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This is to certify that the

dissertation entitled

NOVEL PESTICIDES AFFECTING MITOCHONDRIAL FUNCTIONS '

presented by

Gadelhak Gaber Gadelhak

has been accepted towards fulfillment
of the requirements for

Ph . D degree in Entomology

W2 ltWW

Major professor

 

 

Date §W%_ 27; ’%2

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

LIBRARY

Michigan State

; University

 

 

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TO AVOID FINES return on or before date due.

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DATE DUE DATE DUE DATE DUE

 

 

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MSU Is An Affirmative ActhNEqual Opportunity Institution

mummy”

 

NOVEL PESTICIDES AFFECTING MITOCHONDRIAL
FUNCTIONS

By
Gadelhak Gaber Gadelhak

A DISSERTATION

Submitted to
Michigan State University
In Partial Fulfillment of the Requirement
for the Degree of

DOCTOR OF PHILOSOPHY

Department of Entomology

1992

ABSTRACT

NOVEL PESTICIDES AFFECTING MITOCHONDRIAL
FUNCTIONS

BY

Gadelhak Gaber Gadelhak

'Several new insecticides with novel structures and unknown
modes of action have been studied. Sulfluramid (N-ethyl
perfluorooctane sulfonamide; FinitronTM) causes delayed mortality in
German cockroaches when fed in the diet at 0.01%-1%. lts N-
desethyl analog (NDES) and perfluorooctanesulfonic acid (PFOSA)
were similarly toxic, but somewhat faster acting. These compounds
were less toxic to field strains. Piperonyl butoxide (PBO)
antagonized the toxicity of sulfluramid and NDES. No effect of P80
on PFOSA toxicity was observed. The susceptible strain eliminated
SULF faster than the field strain. Cockroaches injected with or fed
SULF, NDES or PFOSA showed increased 002 production. A clear delay
in respiratory stimulation was observed with SULF but not with
NDES or PFOSA. These results suggestthat SULF acts indirectly, by
conversion to NDES and/or PFOSA.

Sulfluramid was a poor uncoupler against mitochondria from several
sources. NDES was 7- 10 times more active. Despite its ability to

stimulate respiration in vivo, PFOSA did not have any direct

 

 

 

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uncoupling activity but when mixed with some alkylamines, its
uncoupling effect was strongly enhanced. In studies using different
alkylamines, poylamines and phospholipids, only tributylamine' and
dihexylamine were active in this regard. Further study suggests that
PFOSA may act as an ionophore for K+ and the combination with a
lipophilic amine enhances protonophoric activity through ion-pair
formation.

A second compound, EL-436; fenazaquin, {4-[(4-(1,1-dimethyl-
ethyl)phenyl)ethoxy] quinazoline} is an acaricide. Both mitochondrial
and cellular studies proved that the compound acts as a powerful
inhibitor of the mitochondrial electron transport at complex I of the
respiratory chain. Its effects are similar to those of rctenone. The
inhibitory activities of fenazaquin and its more potent analogs were
comparable to that of rotenone with ISO values in the 10-50 uM
range. This action was confirmed in vivo by studying respiratory
inhibition by fenazaquin in cockroaches.

The third compound; EL-499 (2'-bromo-4'-nitro-perflucrocyclohexyl
carboxanilide) is an experimental insecticide. This compound acts as
an extremely powerful uncoupler of oxidative phosphorylation with
activity on insects and vertebrate mitochondria at concentrations
below 10 nM. Plant mitochondria are about 50-fold less sensitive.
Respiratory stimulation and mortality in cockroaches occur atdoses

below 1 ug/g.

TO MY FATHER

 

l woui
members:
Zabik and
PhD progr
Hollingwo

encourage

To all
for being 1
Dr. Daniel

Rahardja,

My gra
Esra and I

 

 

Acknowledgments

I would like to extend my appreciation to my guidance committee
members: Dr. Alfred Haug, Dr. Shealah Ferguson-Miller, Dr. Matthew
Zabik and Dr. Mark Whalon for their advice and support throughout my
PhD program. Special and cordial thanks are to Dr. Robert M.
Hollingworth, my major professor, for his patience and

encouragement.

To all my friends and colleagues, thanks for your friendship and
for being there when I needed you, especially, Dr. Joel M. Wierenga,
Dr. Daniel Herms, Wendy Peiffer, Chris Vandervoort and Utami

Rahardja.

My gratitude and appreciation goes to my big family here, Faiza,
Esra and Ahmed, and to my bigger family back home, my mother, my

brother and my sisters

 

LIST OF T
LIST OF F
LIST OF A

INTRODUI
Chapter 1:

Chapter 2:

Chapter 3; ‘

 

 

Table of Contents

LIST OF TABLES .
LIST OF FIGURES.
LIST OF ABBREVIATIONS

INTRODUCTION . .
Chapter 1: LITERATURE REVIEW.

Mitochondrial functions .

-Uncouplers of oxidative phosphorylation

-New insecticides that act on mitochondrial functions
-References.

Chapter 2: Toxicity and metabolism of the new insecticide
sulfluramid (Finitronm) cockroaches

-|ntroduction .
Materials and methods
Chemicals . .
German cockroaches .
Cockroach bioassays . .
In vivo metabolism studies .

_ Monitoring cockroach respiration in vivo
-Results.
-Discussion .

-References .

Chapter 3: The mitochondrial effects of the new slow acting
compound, sulfluramid (Finitronm) and its metabolites

-lntroduction
-Materials and methods
Materials
Mitochondrial isolation . .
Assay of mitochondrial respiration
In vitro metabolism of sulfluramid
Artificial membrane studies with
cytochrome aaa .
Mitochondrial swelling
-Results . . .
-Discussion .
-References .

v_1

38
39

I- 39

39
40
40
41
42
43
63
69

71

72
72

73
74
75

75
77

93
105

 

Chapter 4

Chapter 5

SUMMARI
Appendx 1

Append-ix 2

 

 

Chapter 4: Inhibition of the mitochondrial respiration
by the quinazolinamine pesticide, fenazaquine (EL-436)

Introduction . .

Materials and Methods
Chemicals .
Mitochondrial isolation . .
Assay of mitochondrial respiration
Sf-9 cell respiration .
Cockroach respiration in vivo

Mite toxicity assays

-Results . . .

-Discussion .

-References .

Chapter 5: The uncoupling activity of the new
polyfluorocaboxyanilideinsecticide, EL-499

-lntroduction . .

- Materials and Methods
Chemicals . .
Mitochondrial isolation
Assay of mitochondrial respiration
ATPase activity .
Cockroach respiration in vivo

-Results and Discussion

-References .

SUMMARY AND CONCLUSIONS
Appendix 1: Voucher specimen data

Appendix 2: The role of prostaglandins in insect reproduction
and the action of formamidines as reproductive toxicants

-Abstract.

-lntroduction .

-Short literature review .
The presence of PG' 5 in insects and mites .
The function of PG' 5 in insects .
Effects of PG' 8 synthesis inhibitors In insects

-Materials and Methods
Tobacco budworm studies
Onion fly Studies . .

In vitro PG synthesis studies

-Resu|ts . . . . . .

-Discussion .

-References .

v11

107

108
108
108
109
109
110
111
111
112
123
126

128

129
129
129
132
133
134
135
135
141

142
147 '

150

151
152
153
153
156
158
159
159
160
161
163
171
174

List of Tables

Table 1: Stoichiometries of proton translccation in electron
transport and oxidative phosphorylation. . . . . 17

Table 2: Percent mortality of susceptible and field (tolerant)
German cockroach males over time using different
concentrations of sulfluramid (SULF) in the diet. . 44

Table 3: Percent mortality of susceptible and field (tolerant)
German cockroach males over time using different
concentrations of N-desethyl sulfluramid (NDES)
inthediet...............46

Table 4: Percent mortality of susceptible and field (tolerant)
German cockroach males over time using different
concentrations of perfluorooctanesulfonic acid (PFOSA)
inthediet...............47

Table 5: Percent mortality of susceptible and field (tolerant)
' German cockroach males over time using two
concentrations of sulfluramid (SULF) in combination with
piperonyl butoxide (P80) in the diet. . . . . . . 52

Table 6: Percent mortality of susceptible and field (tolerant)
German cockroach males over time using three
concentrations of N-desethyl sulfluramid (NDES) in
combination with piperonyl butoxide (PBO)
inthediet. ..............54

Table 7: Percent mortality of susceptible and field (tolerant)
German cockroach males over time using three
concentrations of perfluorooctanesulfonic acid (PFOSA) in
combination with piperonyl butoxide (PBO)
inthediet. ..............55

viii

 

Table 8:

 

Table 9:

 

 

1

Table 10

Table 1

Table 1

Table 8: The rate of 002 production by German cockroach males
following the injection of inhibitors (KCN, rotenone) and .
uncouplers of oxidative phosphorylation (EL—499). Numbers
aremeansofn-2-4.. . . . . . . . . . .58

Table 9: The effects of sulfluramid and NDES on the respiration of
artificial Iiposomes reconstituted with the enzyme
cytochrome oxidase. . . . ~ . . . . . . . . 88

Table 10: The effects of monoamines, polyamines and phospholipids
as possible ion-pairing agents on the uncoupling activity of
perfluorooctanesulfonic acid (PFOSA) using rat liver
mitochondria. . . . . . . . . . . . . .91

Table 11: Structure-activity relations of selected quinazolines on
mitochondrial and Sf-9 cell respiration and toxicity
of mites. . . . . . . . . . . . . . . . 122

Table 12: The presence of prostaglandins in insects of different
orders and their effect on stimulating oviposition. . 155

ix

 

 

Figure 1
I
I

Figure 2.

 

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i
<
I
Figure :
Figure

FiQUTe

FigUre

FiQUre

List of Figures

Figure 1: The number of pesticides submitted to the WHO for
evaluation from 1940 to 1986 compared to the appearance
of resistance in mosquito species.

