 243: 480.
Beeman, R. W. and F. Matsumura. 1973. Chlordimeform: a pesticide
acting upon amine regulatory mechanisms. Nature 242: 273.
Matsumura, F. 1973. Degradation of pesticide residues in the
environment. In "Environmental Pollution by Pesticides".
C. A. Edwards, ed. 484-513, Plenum Press, N.Y.
Doherty, J. D. and F. Matsumura. 1974. A highly ion-sensitive
ATP-phosphorylation system in lobster nerve. Biochem. Biophys
Res. Commun. 57: 987-992.
32
Doherty, J. D. and F. Matsumura. 1974. DDT effect on p incor-
poration from -labelled ATP into proteins from lobster nerve.
J. Neurochem. 22: 765-772.
Benezet, H. J. and
metabolism of
Chem. 22: 427.
Beeman, R. W. and F
chlordimeform
4: 325-336.
F. Matsumura.
mexacarbate by
1974. Factors
microorganisms
influencing the
J. Ag. Food
. Matsumura. 1974. Studies on the action of
in cockroaches. Pesticide Biochem. Physiol.
Conaway, C. C. and F. Matsumura. 1975
utilization of thymidine by TCDD.
Toxicol. 13: 152.
Inhibition of
Bull. Environ.
cellular
Contain.
Nelson, J. 0. and F. Matsumura. 1975. A simplified approach to
studies of toxic toxaphene components. Bull. Environ. Contain.
Toxicol. 13-. 464-470.
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Nelson, J. 0. and F. Matsumura. 1975. Separation and comparative
toxicity of toxaphene components. J. Agric. Food Chem. 23:
984-990.
Doherty, J. D. and F. Matsumura. 1975. DDT effects on certain ATP
related system in the peripheral nervous system of the lobster,
Homarus americanus. Pestic. Biochem. Physio 1. 5: 242-252.
Matsumura, F., R. W. Howard and J. 0. Nelson. 1975. Structure of
the toxic fraction A of toxaphene. Chemosphere 5: 271-276.
Furukawa, K. and F. Matsumura. 1975. Microbial metabolism of
PCB's: studies on the relative degradability of PCB com-
ponents by Alkaligenes sp. J. Agric. Food Chem. 24: 251-256.
Matsumura, F. 1975. Role of microorganisms in altering insecticidal
residues in environment. Proc. North Central Br. Entomol.
Soc. Am. 30: 43-48.
Matsumura, F., H. J. Benezet and K. C. Patel. 1976. Factors
affecting microbial metabolism of y-BHC. J. Pestic. Sci.
1: 3-8.
Matsumura, F. and R. W. Beeman. 1976. Biochemical and physiological
effects of chlordimeform. Environ. Health Perspectives
14: 71-82.
Matsumura, F. 1975. Mechanisms of action of insecticidal and
acaricidal chemical. Proc. Int. Controlled Release Pestic.
Symp. pp. 200-208. Wright State University, Dayton, Ohio.
Matsumura, F. 1974. Microbial degradation of pesticides. In
Survival in Toxic Environments. Kahn, M.A.Q. and J.P. Bederka,
Jr., Eds., Academic Press, N.Y. pp. 129-154.
Conaway, C. C., B. V. Madhukar and F. Matsumura. 1977. P,p'-DDT
studies on induction mechanisms of microsomal enzymes in
rat liver systems. J. Environ. Res. 14: 305-321.
Chandurkar, P. S., F. Matsumura and T, Ikeda. 1978. Identifi-
cation and toxicity of toxicant Ac, a toxic component of
toxaphene. Chemosphere 2: 123-130.
Beeman, R. W. and F. Matsumura. 1978. Formamidine pesticides -
action in insects. In Pesticide and Venum Neurotoxicitv.
Shankland, D.L., R.M'. Hollingworth and T. Smyth, Jr., eds.
Plenum Press, N.Y. pp. 179-188.
Esaac, E. G. and F. Matsumura. 1978. A novel reductive system
involving flavoprotein in the rat intestine. Bull. Environ.
Contam. Toxicol. 19: 15-22.
28
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Manuscripts accepted for publication
Chandurkar, R. S. and F. Matsuraura. 1978. Metabolism of toxicant
B and toxicant C of toxaphene in rats. Bull. Environ. Contam.
Toxicol. In Press.
Ward, C. T. and F. Matsumura. 1978. Fate of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin (TCDD) in a model aquatic environment.
Arch. Environ. Contam. Toxicol.
Esaac, E. G. arid F. Matsumura. 1978. Roles of flavoproteins in
degradation of mexacarbate in rats. Pestic. Biochem. Physiol.
In Press.