(After Georghiou, 1986). . . . . . . . . . 7

Figure 2: Schematic diagram showing the components of the
mitochondrial electron transport chain.
Flavoproteins (FMN, FAD) and iron-sulfur clusters (Fe-S)
transfer the electrons from the donor (NADH) to
coenzyme Q (Q). The b cytochromes interface with
cytochrome c (a soluble protein) which then transfers
the electrons to cytochromes aa3. Some of the inhibitors
of this process are listed. . . . . . . . . . 11

Figure 3: A schematic diagram showing the coupling between the
process of oxidation (electron transfer) and the

phosphorylation of ADP. H+: protons, e‘: electrons,
Aum: the electrochemical potential. . . . . . . 20

Figure 4: Schematic diagram showing the shuttle mechanism that
describes the movement of protons (H+) across the inner
mitochondrial membrane by the anionic uncoupler (HA).
(Modified after McLaughlin and Digler, 1980). . . . 22

Figure 5: The structure of (A) sulfluramid (Finitron) and
(B)fenazaquin (EL-436). . . . . . . . . . 27

Figure 6: Cumulative mortality of German cockroaches of the
susceptible (S) and the field (F) strains as a result of
feeding 0.01% sulfluramid (SULF). Data are means 3; SE
ofn-3-6...............48

Figure 7: Cumulative mortality of German cockroaches of the
susceptible (S) and the field (F) strains as a result of
feeding 0.01% N-desethyl sulfluramid (NDES). Data are
meansiSEofna3-6.. . . . . . . . . . .50

 

 

Figure 8

l

 

Figure 9

Figure

Figure

Flgul

Flgu

Fig

Figure 8: Cumulative mortality of German cockroaches of the
susceptible (S) and field (F) strains as a result of
feeding 0.01% perfonrooctanesulfonic acid (PFOSA).
Data are meansiSEofn-a-G. . . . . . . . 51

Figure 9: Effects of piperonyl butoxide (P80, 0.5%) on the toxicity
of sulfluramid (0.01% bait) in susceptible and field strains
of German cockroaches. . . . . . . . . . . 53

Figure 10: Elimination of 1“C-sulfluramid after injection by males
of susceptible and field-strain German cockroaches. Data
are means 1; SE of n = 3-4. . . . . . . . . . 57

Figure 11: C02 production following the injection of male German
cockroaches with different concentrations of sulfluramid
(SULF), N-desethyl sulfluramid (NDES) and perfluorooctane-
sulfonic acid (PFOSA). Insects were monitored immediately
after injection for 50 min. . . . . . . . . . 60

Figure 12: 002 production following the injection of male German
cockroaches with 4 concentrations Of sulfluramid.
.All treatments were monitored for a period of 9 h. Data are
meansofn-2-4. ............61

Figure 13: 002 production of male German cockroaches after
feeding 2 concentrations of sulfluramid (0.1% & 1 ..0%) All
treatments were monitored for 20 min every 6-8 h for 70 h.
Dataaremeansofn-Z-a . . . . . . . . .62

Figure 14: Possible metabolic routes of sulfluramid by
microsomes or mitochondria . (*) shows the position of the

1“C-label of sulfluramid used in this study. . . . 67

Figure 15: Uncoupling of rat liver mitochondria by sulfluramid
compared to the standard uncoupler CCCP. . . . . 78

X1

 

Figure 1‘

 

Figure

 

Figure

Figure

Figurr

Figul

Flgu

Figure 162- A schematic tracing from a Clark oxygen 'electrode

Figure

Figure

Figure

Figure

Figure

Figure

showing the effects of the N-dese’thylated metabolite
(NDES) in the presence and absence of the ATPase
specific inhibitor, oligomycin, on rat liver
mitochondria. . . . . . . . . . . . . .81

17: The uncoupling effects of sulfluramid (SULF), and the
N-desethylated (NDES), N-methyl (MSULF), N-isopropyl
(ISULF), and unfluorinated (USULF) analogs of sulfluramid,
perfluorooctanesulfonic acid (PFOSA) and

octanesulfonic acid (OSA). . . . . . . . . . 82
18: Metabolism of 14C-sulfluramid by rat liver subfractions
and the effect of NADPH and NADH. . . . . . . 83
19: A schematic diagram showing an artificial liposome

reconstituted with the enzyme cytochrome oxidase (cyt
aa3) and the possible orientation of cytochrome c (cyt c),
and the effects of valinomycin and an uncoupler. (A') on
the movements of the protons [H+]

and potassium [K+].. . . . . . . . . . . . 84

20: A schematic tracing from a Clark oxygen electrode
showing the effects of valinomycin and an uncoupler on an
artificial membrane reconstituted with cytochrome
oxidase. .8...........7

21: A schematic tracing from a Clark oxygen electrode
showing the effect caused by the addition of tributylamine
(TriBA) to perfluorooctanesulfonic acid (PFOSA) and
octanesulfonic acid (OSA). . . . . . . . . . 90

22: The effect of using varying ratios of PFOSA and TriBA on
mitochondrial uncoupling. . . . .. . . . . . 92

x11.

 

Figure I

 

Figure 2

Figure 1

Figure

Figure

Figure

Figuri

Figure 23: Representatives recordings of swelling studies with rat
liver mitochondria.
1) the effects of sulfluramid (A) before the addition of
valinomycin and (B) after the addition of valinomycin.
2) the effects of (A) CCCP, and (B) NDES.
3) The effects. of the PFOSA and TriBA (A) before the
addition of valinomycin and (B) after the addition of
valinomycin...............94

Figure 24: Suggested model explaining the mechanism by which
PFOSA ion-pairs might uncouple oxidative A
phosphorylation.

R3NH+z alkylamine cation. . . . . . . . . . 102

Figure 25: The effect of fenazaquin and rotenone on C02
production by German cockroach males. The effect of
single dose of KCN is also shown. . . . . . . . 113

Figure 26: Representative polarographic traces showing the
effect of CCCP. KCN and fenazaquin on respiration of
the Sf-9 cell line. . . . . . . . . . . . . 115

Figure 27: Representative polarographic traces showing the effect
of fenazaquin on rat liver mitochondria in the presence of
glutamate. The effect is shown with and without the
additiOn of an uncoupier (CCCP). . . . . . . . 117

Figure 28: Representative polarographic traces showing the effect
of fenazaquin on rat liver mitochondria in the presence of
succinate. The effect is shown with and without the
addition of “an unc0upler (CCCP). . . . . . . . 119

Figure 29: The chemical structure of pesticides that showed
uncoupling selectivity. A) karathane (Dinocap, Pesticide
Manual, 1979). B) arylhydrazone
(Holan and Smith, 1986). . . . . . . . . . . 130

x111

 

Figure 1

Figure 2

Figure 8

Figure I

Figure 3

 

Figure 3

Figure 3
Figure 3

FiQUre 3;

Figure 30: The chemical structure of EL-499 with the four analogs
tested in the structure activity relationship. . . 131

Figure 31: The uncoupling activity of EL-499 on Manduca sexta
midgut mitochondria (THMM), rat liver mitochondria
(RLM) and etiolated corn shoot mitochondria (CSM). . 136

Figure 32: The The effect EL-499 and three of its analogs on the
ATPase activity of rat liver mitochondria. . . . . 137

Figure 33: The in vivo effect of injected EL-499 on respiration in
German cockroach males. . . .1 . . . . . . . 138

Figure 34: Mechanism of prostaglandin biosynthesis ( after
Samuelsson, 1987).. . . . . . . . . . . . 162

Figure 35: The effect of aspirin, chlordimeform (CDM) and
demethylchlordimeform (DCDM) on the number of eggs laid
by females of the tobacco budworm
Heliothis virescens. . . . . . . . . . . . 164

Figure 36: The effect of CDM and DCDM on the number of eggs laid
. by the females of the onion fly Delia antiqua. . . 165

Figure 37: The inhibition of prostaglandin synthetase (PG
synthetase)-by formamidines (CDM and DCDM). . . 166

Figure 38: The effect of octopamine on the onion fly oviduct
contractility.
A) Control. B) Octopamine 1 mM. . . . . 167

Figure 39: The effects of both octopamineand prostaglandin E2
(PGE2) on the contractility of the onion fly oviduct.
A) Control B) Octopamine 1 mM.
C) PGF2a1 mM. D) Octopamine 1 mM.

E) PGF2a1 mM. 169

xiv

 

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List of Abbreviations

: Anion

: Adenosine-5'-diphosphate.
1-Anilino-B-naphthalenesulfonate

: Adenosine-5'-triphosphate.

: Butylated hydroxytoluene

: Carbonylcyanide m-chlorophenylhydrazone

: Critical micelle concentration

: Cyanide anion

: Carbon monoxide

: Carbon dioxide

: Dibutylamine

: Diethylamine

: Dihexylamine

: Difference in electrochemical potential for protons

between two bulk phases separated by a membrane.

(kcal/mol or kJ/mol).

: Dimethylsulfoxide

: Deoxyribonucleic acid

: Proton-motive force. The electrochemical potential
gradient for protons between two bulk phases separated
by a membrane. (AuH+/F mV).

: Difference in pH between two sides of a membrane
(pHin - pHout).

 

A‘i’

EDTA

EGTA

FADHz

Fe-s

HMN
H+
lSLHJ
KCN
MFG
'WSU
i4AI
NAI
NEH

(32

Pa
PF

Pr

A‘I’

EDTA

EGTA

FADH2

Fe-S

FMN
H+
ISULF
KCN
MR)
MSULF
NADH
NADPH
NDES
02
USA
PBO
PFOSA
PhB

: Difference in membrane potential between two phases
separated by a membrane (mV). '

: Ethylenediaminetetraacetic acid

: Ethylene-bis-N,N,N',N'—tetraacetic acid

: Field-collected strain of German cockroaches
: Flavin adenine dinucleotide (reduced)

: Prosthetic group of a class of proteins that contain
nonheme iron and acid-labile sulfur

: Flavin mononucleotide (oxidized)

: Proton

:N-lsopropyl analog of sulfluramid

: Potassium cyanide

: Mixed function oxygenase

:N-Methyl analog of sulfluramid

: Nicotinamide adenine dinucleotide (reduced)
: Nicotinamide adenine dinucleotide phosphate (reduced)
:N-Desethylated sulfluramid

:Oxygen

: Octanesulfonic acid

: Piperonyl butoxide

: Perfluorooctanesulfonic acid

: Phenobarbital

 

Pi

R3NH’
RCR
RLM

RR

81-9

SULF

TCA

TetBA
TriBA
TWIEA
TrHiA
Twtien

USULr

 

Pi

RaNH"
RCR
RLM

Sf—9

SULF

T

TCA

TetBA

TriBA
TriEA

TriHA

Tween 20

USULF

: Inorganic orthophosphate

:Gas constant.

: Trialkylamine cation.

: Respiratory control ratio

: Rat liver mitochondria.

' .Respiratory ratio (a parameter used to characterize the
tsiguhégess of coupling ofliposomes respiration in this

: Susceptible strain of German cockroaches

:Cell line from pupal ovaries of the fall armyworm
Spodoptera frugiperda

: Sulfluramid

: Absolute temperature (°K)

: Tricarboxylic acid cycle

: Tetrabutylammonium ion

: Tributylamine

: Triethylamine

: Trihexylamine

: Polyoxyethylenesorbitan monolaurate

: Unfluorinated analog of sufluramid

xvii

INTRODUCTION

 

 

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Introduction

Thelmajor classes of insecticides now in use mainly act on targets in
the nervous system. These insecticides are steadily declining in
number and users are also suffering a continuous problem due to the
excessive use of these insecticides which leads to resistance. The
search for new pesticides and pesticidal targets is increasingly
challenging because a new pesticide must meet many requirements
before registration by EPA. In addition to good activity against
economically important insects, the most important requirements are
to be environmentally friendly and safe to users and consumers.
Relatively few biochemical targets have been found to produce
effective pesticides. Mitochondrial respiration is one well-validated
site for pesticide action. New compounds that affect the respiratory
system and primarily the mitochondrial functions are therefore of
interest. If these compounds have enough non-target safety, they
will provide a solution to many environmental and agricultural
problems.

Resistance is an increasing problem, and since resistance often
involves selection of an insensitive site of action (binding site),
uncouplers of mitochondrial oxidative phosphorylation are of special
interest. This group is unique in that it does not require a binding
site to be active. Their mechanism of action depends totally on its
physicochemical characteristics (McLaughlin and Digler, 1980). The
only drawback of this group is the universality of mitochondria in
eukaryotic organisms. It would be of great use if such a compound

could be designed to be selectively toxic to pests. One way in which

 

  

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an insecticide can be made more selective is by conversion of the
active material to a derivative (propesticide) (Neuman and Drabek,
1986).