29
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GLOSSARY
adrenomimetic: mimicking the actions of norepinephrine or
dopamine.
algae: primitive plants, one or many celled, usually aquatic and
capable of growth in mineral materials via energy from the
sun and the green coloring material, chlorophyll.
anaerobic: refer to reaction systems carried out in the absence
of oxygen.
antagonism: reduction of negation of the effects of one drug by
another drug.
bioassay.- assay method utilizing biological organisms to assess
potential environmental toxins.
biogenic amines: amino acid-derived chemicals, including
norepinephrine, dopamine, octopamine, and serotonin, which
function as intercellular messengers (transmitters) within
the nervous system or between nerve and muscle cells, or
nerve and gland cells.
biotransformation: conversion of a foreign chemical, such as an
insecticide, to new products (metabolites) in living
organisms. These products are usually with lower or higher
biological activity.
cholinergic: a type of nerve cell, whose outgoing chemical
messages are mediated by the transmitter, acetylcholine.
cyclic AMP: an intracellular "second messenger" present in
message-receiving cells, whose production is stimulated by
the primary messengers (e.g. biogenic amines) and which
is directly responsible for the physiological actions of
the primary messengers.
dechlorination: elimination of a chlorine atom.
dehydrochlorination: elimination of a HCl moiety from a chemical,
endocrine: involving long-range chemical messengers, e.g.
blood-borne hormones.
flavin: an enzyme cofactor required in the catalytic conversion
of substances to metabolites by certain enzymes.
flavoprotein: a protein that contains in its structure a flavin
group, such as FAD, FMN.
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ganglia: nerve centrs, consisting of masses of nerve cells,
where nervous information is analyzed and interpreted.
heme: a biochemical that contains iron such as cytochromes and
hemoglobin.
homogenate: a suspension of breakdown-cells in buffer solution.
The cells are breakdown by means of certain equipment,
such as blender.
hydrolytic: breaking a molecule to more than one part, e.g.
ester is hydrolyzed to acid and base.
larvae: immature forms of insects which undergo a complete type
of development.
LCcrv: lethal concentration of some toxic substance which will
kill 507o of the assayed population.
metabolism: alteration of chemicals by a biological organism
usually in response to intake of nutrients.
microsomes: small particles inside the living cells, obtained
by preparing cell homogenate and centrifugation at
high speed in vacuum.
monoamine oxidase (MAO): an enzyme which destroys biogenic amines
and which is important in regulating biogenic amine levels.
NADPH: an enzyme cofactor required for many oxidative as well as
reductive conversion of insecticides in living organisms.
Its chemical name is: nicotinamide adenine dinucleotide
phosphate.
N-demethylation: elimination of a methyl group.
non-target organisms: organisms which are susceptible to pesti-
cide poisoning(s) even though they are not the pest species
themselves.
octa: containing or having eight parts or units.
oxidative: a process by which a given chemical is oxidized,
e.g. incorporation of an oxygen atom into the chemical.
receptor: a specialized area of a message-receiving cell, which
binds the chemical messenger, and transmits the message to
the rest of the cell, e.g. by dispatching the internal
messenger, cyclic AMP.
reductive: a process by which a given chemical is reduced, e.g.
elimination of an oxygen atom or incorporation of a hydrogen
atom into the chemical.
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reserpine: a sedative which produces sedation and several other
behavioral effects by depleting biogenic amines from the
central nervous system.
Sephadex G-75: a carbohydrate polymer used in separation and
isolation of biochemical, e.g. proteins, according to their
molecular weight.
supernatant: the clear solution that results from centrifugation
of the cell homogenate.
synergism: facilitation or enhancement of the effects of one
drug by another drug.
Thunberg tubes: glass tubes with a side arm and ground-glass
stopper that are used to carry out reactions under anaerobic
conditions.
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TECHNICAL REPORT DATA
(Please read Instructions an the reverse before completing)
REPORT NO.
EPA-600/1-78-065 I
TITLE AND SUBTITLE
MECHANISMS OF PESTICIDE DEGRADATION
AUTHOR(S)
Fumio Matsumura
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Entomology
University of Wisconsin
Madison, Wisconsin 53706
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
ANIZATION CODE
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
R-801060
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This research project was initiated with the overall objective of determining
(1) the chemical structures of toxic components of toxaphene, (2) to study anaerobic
metabolism to degrade toxaphene and other pesticides, and (3) to understand toxic
action mechanism of chlordimeform.
As a result of intensive efforts the molecular structures of three of the most
toxic principles of toxaphene were identified. Together these comprise at least 70%
of toxaphene's toxicity toward mice. This is the first time that the structure of
toxic components of toxaphene became apparent despite the widespread use (over 1
billion pounds, which is comparable to DDT) of toxaphene in the last 3 decades.
Toxaphene on the other hand degrades relatively faster than other chlorinated
pesticides such as DDT and dieldrin. The reason for it is that toxaphene is susceptible
to reductive degradative forces.
Chlordimeform was found to affect amine regulatory mechanisms in animals. Such
actions explain some of the subtle effects of this pesticide on animals. Inasmuch
as that biogenic amines are known to play many important biological roles such as
controlling emotion, behavior and circulatory functions of the body.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
pesticides
toxicity
chlorohydrocarbons
molecular structures
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b.IDENTIFIERS/OPEN ENDED TERMS
toxaphene
chlordimeform
19 SECURITY CLASS (This Report]
UNCLASSIFIED
2ff SECURITY CLASS (This, page!
UNCLASSIFIED
COS AT I Field/Group
07 C
06 T
21 NO. OF PAGES
40
22 PRICE
EPA Form 2220-1 (Rev. 4-771 PREVIOUS EDITION > s OBSOLETE
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