A compound marketed recently, sulfluramid (Finitronm), shows some
of these characteristics. The mammalian toxicity of this compound is
significantly lower than its toxicity against insects. This is one of two
compounds studied in this dissertation. Bioassays of the activity of
sulfluramid and its analogs have been conducted to lay the.
foundation for more detailed in vivo and in vitro studies. The goal of
these studies is to define and establish the mechanism(s) of action of
this new compound. The toxicity and metabolism of sulfluramid are
discussed in Chapter 2 where we show that it is a slow-acting
insecticide and that is being metabolically activated. The chemical
structure of this compound suggested that it could be acting as an
uncoupler of mitochondrial oxidative phosphorylation. This
hypothesis was tested and is discussed in chapter 3 along with a
structure-activity study of some sulfluramid analogs. The mechanism
by which these metabolites may exert their action is also discussed.
The second compound that is being tested, primarily as an acaricide.
is fenazaquin or EL-436. It belongs to a new family of experimental
compounds called quinazolines. The mode of action of this compound
is studied in chapter 4. The compound's effects were studied on
respiration in German cockroaches and a Spodoptera frugr'perda
ovarian cell line (Sf-9) and in rat liver mitochondria. This compound
was found to act as an inhibitor of the electron transfer system at

complex I (rotenone-like activity). Structure-activity studies were

 

 

conducte.
toxicity.

Anorher

 

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milochori
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taken in

conducted on mitochondrial inhibition, cell respiration and mite
toxicity.

Another compound included in this study is the experimental
insecticide EL-499. It is a perfluorocyclohexylcarboxanilide and its
mode of action is covered in chapter 5. This insecticide was tested on
mitochondria from insects, mammals and plants. It is a powerful
uncoupler of oxidative phosphorylation. When injected, the
compound increased C02 production in German cockroaches.

As a part of an enrichment study, the effects of formamidines on
prostaglandin synthesis was studied (Appendix II).

This work is preceded by a literature review that emphasizes
knowledge of mitochondria, bioenergetics and the actions of
uncouplers of oxidative phosphorylation. This review will help the
reader to follow the rationale behind the experimental approaches

taken in this study.

Chapter 1
LITERATURE REVIEW

LITERATURE REVIEW

Pesticides are one of the most effective means to increase and
protect agricultural production through controlling pests such as
insects, acarines and rodents. Despite 'concerns regarding their
potentially harmful effects on human and environmental health, they
will continue to be used in large amounts for the foreseeable future.
For several reasons, the agricultural industry is now suffering the
loss of many conventional pesticides that have been in common use.
This loss is mainly because of the development of resistance,
environmental impact and non—target organism toxicity. The rate of
loss of older pesticides in some areas is faster than the rate of
replacement. Georghiou (1986) illustrated the magnitude of the
problem when he related resistance in mosquitos to the number of
insecticides submitted to the WHO for testing between 1940-1984 as
shown in Figure 1. While resistance has increased steadily, the
number of new experimental insecticides has diminished'greatly in
the last 20 years. This declining availability of effective compounds
makes the search for new types of pesticides that are safer and
environmentally friendly increasingly urgent.

Since pesticides are carefully chosen to have a high degree of
toxicity towards the target. pests, it is rational to expect that they
may have other side effects on non-target organisms including man.
One of the major goals of pesticide toxicology, besides pinpointing the
mode and the site of action of a compound, is to evaluate and lessen
the hazards that might occur in the environment because of using
such potent compounds. However, to try to find or design a

compound that will not injure non-target organisms is not an easy

in.

 

   
   

 

 

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task. This is particularly true with insecticides since there are many
similarities between insect and man at the biochemical level
(Hollingworth, 1975). At the same time, pest populations, especially
insects and fungi, have developed defences against these compounds
in the form of increased metabolism or a changed site of action that
allow them to tolerate higher doses of the compound that have been
effective against them in the past. This is termed resistance. Other
populations under repeated selection pressure have progressed
further and developed multi-resistance, in which two or more
resistance genes are present enabling them to resist compounds from
several different groups of pesticides. This broad range of defensive
mechanisms may render them insensitive to new insecticides even
before they are marketed, and is one of the major challenges facing
crop production and human health protection in the coming decades
(National Research Council, 1986).

The four major classes of organic insecticides (organochlorines,
organophosphates, carbamates and pyrethroids) developed since
1945 are all neurotoxicants which act on targets present in both
insects and vertebrates (Hammock er al, 1986). The search for new
targets for insecticides acting outside the nervous system is rather
important to increase selective toxicity and to delay the onset of
resistance. One familiar group! of toxicants with good potential as
insecticides that act through different mechanisms is the uncouplers
and inhibitors of mitochondrial oxidative phosphorylation. Oxidative
phosphorylation is defined as the process in which ATP is formed as
electrons are transfered from NADH (or iFADHz) to 02 by a series of

electron carriers (Stryer, 1988). Uncouplers are those compounds

that uncouple the phosphorylation (ATP-synthesizing) system from
the electron transfer process inside the mitochondria. This is done by
diverting the energy derived from electron transfer and conserved in
the form of a transmembrane electrochemical gradient (AuH+) (H+
outside) to release heat instead of the formation of ATP.
Mi n ti 1 n i n °

Mitochondria are the subcellular organelles which are the centers
of bioenergetics of all eukaryotic cells. They consist of an outer
membrane which is highly permeable, an inner membrane of much
more selective permeability and an inner matrix. The inner
mitochondrial membrane contains most of the enzymes for both
electron transport and phosphorylation reactions, while the matrix
has nearly all the enzymes that catalyze the TCA cycle. The electron
transfer chain is grouped into four complexes that modulate the
transfer of two electrons from NADH (or FADHz) to molecular oxygen
(Figure 2). The electron transfer chain is composed of NADH:
ubiquinone oxidoreductase (complex I), succinate: ubiquinone
oxidoreductase (complex 11), ubiquinol: ferrocytochrome c
oxidoreductase (complex III), and ferricytochrome c: oxygen
oxidoreductase (cytochrome oxidase or complex IV). This concept has
been described by many authors, e.g. Chance and Williams, (1956);
Chance, (1967); Williams, (1973); Ferguson-Miller et al, (1979) and
Hatefi, (1985).
During the transport through complexes 1, III and IV, electrons
undergo large decreases in ,redox potential (_>_300 mV) as reported by
Dutton et al, (1970) and Erecinska et al, (1974). The energy released

from the .three sites is used to drive the transport of protons from

 

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12

the matrix across the inner membrane against their concentration
gradient. The mechanism of proton translocation by complex I is not
well understood but Mitchell (1979)'proposed that the flavin moiety
of the complex assembly ejects protons from the matrix as it is being
sequentially Oxidized and reduced. For complex 11, the transport
occurs via coenzyme Q or cytochrome b of the inner membrane
(Wikstrom et al,‘ 1976). The terminal site, cytochrome oxidase, which
is responsible for transferring electrons to oxygen is also proven to
be redox-driven proton pump (Wikstrom, 1977; Malstrom, 1985).
Although the chemiosmotic theory has been widely accepted and
has been the base for current understanding of mitochondrial
bioenergetics, it still cannot offer a complete explanation for certain
characteristics of the electrochemical gradient (Allin) generated
across the mitochondrial membrane. This gradient can be separated

into an electrical (N?) and a chemical (ApH) component.
Aum = F (A‘P) - 2.3 RT (ApH)

A‘I’ = membrane potential
ApH = pH difference between the inside and the outside of the
membrane

Some of these anomalies are: (l) The rate of electron transport and
ADP phosphorylation do not coincide with the magnitude of Apr“.
Nicholls (1974), suggested that the mitochondrial proton conductance
(leak) increases at higher values of Ap (proton motive force, Ap =
Au,“ IF mV). (2) At equilibrium, ATP energy is not proportional to
the electrochemical gradient (Aum). This was confirmed by finding
that stoichiometries of the proton pumps decrease (slip) at high Ap

(Pieterbon et a1, 1981; Zorati et a1, 1986). Although recently, it has

13'

been reported that all relative proton Stoichiometries of the
respiratory chain and of ATP-synthesis are constant across a range of
values of AP (Hafner and Brand, 1991).

(3) The suggestion that the proton pumps of the electron transport
chain are directly coupled to the ATP synthetase system rather than
indirectly through a transmembrane H+ gradient. For further detailed
understanding of these points refer to Sorgato et al, (1978);
Westerhoff et al, (1981); Mitchell, (1981) and Westerhoff et al, (1984
b). Some untested explanations for these points were suggested by
Williams (1978, 1984) and Ferguson (1985) by proposing that
protons are not ejected, but instead are produced and locally
controlled in the environment of the inner mitochondrial membrane.
Westerhoff et al (1984 a and b) have also suggested that the primary
(electron-transfer chain sites I, II, and III) and secondary (H+-
ATPase translocator) proton pumps are spatially situated to be close
so as to create functional proton compartments called the "Proton
Domains". These protons are directly transferred from the proton
pumps to the F0 of the ATP synthetase. In addition, the volume of
this proton domain is small enough that only few protons would have
to be transported to attain a sufficient electrochemical gradient to
drive ATP synthesis. All of these suggestions are yet to be confirmed.
For further discussion of these unresolved issues sec Rottenberg,

(1985).

14

ATP synthesis in mitochondria is catalyzed by the FtFo-ATP
synthetase (H+-translocating ATPase) which is a mushroom-like
structure situated on the inner side of the mitochondrial membrane
facing the matrix. The F0 component of this enzyme is a
transmembrane protein that is composed of 3 subunits known as a,
b, and c (Alfonzo and Racket, 1979). The F1 component isa soluble
protein facing the matrix (Racker, 1968) and is comprised of 5
subunits (Kagawa, 1978 and Maloney, 1982) known as alpha3, beta3,
gamma, delta, and epsilon (Amzel and Pederson, 1983). The F1
protein is connected to the Fe one via an oligomycin-sensitizing
protein (Senior, 1979) and the gamma subunit of the F1 (Pederson and
Carafoli, 1987a). The function of the Fe moiety of the enzyme is
believed to be a proton channel (Seren at al, 1985) while the F1
protein catalyzes both ATP synthesis and ATP hydrolysis (Boyer at
al, 1982). Oligomycin, an antibiotic that binds to the Fe protein,
irreversibly inhibits the F1Fo-ATP synthetase (Bertina ef al, 1974).
Also carbodiimides bind to a specific protein of the F0 component.
This appears to be the basis of the toxic action of the new pesticide
diafenthiuron (Ruder et al, 1991).

The mechanism by which ATP is synthesized is a complicated one
and not all the aspects of this mechanism are understood. Based on
Mitchell's chemiosmotic theory, the energy created by the flow of
electrons through the electron transfer chain is used indirectly to
drive ATP synthesis by the creation of a proton gradient across the
inner membrane (Hutton and Boyer, 1979). Both ADP and Pi bind to
high affinity catalytic sites on the beta subunit or at the alpha-beta

interface (Pederson and Carafoli, 1987b). ATP synthesis proceeds on

15

the enzyme without the need for external energy (Grubmeyer et al,
1982; Eisenberg and Hill, 1985). The movement of protons down their
concentration gradient back across the inner mitochondrial
membrane is believed to protonate some functional groups on the Fo
protein (Penefsky, 1985). This protonation would cause
conformational changes in the F1 protein. These conformational
changes are believed to facilitate the dissociation of ATP at one
catalytic site and the binding of both ADP and Pi at another (Boyer,
1975; Boyer at al, 1977; Boyer, 1979; and O'Neal and Boyer 1984).
So, ATP synthesis happens by the reversal of ATP hydrolysis and
protons do not have an actual part in the chemistry of the process
except for the induction of the initial conformational changes that
will cause other conformational changes at the substrate and/or the
product binding site.

Electron transport and ATP biosynthesis are closely coupled. In
typical respiring mitochondria, the level of ADP as the ultimate
energy acceptor is rate limiting on respiration. To measure the
tightness of this coupling, the ratio of the rate of oxygen consumption
during ATP synthesis (state 3; ADP present) to the same rate during
resting (state 4; ADP absent) is used. This ratio is called the
respiratory control ratio (RCR) (Chance and Williams, 1956). The
higher this ratio, the higher the degree of coupling. Another ratio
that is also used in characterizing mitochondrial efficiency is the
ADP:0 ratio, which indicates the number of ATP molecules
synthesized per pair of electrons oxidized. For electrons originating
from NADH, this ratio generally approaches 3.0. The exact

stoichiometry of such a reaction is still controversial. The

16

stoichiometry of both proton ejection (H+/O) and the number of
protons used to produce one ATP (H+/ATP) differ between authors.
The estimation of two was 6 (Mitchell and Moyle, 1984), 11 (Beavis
and Lehninger, 1986 a and b; Beavis, 1987 a and b), 12.(Vercesi et al,
1978; Lehninger, 1984) and 13 (Lemaster, 1984). Table 1

summarizes the history of these Stoichiometries. The H+IATP
stoichiometry is now widely accepted to be 3.0 (Brand et al, 1976).
The reason for these inconsistencies is that under the typical
conditions of mitochondrial incubations in vitro, substrate oxidation
is presumably not completely coupled to ADP phosphorylation. This
incomplete coupling, as predicted by Mitchell (1961), arises from
secondary proton transport pathways or energy leaks in the electron
transport chain. While proton ejection is energy dependent, the leak
could be dependent on the electrical gradient (Zoratti et al, 1986) or
the pH gradient ( Krishnamoorthy and Hinkle, 1984). It has also been
reported that H“ may pass through Fo without ATP formation, or they
might traverse the protein domain of the electron transport chain
without causing reverse electron transfer (Zoratti er al, 1983;
Pietrobon and Caplan, 1986 a and. b). This non-linearity between
mitochondrial respiration and Aug+ has also been reported by O'Shea
et al (1984), Brown and Brand, (1986) and Duszynski and Wojtzak,
(1985).

Another important aspect is that genetic and chemically-induced
deficiencies in electron transport and oxidative phosphorylation have
been linked to many degenerative diseases, e.g. ischemic heart
disease, Parkinson's disease, Alzheimer's disease, Huntington's

disease and aging. The involvement of both mitochondrial DNA and

17

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18

nuclear DNA in the biogenesis of the energy conservation
components make their genetics very complex. In addition, the
mitochondrial DNA mutations observed in these cases are closely
related to the abundance of the oxygen free radicals generated
during electron transfer from reduced flavin dehydrogenases, CoQ
and cytochrome b to oxygen. These two concepts along with
dependance of many tissues on mitochondria as the major source for
ATP make the oxidative phosphorylation paradigm of great
importance in understanding important genetic and degradative
diseases (Lestienne et al, 1990; Wallace, 1992).
r f ' iv h h l i it

Many compounds are known to disrupt mitochondrial functions.
Some of these compounds are inhibitors of either the electron
transfer proteins or the FlFo-ATPase system (as shown in Figures 2
and 3). Others, called uncouplers, are known to dissipate the
transmembrane electrochemical gradient. The energy released by the
electron transport chain is then diverted by uncouplers to heat
without being utilized in ATP synthesis. This uncouples electron
transport from ATP biosynthesis. Electron transport is freed from
control by the acceptor (ADP) level and runs at maximum rate. A
corresponding increase in 02 consumption is then observed.
Uncouplers are also known to activate the FlFo-ATPase system
causing the destruction. of existing ATP since the Au,“ is dissipated
by uncouplers, the driving force for ATP biosynthesis is reversed and
ATP hydrolysis occurs. ‘
Uncouplers must (a) be weak acids to allow proton transport by

association/dissociation, (b) be lipophilic to concentrate in the

 

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21

. mitochondrial membrane, and (c) contain conjugated aromatic
system and/or shielding of the ionizable group by hydrophobic
groups to diffuse or shield the anionic charge and thus stabilize the
anionic form in the lip0philic environment (Terada, 1981 & 1987).
Their mechanism of action depends totally on these characteristics.
According to the chemiosmotic theory, protons are pumped across
the inner mitochondrial membrane prior to the formation of ATP.
Uncouplers circumvent this process by acting as protonophores that
shuttls the ejected protons back to the inside (Figure 4). The anionic
form of the uncoupler (A') moves from right to left within the inner
membrane along both its electrical and concentration gradients. This.
will drive the reaction towards the formation of the dissociable acid
(HA) at the left interface by removing protons from the acidic
aqueous phase. The increase in the HA1 concentration at this interface
will'create a concentration gradient that will move HA to the right-
hand interface. Because of the higher pH at this interface, the acid
will tend to dissociate yielding a proton (H+) to the aqueous phase
inside the mitochondrial matrix and rendering the uncoupler anion
(A') free to shuttle another proton (McLaughlin and Dilger, 1980).
This protonophoric mechanism of action is purely physicochemical
and does not involve a specific binding site for the uncoupler. The
uncoupler acts catalytically. This makes uncouplers unlike most other
toxicants which have specific binding sites such as enzymes and
neuroreceptors associated with their action. There is some evidence
. that specific binding sites for uncouplers may exist within the FtFo-
ATPase based on the photoaffinity labeling studies by Hanstein and

Hatefi (1976) and Parttis er al (1984) and on studies with uncoupler-

22

//////////////////'e

HA > HA

69

   
 

 

 

 

 

   

A
H+ H“
V
A-< A"
W

Membrane

Figure 4: Schematic diagram showing the shuttle mechanism that
describes the movement of protons (H+) across the inner
mitochondrial membrane by the anionic uncoupler (HA).
(Modefied after McLaughlin and Digler, 1980).

23

resistant bacterial mutants (Decker and Lang, 1978; Ito and
Ohnishi,l981; Sedgwick er al, 1984; Jones and Beechey, 1987; Quirk
et al, 1989). However, such conclusions based on both photoaffinity
labeling and bacterial resistance have been criticized (Terada, 1981;
Ferguson, 1985). Thus, as shown with the detailed mechanism of
oxidative phosphorylation itself, the question of the existence and
role of uncoupler binding sites remains unclear. To date, none have
been identified satisfactorily and the simple protonophoric action of
lipophilic weak acids remains the best explanation for uncoupling
(Miyoshi and Fujita 1988; and Gno et al., 1991).

A problem with the use of uncouplers as pesticides is the common
presence of mitochondria in all eukaryotes which tends to make
uncouplers universal in their toxic action i.e. they tend to be non-
selective. A number of current pesticides do act by uncoupling
oxidative phosphorylation in pests, especially rodents, fungi,
mollusks and mites e.g. dinitrophenols (Corbett et al, 1984),
diarylamines (Dreikorn,l984), hydroxybenzonitriles (Corbett et al,
1984) and salicylanilides (Ilivicky and Casida, 1969). Although, the
main disadvantage of this group is their lack of selectivity between
pests and non-target species, the probable absence of a binding site
in their action may render them difficult to develop resistance
against since this resistance often is due to the presence of an
insensitive site of action (Oppenoorth, 1985). In keeping with this,
there do not appear to be well-established instances of the
development of resistance to pesticides acting as uncouplers in

higher organisms (Dr. G. P. Georghiou, personal communication).

24

Although mitochondrial energetics is common to multicellular
organisms, there is evidence that the properties of mitochondria
differ between organisms. One of the major differences reported was
between poikilotherms (reptiles) and homoeotherms (mammals and
birds) in the rate of oxygen consumption, where a. resting mammal
may consume oxygen 4-5 times faster than a reptile of the same
body size at the same body temperature (Dawson and Hulbert, 1970).
This was further elaborated by Brand et al (1991) by finding that the
proton permeability of the liver inner mitochondrial membrane of a
rat is greater than in a bearded dragon. No studies of that sort seem
to have been done to establish a difference between insect and
mammalian mitochondria.

The idea of selective uncouplers was emphasized by Holan and Smith
(1986) when they examined the selectivity of a series of
arylhydrazones in the mitochondria of sheep blowfly (Lucilia
caprina) flight muscle. One of these compounds was 7.3 times higher
in its uncoupling activity on the blowfly mitochondria than the rat
liver mitochondria. Ilivicky and Casida (1969) have also suggested
that uncouplers may vary in potency between mitochondria from
different sources by a factor of lO-fold or more. However, such
comparisons must be made with great care e.g. with an equal
number of respiratory assemblies in each incubation. By contrast, no
significant difference in uncoupling potency have been reported
between mitochondria from housefly flight muscle and rat liver
(Miyoshi and Fujita, 1988). The conclusion drawn was that all weakly
acidic uncouplers act as ionophores without significance difference in

potency between mitochondria from insects and mammals and the

25

question of the existence and significance of specificity in uncoupling
remains open. However, the possible advantages in decreased
toxicity and reduced resistance potential of selective uncouplers
makes the topic one of both practical and theoretical significance.
Nw'n ii h mi hnrilfnin°

One of the newly registered insecticides for urban pest control is
sulfluramid (Figure 5-A). The compound is registered under the
name "Finitronm" by Griffin Chemical Co. (Valdosta, GA). This
compound is a polyfluorinated alkylsulfonamide that is fairly
lipophilic with weakly acidic proton but lacks an aromatic moiety.
'The structure of the compound suggested the possibility of it being
an uncoupler. Several aspects of the action of sulfluramid have been
studied in this work. The toxicity and metabolism of the compound is
approached in chapter 2 of this dissertation. The mitochondrial
respiration, swelling and a structure-activity relations are covered in
chapter 3.

A second compound was also included in this work. This
compound is a quinazoline ether (Figure S-B). The compound is being
developed under the name of fenazaquin by DowElanco as an
acaricide-insecticide(lndianapolis, IN). It may also be referred to as
EL-436 in this study. The mode of action for this compound has been
studied on mitochondria (rat liver), the Sf-9 cell line (Spodoptera
frugr'perda ovarian cell line) and on the respiration of the German
cockroaches, Blattella germanica (L.) (Chapter 4). The compound acts
as an inhibitor of electron transfer at complex I of the respiratory
chain. Structure-activity studies have also been conducted with this

compound.

26

The third compound, EL-499 (Figure 30), is an experimental
insecticide developed by DowElanco Co. (Indianapolis, IN) against soil
or household insects. The compound is a powerful uncoupler of
mitochondrial oxidative phosphorylation. EL-499 is studied in vitro
on mitochondria from different sources. It is also studied in viva on
German cockroaches and was found to lethally increase C02

production in a dose dependent fashion.

27

O C H
cF3-CFz-CFz-CFg-CFz-CFg-CFg-CFz‘IS"N

O H

Figure 5: The structure of both (A) sulfluramid (Finitron) and
(B) fenazaquin (EL-436).

28

References

Alfonzo, M. and Racker, E. 1979. Components and mechanism of
action of ATP-driven proton pump. Can. J. Biochem. 57: 1351-

1358.

Beavis, A. D. 1987a. Upper and lower limits of the charge .
translocation stoichiometry of mitochondrial electron transport.

J. Biol. Chem. 262: 6165-6173.

Beavis, A. D. 1987b. Upper and lower limits of the charge
translocation stoichiometry of cytochrome c oxidase. J. Biol.

Chem. 262: 6174-6181.

Beavis, A. D. and Lehninger, A. L. 19860. Determination of the upper
and lower limits of the mechanistic stoichiometry of
incompletely coupled fluxes. Stoichiometry of incompletely
coupled reactions. Eur. J. Biochem.158: 307—314.

Beavis, A. D. and Lehninger, A. L. 1986b. The upper and lower limits
of the mechanistic stoichiometry of mitochondrial oxidative
phosphorylation. Stoichiometery of oxidative phosphorylation. Eur.
J. Biochem.158: 315-322.

Bertina, R. M.; Steenstra, J. A. and Slater, E. C. The mechanism of
inhibition by oligomycin of oxidative phosphorylation in
mitochondria. 1974. Biochem. Biophys. Acta 368: 279-297.

Boyer, P. D. 1975. Energy transduction and proton translocation by
adenosine triphosphate. FEBS Lett. 50: 91-94.

Boyer, P. D. 1979. The binding-change mechanism of ATP synthesis.
In Membrane Bioenergetics. Eds. Lee, C. P.; Schatz, G. and Emster,
L. pps., 461-479. Adison Wesley, reading, Massachusetts.

Boyer, P. D. 1984. Energy-linked changes of enzyme conformation in
ATP formation and other bioenergetic processes. In Symposium on
Frontiers in Bio-Organic Chemistry and Molecular Biology. Ed.
Ovchinnikov, Y. A., pps. 1-8 Elsevier, Ameterdam.

29

Boyer, P. D.; Chance, B.; Emster, L., Mitchell, P.; Racker, E. and
Slater, E. C. 1977. Oxidative phosphorylation and
photophosphorylation. Ann. Rev. Biochem. 46: 955-1026.

Brand, M. D. 1990. The proton leak across the mitochondrial inner
membrane. Biochem. Biophys. Acta 1018: 128-133

Brand, M. D.; Couture, P.; Else, P. E.; Withers, K. W. and Hulbert, A. J.
1991. Evolution of energy metabolism. Proton permeability of the
inner membrane of liver mitochondria is greater in a mammal
than in a reptile. Biochem. J. 275: 81-86.

Brand, M. D., Reynafarje, B. and Lehninger, A. L. 1976. Stoichiometric
relationship between energy-dependent proton ejection and
electron transport in mitochondria. Proc. Natl. Acad. Sci. (USA).

73: 437-441.’

Chance B. 1967. The reactivity of haemoproteins and cytochromes.
Biochem. J. 103: 1-18.

Chance B. and Williams, G. R. 1956. The respiratory chain and
oxidative phosphorylation. Adv. Enzymol. 17: 65-134.

Corbett, J. R.; Wright, K. and Baillie, A. C. 1984. The biochemical
mode of action of pesticides. 2nd edn. Academic Press, London.

Cross, R. L.; Grubmeyer, C. and Penefsky, H. S. 1982. Mechanism of
ATP hydrolysis by beef heart mitochondria] ATPase. Rate
enhancements resulting from cooperative interactions between
multiple catalytic sites. J. Biol. Chem. 257: 12101-12105.

Dawson, T. J. and Hulbert, A. J. 1970. Standard metabolism, body
temperature and surface areas of Australian marsupials.. Amer. j.
Physiol.218: 1233-1238.

Decker, S. J. and Lang, D. R. 1978. Membrane bioenergetic parameters
in uncoupler resistant mutants of Bacillus megaterium. J. Biol.
Chem. 253: 6738-6743.

Dreikom, B. A. and O'Dohesty, G. O. P. 1984. The discovery and
development of bromethalin, an acute rodenticide with a unique
mode of action. ACS Symposium. Ser. 255: 45-63.

30

Duszynski, J. and Wojtzak, L. 1985. The apparent non-linearity of the
relationship between the rate of respiration and the protonmotive
force of mitochondria can be explained by heterogeneity of
mitochondrial preparations. FEBS Lett. 182: 243-248.

Dutton, P. L.; Wilson, D. F. and Lee, C. P. 1970. Oxidation-reduction
potentials of cytochromes in mitochondria. Biochemistry 9:

5077-5082.

Eisenberg, E. and Hill, T. L. 1985. Muscle contraction and free energy
transduction in biological systems. Science (Washington, D. C.)
227: 999-1006.

Erecinska, M.; Veech, R. L. and Wilson, D. F. 1974. Thermodynamic
relationships between the oxidation-reduction reactions and the
ATP synthesis in suspensions of isolated pigeon heart
mitochondria. Arch. Biochem. Biophys. 160: 412-424.

Ferguson, 8. J. 1985. Fully delocalized chemiosmotic or localized
proton flow pathways in energy coupling? A scrutiny of
experimental evidence. Biochem. Biophys. Acta 811: 47-95.

Ferguson-Miller, S.; Brautigan, D. L. and Margoliash, E. 1978.
Definition of cytochrome c binding domains by chemical
modification. III. Kinetics of reaction of carboxydinitrophenyl
cytochromes c with cytochrome c oxidase. J. Biol. Chem. 253:
149- 159.

Georghiou, G. P. 1986. The magnitude of the resistance problem.
Pesticide Resistance: Strategies and Tactics for Management.
National Academy Press, Washington, D. C.

Gno, Z.; Miyoshi, H.; Komyoji, T.; Hoga, T. and Fujita, T. 1991.
Uncoupling activity of a newly developed fungicide, fluazinam [3-
chloro-N-(3-chlor-2,6-dinitro-4-trifluoromethylphenyl)-5-
trifluoromethyl-2-pyridinamine]. Biochem. Biophys. Acta 1056:
89-92. '

Grubmeyer, C.; Cross, R. L. and Penefsky, H. S. 1982. Mechanism of
ATP hydrolysis by beef Heart mitochondrial ATPase. Rate
constants for elementary steps in catalysis at a single site. J.
Biol. Chem. 257:12092-12100.

31

Hafner, R. P. and Brand, D. B. 1991. Effect of protonmotive force on
the relative proton stoichometries of the mitochondrial proton
pumps. Biochem. J. 275: 75-80.

Hammock, BB. and Soderland, D. M. 1986. Chemical strategies for
resistance management. Pesticide Resistance: Strategies and
Tactics for Management. National Academy Press, Washington,
DC.

Hanstein, W. G. 1976. Uncoupling of oxidative phosphorylation.
Biochem. Biophys. Acta 456: 129-148.

Harold, F. M. 1986. The Vital Force: A study of bioenergetics. W. H.
Freeman and company. New York.

Hatefi, Y. 1985. The mitochondrial electron transport and oxidative
phosphorylation system. Ann. Rev Biochem.54: 1015-1069.

Hinkle, P. C. and Yu, M. L. 1979. The phosphorus/oxygen ratio of
mitochondrial oxidative phosphorylation. J. Biol. Chem. 254:
2450-2455.

Holan, G. and Smith, D. R. G. 1986. A new selective insecticidal
uncoupler of oxidative phosphorylation. Experientia 42: 558-560.

Hollingworth, R. M. 1976. The biochemical and physiological basis of
selective toxicity. Insecticide biochemistry and physiology. Ed.
Wilkinson, C. F. pp.: 431-506. Plenum Press, New York and
London.

Hollingworth, R. M. 1975. Strategies in the design of selective insect
toxicants. Pesticide selectivity. Ed. Street, J. C. pp.:67-111.
Marcel Dekker, New York.

Hutton, R. L. and Boyer, P. D. 1979. Subunit interaction during
catalysis. alternating site cooperativety of mitochondrial
adenosine triphosphataseJ. Biol. Chem. 254: 9990-9993.

Ilivicky, J. and Casida, J. E. 1969. Uncoupling action of 2,4-
dinitrophenols, 2-trif1uoromethylbenzimidazoles and certain
other pesticide chemicals upon mitochondria from different
sources and its relation to toxicity. Biochem. Pharmc.18: 389-
401.

32

Ito, M. and Ohnishi, Y. 1982. Isolation of Escherichia coli mutants
which are resistant to an inhibitor of H-ATPase, tributyltin and
also to uncoupler of oxidative phosphorylation. FEBS Lett.l36:
225-230.

Jones, M. R. and Beechey, R. B. 1987. Escherichia coli mutants
resistant to uncouplers of oxidative phosphrylation. J. Gene.
Microbial. 133: 2759-2766. -

Kagawa, Y. 1978. Reconstitution of the energy transformer, gate and
channel subunit reassembly, crystalline ATPase and ATP
synthesis. Biochem. Biophys. Acta 505: 45-93.

Krishnamoorthy, G. and Hinkle, P. C. 1984. Non-ohmic proton
conductance of mitochondria and Iiposomes. Biochemistry 23:
1640-1645.

Lehninger, A. L. 1984. Proton translocation coupled to mitochondrial
electron transport. Biochem. Soc. Trans. 12: 386-388.

Lemaster, J. J. 1984. The ATP-to-oxygen Stoichiometries of
oxidative phosphorylation by rat liver mitochondria. An analysis
of ADP-induced oxygen jumps by linear nonequilibrium
thermodynamics. J. Biol. Chem. 259: 13123-13130.

Lestienne, P; Nelson, J.; Riederer, P; Jellinger, K. and Reichmann, H.
1990. Normal mitochondrial genome in brain from patients with
Parkinson's disease and complex I defect. J. Neurochem. 55: 1810-
1812

Maloney, P. C. 1982. Energy coupling to ATP synthesis by the proton-
translocating ATPase. J. Membr. Biol. 67: 1-12.

Malstrom, B. G. 1985. Cytichrome c oxidase as a proton pump. A
transition-state mechanism. Biochem. Biophys. Acta 811: 1-12.

McLaughlin, S. G.A and Digler, T. P. 1980. Transport of protons across
membranes by weak acids. Physiol. Rev. 60: 825-863.

Mitchell, P. 1961. Coupling of phosphorylation to electron and
hydrogen transfer by a chemi-osmotic type of mechanism. Nature
(London) 191: 144-148.

33

Mitchell, P. 1975. Proton translocation mechanisms and energy
transduction by adenosine triphosphates: an answer to criticisms.
FEBS Lett. 50:95-97.

Mitchell, P. 1976. Possible molecular mechanisms of the proton
motive function of cytochrome systems. J. Theor. Biol. 62: 327-
376.

Mitchell, P. 1979a. Keilin's respiratory chain concept and its
chemiosmotic theory. Science (Washington, D. C.) 206: 1148-
1159.

Mitchell, P. l979b. Compartmentation and communication in living
systems. Ligand conduction: a general catalytic principle in
chemical, osmotic and chemiosmotic reaction systems. Eur. J.
Biochem. 95: 1-20.

Mitchell, P. 1981. From black-box bioenergetics to molecular
changes: Vectorial ligand-conduction mechanisms in
biochemistry. In Chemiosmotic proton circuits in biological
membranes. In the honor of Peter Mitchell. (Eds, Skulachev, V. P
and Hinkle, P. C.). pps.: 611-633. Adison- Wesley, Canada.

Mitchell, P. 1985. The correlation of chemical and osmotic forces in
biochemistry. J. Biochem. 97: 1-18.

Mitchell, P. and Moylc, J. 1967. Respiratory-driven proton
translocation in rat liver mitochondria. Biochem. J. 105: 1147-
1162.

Miyoshi H. and Fujita, T. 1988. Quantitative analysis of the
uncoupling activity of substituted phenols with mitochondria
from flight muscles of house flies. Biochem. Biophys Acta 935:
312-321.

Nicholls, D. G. 1979. Brown adipose tissue mitochondria. Biochem.
Biophys. Acta 549: 1-29.

Nicholls, D. G. 1982. Bioenergetics: An introduction to the
chemiosmotic theory. Academic press, London.

O'Neal, C. C. and Boyer, P. D. 1984. Assessment of the rate of bound
substrate interconversion and of ATP acceleration of product

34

release during catalysis by mitochondrial adenosine
triphosphate.]. Biol. Chem. 259: 5761-5767.

Oppenoorth, F. J. 1985. Biochemistry and genetics of insecticide
resistance. In Comprehensive Insect Physiology, Biochemistry and
Pharmacology. vol. 12, Eds. Kerkut, G. A. and Gilbert, L. 1.
Pergamon Press, Oxford and New York.

O'Shea, P. S.; Petrone, G.; Casey, R. P. and Azzi, A. 1984. The current-
voltage relationships of liposomes and mitochondria. Biochem. J.
219: 719-726.

Parttis, M. D.; Griffiths, D. G. and Beechey, R. B. 1984. Discrimination
between the binding sites of modulators of the H-translocating
ATPase in the rat liver mitochondrial membrane. Arch. Biochem.
Biophys. 232: 610-615.

Pederson, P. L. and Carafoli, E. 1987a. Ion motive ATPase. 1. Ubiquity,
properties and significance to cell function. Trends Biochem. Sci.
12: 146-150.

Pederson, P. L. and Carafoli, E. 1987b. Ion motive ATPase. 11. energy
coupling and work output. Trends Biochem. Sci. 12: 186- 189.

Penefsky, H. S. 1985. Mechanism of inhibition of mitochondrial
adenbsine triphosphate by dicyclohexylcarbodiimide and
oligomycin: relationship to ATP synthesis. Proc. Nat. Acad. Sci.
(USA) 82: 1589-1593.

Pietrobon, D.; Azzone, G. F. and Waltz, D. 1981. Effect of funiculosin
and antimycin-A on the redox-driven H+- pumps in mitochondria:
on the nature of 'leaks’. Eur. J. Biochem. 117: 389-394.

Pietrobon, D. and Caplan, S. R. 1986 a. Double-inhibitor and
uncoupler-inhibitor titeration. 1. Analysis with a linear model of
chemiosmotic energy coupling. Biochemistry 25: 7682-7690.

Pietrobon, D. and Caplan, S. R. 1986 b. Double-inhibitor and
uncoupler-inhibitor titeration. 2. Analysis with a nonlinear
model of chemiosmotic energy coupling. Biochemistry 25: 7690-
7696.

35

Quirk, P. G.; Jones, M. R.; Haworth, R. S.; Beechey, R. B. and Campbell,I.
D. 1989. Uncoupler resistance in Escherichia coli: the role of
cellular respiration. J. Gene. Microbiol. 135: 2577-2587.

Racket, E. 1968. The membrane of the mitochondrion. Scientific
American 218: 32-39.

Rottenberg, H. 1985. Proton-coupled energy conservation:
Chemiosmotic and intramembrane coupling. Modern Cell Biology
4: 47-83.

Ruder, F. J.; Guyer, W.; Benson, J. A. and Kayser, H. 1991. The thiourea
insecticide/acaric'ide diafenthiuron has a novel mode of action -
inhibition of mitochondrial respiration by its carbodiimide
product. Pestic. Biochem. Physiol. 41: 207.

Seren, S. ; Caporin, G.; Galiazzo, F.; Lippe, G.; Ferguson, 8. J. and
Sorgato, M. C. 1985. Current-voltage relationships for proton flow
through the F0 sector of the ATP-synthesis, carbonylcyanide-p-
trifluoromethoxyphenylhydrazone or leak pathways in
submitochondrial particles. Eur. J. Biochem. 152: 373-379.

Sidgwick, E. G., Hou, C. and Bragg, P. D. 1984. Effects of uncouplers on
the bioenergetic properties of a carbonylcyanide m-
chlorophenylhydrazone-resistant mutant Escherichia coli UV6.
Biochem. Biophys Acta 767: 479-492.

Sorgato, M. C.; Ferguson, S. J.; Kell, D. B.; and John, P. 1978. The
proton motive force in bovine heart submitochondrial particles.
Magnitude, site of generation and comparison with the
phosphorylation potential. Biochem. J. 174: 237-256.

Stryer, L. 1988. Biochemistry. 2nd eddition. W. H. Freeman and Co.
New York.

Terada, H. 1981. The interaction of highly active uncouplers with
mitochondria. Biochem. Biophys. Acta 639: 225-242.

Vercesi, A.; Reynafarje, B. and Lehninger, A. L. 1978. Stoichiometry
of H+ ejection and Ca2+ uptake coupled to electron transport in rat
heart mitochondria. J. Biol. Chem. 253: 6379-6385.

36

Wallace, D. C. 1992. Mitochondrial genetics: a paradigm for aging and
degenerative diseases?. Science 256: 628-632.

Westerhoff, H. V. and Chen, Y. D. 1985. Stochastic free energy
transduction. Proc. Nat. Acad. Sci. (USA) 82: 3222-3226.

Westerhoff, H. V.; Colen, A. M. and Van Dam, K. 1984a. Metabolic
control by pump slippage and proton leakage in 'delocalized' and
more localized chemiosmotic energy-coupling schemes. Biochem

Soc. Trans. 11: 81-85.

Westerhoff, H. V.; Melandri, B. A.; Venturoli, G.; Azzone, G. F. and Kell,
D. B. 1984b. Mosaic protonic coupling hypothesis for free energy
transduction. FEBS Lett. 165: 1-5.

Westerhoff, H. V.; Melandri, B. A.; Venturoli, G.; Azzone, G. F. and Kell,
D. B. 1984c. A minimal hypothesis for membrane-linked free-
energy transduction. Biochem. Biophys. Acta 768: 257-292.

Westerhoff, H. V.; Simonetti, A. L. M. and Van Dam, K. 1981. The
hypothesis of localized chemiosmosis is unsatisfactory. Biochem.
J. 200: 193-202.

Wikstrom, M. 1977. Proton pump coupled to cytochrome c oxidase in
mitochondria. Nature (London) 266: 271-273.

Williams, R. J. P. 1961. Possible functions of chains of catalysts. J.
Theor. Biol. 1: 1-17.

Williams, R. J. P. 1973. Electron transfer and oxidative energy.
Biochem. Soc. Trans. 1: 1-26.

Williams, R. J. P. 1978. The multifarious couplings of energy
transduction. Biochim. Biophys. Acta 505: 1-44.

Williams, R. J. P. 1984. Electron flow in membranes. Biochem. Soc.
Trans. 12: 396-399.

Zoratti, M.; Favaron, M.; Pietrobon, D. and Azzon, G. F. 1986. Intrinsic
uncoupling of mitochondrial proton pump. 1. Non-ohmic
conductance cannot account for the nonlinear dependance of
static head respiration on Am“. Biochemistry 25: 760-767.

37

Zoratti, M.; Pietrobon, D. and Azzon, G. F. 1983. Studies on the
relationship between ATP synthesis and transport and the proton
electrochemical gradient in rat liver mitochondria. Biochem.

Biophys. Acta 723: 59-70.

Chapter 2

Toxicity and Metabolism of the New Insecticide
Sulfluramid (FinitronTM) in Cockroaches

39

INTRODUCTION

Vander Meer et a1 (1986) described the serendipitous discovery
of a new class of insecticides, while searching for new compounds
that could be used against the imported fire ant Solenopsis invicta.
Fluorinated surfactants were initially used to overcome the solubility
problem of another experimental pesticide but were soon found to
have their own innate delayed toxicity against fire ants. This new
group is the perflourinated sulfonamides with a formula
CnF2n+1SOzNR1R2 . Sulfluramid, which has n = 8, R1 = H and R2 =
C2H5, was one of the screened compounds that has both high activity
and delayed toxicity making it a candidate for further development.
These compounds act as stomach poisons in insects but have
relatively low contact toxicity. Acute mammalian toxicity is quite low
with an oral LD50 of 500-5000 mg/kg depending on the carrier.
Sulfluramid (Finitronm, by Griffin Corporation) is now being
marketed as bait formulations (1.0% a.i., Schal, 1992) for cockroach
and ant control.
This chapter will look at the toxicity and metabolism of sulfluramid
(SULF) and related metabolites (NDES, PFOSA) against the German

cockroach Blattella germanica (L.).

Materials and Methods
1. Chemicals
Sulfluramid (SULF) , N-desethyl sulfluramid (NDES) , and
perfluorooctane sulfonic acid (PFOSA) were obtained from Griffin
Corp. (Valdosta, GA). SULF and NDES are a mixture of 95% linear

carbon chain and 5% branched isomers with 98% C8 chain length and

40

2% C6. PFOSA is a 99% C8 chain and is a mixture of 70% linear and
30% branched isomers. [N-ethyl-l-14C]Sulfluramid (14.1 mCi/mmol)
was also obtained from Griffin Corp. Oligomycin, ADP, CCCP and other

reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

2. German cockroaches

Susceptible and field collected (C.I. strain) insects were provided
by S. C. Johnson & Son (Racine, WS). Insects were maintained without
insecticide selection throughout the study under a photoperiod of
12:12 (L:D) at 25-28°C and were supplied with dry dog chow (Purina)
ad lib.

3. Cockroach bioassays:

Initial bioassays were run using a bait formulation. The bait was
made by mixing ground dog chow (Purina) thoroughly with the
desired compound concentration in 50% acetone/water. The mixture
was left for 3-5 min to evaporate most of the acetone. Mixtures were
then packed in a plastic test-tube rack (holes were 1.0" long, 0.25"
diameter) without excessive pressure and left in a well ventilated
area for 24 h till dry and firm. The dried pellets (1.46 i 0.07 g) were
then pushed out of the rack and put in open petri dishes for another
24 h to ensure dryness.

Insects were anesthetized by exposure to C02 for 10-20 sec and 20
adult males were taken for each treatment and held in 1.5 gallon
glass containers. Insects were starved for 24 h (with a supply of
water) before the introduction of the bait pellet. Bait concentrations

were 0.0001, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1.0% by weight for

W?

 

 

41

sulfluramid, NDES and PFOSA. An inhibitors of mixed function
oxygenases (piperonyl butoxide, PBO, 0.5%) and inducers of the same
enzyme systems [phenobarbital, (PhB) or butylated hydroxytoluene,
(BHT), 0.2%] were also used and mixed in the bait with the previous
insecticide concentrations. During the course of the experiment,
insects were held in the 1.5 gallon glass containers with hides and
provided bait and water freely. Mortality was observed daily for 11-
12 days and dead insects were removed. Experiments were
replicated 3-5 times and mortality was corrected using Abbott's
formula (Abbott, 1925). Experiments were terminated and excluded

from analysis if control mortality reached 20%.

4. In vivo metabolism studies‘

Initial purification of the [N-ethyl-l-14C]sulf1uramid twas
conducted using thin layer chromatography where 100 ul (~50 uCi)
solutions were spotted as a band on a Whatman LKF6 TLC plate
(Whatman, Maidstone, England). The plate was developed in
acetone/toluene 25:75. The developed plate was visualized using
autoradiography. The area corresponding to sulfluramid was then
scraped off the plate and eluted with acetone. Silica particles were
removed by centrifugation and the clear acetone was evaporated
under nitrogen. The white residue was then dissolved in DMSO to be
stored as a stock in solution at -20°C. Individual male cockroaches in
replicates of four were injected with 4 gag/insect (in 1 pl of DMSO : 1%
Tween 20 in water, 2:8) of the radioactive sulfluramid and held in 16
x 100 mm glass test tubes. Cockroaches were removed and

homogenized using a VirTishear homogenizer (The Virtis Co. Inc.,

42

Gardiner, NY) in 3 ml of acetonitrile : methanol, (4:1) every three
hours for 24 hours (susceptible insects were all dead by then). The
suspension was centrifuged for 10 min and 2 ml were taken for
scintillation counting. To identify the nature of the radioactive
compounds, 50 [.11 samples were also spotted on a LK5DF TLC plate,
developed in acetone/toluene 25:75, visualized by autoradiography,
scraped and counted as mentioned previously. Counting was done
using BetaTrac 6895 liquid scintillation counter (TM Analytical, Elk

Grove Village, IL). Experiments were repeated three to four times.

5. Monitoring cockroach respiration

Male cockroaches of the susceptible strain were immobilized by
cooling for 3-5 min in a freezer before injection with test compounds.
Injection was between the first and second abdominal segments
using a syringe with a finely drawn glass needle. Injection was done
with the needle inserted towards the head and ‘rjust beneath the
cuticle. The injection volume was '1 ul delivered slowly to prevent
excessive bleeding. Sulfluramid, NDES and PFOSA were dissolved in
DMSO (200 pl) and added to water (800 ul) containing Tween-20
(1%) as an emulsifier just before injection. Insects were tested in
groups of four confined in a small piece of plastic tubing (12x50 mm)
with both ends sealed using muslin. Insects were then monitored for
C02 production in a 1 liter chamber of a Li-6200 C02 analyzer (LiCor
Inc., Lincoln, NE). The instrument was calibrated daily using a known
mixture of CO2 in air (507 ppm). Injected insects were left for 10 min
to recover before beginning respiration monitoring. Carbon dioxide

levels in the chamber were then measured every 5 min for 50 min.

43

The same methodology was followed to monitor C02 for certain SULF
concentrations over longer periods of times (up to 12 h)
A second series of studies was conducted to examine the effects on
respiration after exposure of the insects to SULF through feeding as a
more realistic means of presenting the toxicant. Diet pellets were
prepared from ground dog chow as described previously to contain
either 0.1% or 1% active ingredient by weight. Adult male insects
were placed in 6 oz cups in groups of four and were starved for 24‘ h
before bait introduction. Respiration was monitored at 6-8 h
intervals for 70 h. Before each monitoring, insects were immobilized
by cooling for 5-7 min. Carbon dioxide was monitored as before for
20 min, the insects were removed from the apparatus and placed
back in the 6 oz cups that contained the bait pellets, harborage and
water. .

RESULTS
The symptoms of poisoning were slow in onset and terminated in
hypoactivity, prostration, and death. Toxicity studies using different
ascending concentrations of SULF revealed the slow acting nature of
this compound (Table 2). In the susceptible strain, sulfluramid
caused 100% mortality within 10 days at 0.01% while it took less
than 6 days to reach 100% mortality for higher concentrations (6
days. for 0.03% and 0.1%; 4 days for 0.3% and 1.0%). Lower
concentrations (0.0001%, 0.001% and 0.003%) did not reach more
than 23.9% mortality within the duration of the experiment. In the
field (tolerant) strain, lower concentrations caused similar mortality
but higher concentrations caused lower mortality than in the

susceptible strain and 100% mortality was not reached even at 1%.

44

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In the case of the N-desethylated metabolite (NDES), the first
concentration to cause 100% mortality (in 11 days) was 0.01% in the
susceptible strain (Table 3). Generally, NDES and SULF cause similar
levels of mortality but NDES'tends to be faster acting especially at
high doses and somewhat more toxic at lower doses (0.001-0.003%).
In the field stain, 100% mortality was reached but only at 1.0% after
five days, compared to two days for the susceptible strain. Again the
comparison between SULF and NDES revealed no remarkable
differences.

For perfluorooct‘ane sulfonic acid, (PFOSA), 100% mortality was
reached after 9 days at the 0.003% concentration which makes it
significantly more potent than either SULF or NDES at that particular
concentration (Table 4). At 0.01%, it took only 5 days to reach 100%
mortality while it took 10 days for both SULF and NDES at the same
concentration. Clearly, PFOSA is more toxic than either SULF or NDES
to the susceptible strain. The overall mortality from PFOSA after 11
days for all concentrations (except for 1.0%) was also higher in the
field strain than that seen with SULF and NDES. However, the field
strain was significantly more tolerant of PFOSA than the susceptible
strain and, notably, 100% mortality was not observed.

In an attempt to show the difference between the susceptible and
the field strains, data for one concentration (0.01%) are compared
graphically (Figure 6). The field strain shows lower mortality than
the susceptible and never reached 100% mortality when SULF was
used . After six days, the difference between strains was highly
significant (P> 0.0001). The same difference was also highly
significant for both NDES and PFOSA (Figures 7 and 8) with the

46

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48

 

 

120-

100-
> 1
l:
..l 80-
<
.—
C «1
O
I
m 60'
>
I: q
<
S
S 40-
D
0 4
3' 20. / —-— SULF 0.01 s

—*_ SULF 0.01 F
/
0" I r I I ' I I I ' fl
0 2 4 6 8 10 12 14

TIME (Days)

Figure 6: Cumulative mortality of German cockroaches of both
susceptible (S) and the field (F) strains as a result of
feeding 0.01% sulfluramid (SULF). Data are means i SE of
n = 3-6.

49

susceptible strain reaching 100% mortality earlier in the case of
PFOSA.

Another series of experiments was conducted to see if the potency of
sulfluramid and its analogs would be affected by the presence of
piperonyl butoxide in the diet (PBO, 0.5%) as an inhibitor of mixed
function oxygenases (MFO's) (Tables 4, 5 and 6). Given alone, PBO had
only a marginal toxicity at this dose. For the susceptible strain, both
SULF concentrations (0.01% and 0.1%) had lower mortality when
combined with PBO than alone. Antagonism was most evident at the
lower dose. In the field strain, there was no clear difference between
treatments as a result of PBO addition at 0.1% SULF, but antagonism
was seen at 0.01% (Table 5). These differences are shown graphically
in Figure 9. The same approach was taken for NDES but three NDES
concentrations were uSed in order to observe potential synergism by
PBO. The effect of NDES on the susceptible strain was typical. PBO
reduced the mortality when combined with NDES especially at the
higher concentration. (0.01%, Table 6). No evidence for synergism by
PBO was seen. There was no clear evidence for antagonism of NDES
by PBO in the field strain. Instead, a degree of synergism was seen at
both 0.001% and 0.01% NDES. For PFOSA, differences between PBO
and control treatments were small and no clear effect was observed
for PBO in either strains (Table 7).

The induction of MFO's was another rational way to assess the
significance of metabolism in the toxicity of SULF. Butylated
hydroxytoluene (0.2%) and phenobarbital (0.2%) as inducers of MFO's

were incorporated in the diet with two SULF concentrations (0.01%

120 1

100q

80-

60'-

4o-

% CUMULATIVE MORTALITY

20"

 

50

W

—l— NDES 0.01 8
—¢'— NDES 0.01 F

 

 

TIME (Days)

Figure 7: Cumulative mortality of German cockroaches of both
susceptible (S) and the field (F) strains as a result of .
feeding 0.01% N-desethyl sulfluramid (NDES). Data are

means :1; SE of n - 3-6.

51

 

 

120 -

100 -*
> 1
t.
_l
< 80 "
.—
a:
O
2
m 60 '-
2
l- 1
<
S
2 4o -
D
O
32 20 a

+ PFOSA 0.01 S
+ PFOSA 0.01 F
O ' I I I ' I r I ' T Y I . 1
0 2 4 6 8 10 12 14

TIME (Days)

Figure 8: Cumulative mortality of German cockroaches of both
susceptible (S) and the field (F) strains as a result of
feeding 0.01% perfluorooctanesulfonic acid (PFOSA). Data are

means i SE of n = 3-6.

52

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’ 100 .. —D— Susc. no PBO
—&— Field. no PBO
>_ ‘ —'— Susc. + PBO
: —A-— Field + PBO

..J 80 -
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a:
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Time (days)

Figure 9: EffeCts of Piperonyl Butoxide (PBO, 0.5%) on the
Toxicity of Sulfluramid (0.01% bait) in Susceptible
and Field Strains of German Cockroaches.

54

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56

and 0.1%). No significant difference in mortality was recorded due to
the use of these inducers (experiments were replicated only once).
To study if SULF was being metabolized during the course of
treatment, insects were injected with 4 ug of radiolabeled compound
and the loss of parent compound was observed over 24 h. The study
was done on individuals from both the susceptible and the field
strains (Figure 10). The study was limited to observations of the
survival of the parent (”C-SULF) because of probable loss of ‘the N—
ethyl label by MFG-mediated N-dealkylation. No metabolites were
observed on TLC analysis of body extracts. The rate of removal of
SULF was relatively slow but linear (approximately 50% in 24 h in
the susceptible strain). The rate of metabolism is slower in the field
strain with about 25% removed in 24 h. The difference between the
two strains in terms of the rate of metabolism is significant at P >
0.001. ,

If SULF acts as a mitochondrial uncoupler, an increase in respiration
should be seen in vivo at doses causing lethal effects. A series of
experiments were designed to monitor C02 production of live insects
using a LiCor C02 monitor typically used to study photosynthesis. In
initial tests to establish this method, insects were injected with
solvent (DMSO/Tween), inhibitors of oxidative phosphorylation (KCN
or rotenone), or a known uncoupler of oxidative phosphorylation (the
carboxanilide, EL-499, Appendix II) as shown in Table 8. This
method proved to be a convenient and sensitive one for monitoring
respiration in insects. The uncoupler caused a clear (4 to 5-fold)
increase in C02 production while rotenone and KCN both decreased

C02 production compared to controls.

57

 

 

5 -
4
\ i

3
EL
. i
e a .
G
:r.
c
0 .4
O
C
O
o

2 d

0 Susceptible Strain
I Field Strain
1 l V I 1
o 10 20 30

Time (h)

Figure 10: Elimination of injected 1“C-sulfluramid by males of

susceptible and field-strain German cockroaches. Data
are means 3; SE of n = 3-4.

58

Table 8: The rate of C02 production by German cockroach males
following the injection of inhibitors (KCN, rotenone) and an uncoupler

(EL-499) of oxidative phosphorylation. Numbers are means of
n = 2-4.

 

 

Treatments Rate“
(ppm/min)
Instrument Background 0.09
Uninjected insects 1.8
Solvent (DMSO/TWEEN) 3.0
KCN (20 uglinsect) 0.3
Rotenone (0.5 uglinsect) 1.6
EL-499 (0.25 uglinsect) 13.6

 

(*) Insects were monitored for 50 min immediately after injection.

59

After establishing the method, a series of experiments were
conducted involving injection of insects with SULF, NDES and PFOSA
for C02 monitoring to observe any increase in the output and
establish a dose—response relationship. In this case, insects were
monitored immediately after injection and only for 50 min.

SULF did not increase C02 production within 50 min, even at a higher
concentration of 10 ug/insect (Figure 11). Both NDES and PFOSA were
able to increase C02 production in a dose-related manner with PFOSA
being slightly more potent than NDES. The maximum stimulation was
about four-fold. PFOSA doubled the respiratory rate at about 0.3
uglinsect while 0.6 ug/insect was needed for NDES to cause the same
effect.

SULF was then studied using the same methodology but with
monitoring of C02 over longer periods of time. As shown in Figure 12,
SULF increased C02 production if given enough time after injection.
The rate was at a maximum after 2 h when 5.0 uglinsect were
injected, and after 3 h with 3.0 ug/insect. It took more time to see a
significant increase at lower doses. In the case of both 1.0 and 0.5
ug/insect, a wide peak was reached about 6 h after injection. The
decrease in C02 production after the peak in each case corresponded
with incipient mortality in the insects.

To confirm that SULF causes an increase in C02 production when fed
in the diet at realistic use rates, insects were fed two different
concentrations (0.1% and 1.0%). The C02 production was monitored
over several days at intervals of 6-8 h. At a dietary concentration of
1.0%, C02 production was significantly increased after 8 hours and

reached a maximum stimulation (three-fold) after 20-24 h. The

60

 

 

 

100-
so-
75
'g 60-
E
E
O.
3
40'
N
o
O
20-
q
o . . . a . 2H--.

 

10

Concentration
(uglinsect)

Figure 11: C02 production following the injection of male German
cockroaches with different concentrations of sulfluramid
(SULF), N-desethyl sulfluramid (NDES) and perfluorooctane-
sulfonic acid. (PFOSA). Insects were monitored immediately

after injection for 50 min.

61

605-

Control (solvent)
0.5 uglinsect
1.0 ugflnsect
3.0 ug/insect
5.0 ug/insect

 
 
 
 
  

 

 

 

E
E
2'
E
n.
e
N
O
0
O
10 " o
O O .
d . . . O
o ' I l I I l
0 2 4 6 8 10
Time (h)

Figure 12: 002 production following the injection of male German
cockroaches with 4 concentrations of sulfluramid '
plus the solvent. All treatments were monitored for a

period of 9 h. Data are means n . 2-4.

62

301

—°— (INTRO.
—D— SULF 0.1 °/o
—+_ SULF 1.0%

 

 

002 (ppm/glmln)

 

0 20 40 60 80

Time (hrs)

Figure 13: C02 production of male German cockroaches after
feeding 2 concentrations of sulfluramid (0.1% & 1.0%) . All
treatments were monitored for 20 min every 6-8 h for 70 h.

Data are means of n - 2—3.

63

subsequent decline in C02 production is correlated with death of the
insect. The maximum occurred after 40-45 h for the 0.1% dose

(Figure 13).

DISCUSSION
The results described in this study demonstrate the delayed action of
SULF when introduced through the diet. Slow action was reported by
Reid et al (1990) when observing the cumulative mortality of
German cockroaches due to the topical and dietary application of
SULF. Their study indicates that SULF toxicity continues for several
days in both applications and that topical application caused faster
kill because acetone facilitates compound penetration in this case.
Delayed toxicity was also observed by Nan-Yao Su and Scheffran
(1991) when SULF was used against subterranean termites both
topically and in the diet. Although they reported some feeding
deterrence at high concentrations, SULF mortality increased for up to
8 weeks. The results reported by Appel and Abd-Elghafar (1990)
also suggested that food deterrence was an obstacle in their Ebeling's
choice-box studies. In the mean time, SULF behaved typically on
both male'and female German cockroaches. In addition, SULF
increased the percentage of dropped-oothecae in a dose- dependent
manner.
' We found that NDES was just as toxic as SULF and PFOSA was even
more toxic. Since these are potential metabolites, it clearly suggests
that SULF could act indirectly through one or both of these

compounds as the primary toxicant. All these compounds increased

64

the respiration rate in vivo and the order of potency was PFOSA >
NDES>SULF.

All published studies were done using an insecticide-susceptible
strains of the German cockroaches. Our study has extended to
investigate the toxicity of SULF and its metabolites on a collected-
field strain. This strain showed a significant tolerance to SULF. The
mortality did not reach 100% even at the commercial bait rate of 1%
which suggest that the strain may be heterogeneous with some
members were quite sensitive and others were much more resistant.
The field strain was also significantly tolerant to both NDES and
PFOSA.

Increased respiration after feeding or injection indicates possible
action of SULF and/or its metabolites as uncouplers. This fits with the
in vitro observation of uncoupling by NDES in rat kidney
mitochondria (Schnellmann, 1990) (Chapter 3).

Metabolic activation appears to play an important role in the. toxicity
of sulfluramid as judged by the delay in the C02 production after
injection of sulfluramid and by the PBO results. Schnellmann (1990)
also observed that NDES is more active as an uncoupler than SULF.
The addition of PBO in the diet antagonized the action of SULF and
reduced mortality when compared to treatments without PBO.
Antagonism was observed when NDES was used with the susceptible
strain which suggests that further metabolic activation may occur via
MFO. However, the synergism of NDES by PBO in the field strain
suggests that metabolic differrences may exist between the strains
and that further metabolism of NDES by MFO is not critical for
toxicity. The lack of effect of PBO when combined with PFOSA is

65

reasonable since It is unlikely for PFOSA to be a substrate for MFO
action. It is surprising that neither phenobarbital nor BHT enhanced
toxicity although they are known to induce MFO in other insects
(Terriere and Yu 1976). It may be that the dose used was insufficient
to cause induction and it could also be that MFO carry out both
activation and detoxication and hence toxicity is not shifted by these
inducers.

The fact that SULF is removed more slowly in field strain than
susceptibles and that PBO has a lower antagonistic effect on the field
strain suggest that a lower rate of activation may be a partial
explanation for tolerance. Our results show that injected SULF
disappeared about twice as fast in susceptible compared to resistant
roaches. Several metabolic routes are possible (Figure 14), but
Manning 'et al, (1991) suggested that the main rout for metabolism in
mammals is by the elimination of the N-ethyl group of sulfluramid in
the form of C02 and the major metabolite identified was NDES. Their
study was actually done on rats and they indicated that the reaction
is much faster than we report here in cockroaches.

In conclusion, sulfluramid is an effective slow acting insecticide.
Symptoms give little indication of the mechanism of action but an
uncoupling action is supported by the increased respiration after
sulfluramid injection or feeding. NDES is just as toxic and PFOSA is
even more toxic than SULF. This and other evidence suggests that
SULF acts indirectly through one or both of these metabolites. The
field-collected strain was more tolerant to SULF than the susceptible
one and is also tolerant to both NDES and PFOSA. SULF is removed

more slowly in the field-collected strain than the susceptible one and

66

 

 

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68

a lower rate of activation may be a partial explanation for tolerance.
However, it can not-be the sole explanation since the probable active
metabolites,NDES and PFOSA were also tolerated by the field strain.
Further metabolism studies are needed to determine the rates and
routes of sulfluramid metabolism in the two strains. The mechanism
of toxicity of PFOSA also deserves study since, although it enhances
respiration, it is a very strong acid it is unlikely to act as a
protonophoric uncoupler. The possible role of the reported repellency

of sulfluramid in tolerance is also of interest.

69

References

Abbott, W. S. 1925. A method of computing the effectiveness of an
insecticide. J. Econ. Entomol. 18: 265-267.

Appel, A. G. and Abd-Elghafar, S. F. 1990. Toxicity, sublethal effects
and performance of sulfluramid against the German cockroach
(Dictyoptera: Blattellidae). J. Econ. Entomol. 83: 1409-1414.

Manning, R. 0.; Bruckner, J. v.; Mispagel, M. E. and Brown, J. M. 1991.
Metabolism and disposition of sulfluramid, a unique
polyfluorinated insecticide, in the rat. Drug metabolism and

disposition. 19: 205-211.

Reid, B. L.; Bennett, G. W. and Barcay, S. J. 1990. Topical and oral
toxicity of sulfluramid, A delayed-action insecticide, against the
German cockroach (Dictyoptera: Blattellidae). J. Econ. Entomol.
83: 148-152.

Schal, C. 1992. Sulfluramid resistance and vapor toxicity in field-
collected German cockroaches (Dictyoptera: Blattellidae). J. Med.
Entomol. 29: 207-215.

Schnellmann, R. G. 1990. The cellular effects of a unique pesticide
sulfluramid (N-ethylperfluorooctane sulfonamide) on rabbit renal
proximal tubules. Toxic. in vitro 4: 71-74.

Schnellmann, R. G. and Manning, R. O. 1990. Flourooctane
sulfonamide: A structurally novel uncoupler of oxidative
phosphorylation. Biochem et Biophys. Acta 1016: 344-348.

Su, N. and Scheffrahn, R. H. 1991. Laboratory evaluation of two slow-
acting toxicants against Formosan and Eastern subterranean
termites (Isoptera: Rhinotermitidae). J. Econ. Entomol. 84: 170-
175.

70

Terriere, L. C. and Yu, S. J. 1976. Microsomal oxidases in the fleshfly
Sarcophaga bullata. Parker) and the black blowfly (Phormia regina
(Meigen). Pestici. Biochem. Physiol.6: 223-228.

Vander Meer, R. K., Lofgren, C. S.and Williams, D. F. 1985.
Fluoroaliphatic sulfones: A new class of delayed-action
insecticides for control of Solenopsis invecta (Hymenoptera:
Formicidae). J. Econ. Entomol. 78: 1190-1197.

Chapter 3

The Mitochondrial Effects of the New Slow Acting
Compound Sulfluramid (FinitronTM) and its
Metabolites.

72

Introduction

Perfluoroaliphatic sulfonamides are a new family of pesticides
that are being developed to control major household and other social
insects (cockroaches and ants) (Vander Meer et al 1985). They are
also active against the red imported fire ant Solenopsis invicta
(Vander Meer et al, 1986; Williams et a1 1987). The first member of
this family to be commercialized is sulfluramid or ."FinitronTM" (Griffin
Chemical Co.).
Derivatives of perfluoroalkanecarboxylic and perfluoroalkane-
sulfonic acids are widely used in the industry because of their
unique properties as surfactants. They are used as lubricants,
surfactants, plasticizers and aqueous film-forming foam fire
extinguishants (Guenthner and Victor 1962; Shinoda and Nomura
1980). In a previous paper (Chapter 2) we have shown that
sulfluramid, NDES and PFOSA all strongly stimulate respiration in
vivo in cockroaches, although sulfluramid does so only after a delay.
There is strong evidence that SULF acts only after metabolic
activation, presumably to NDES, PFOSA or both.
This study is conducted to investigate the action for this new family

of insecticides on mitochondria.

Materials and Methods
1. Materials:
Sulfluramid (N-ethyl heptadecafluoro-l-octanesu1fonamide, SULF),
the N-methyl (MSULF) and N-isopropyl (ISULF) analogs of
sulfluramid, its unfluorinated analog (USULF), N-desethylated
sulfluramid (NDES), perfluorooctanesulfonic acid (PFOSA) and

73

octanesulfonic acid (OSA) were provided by Griffin Corporation
(Valdosta, GA). SULF 'and NDES are a mixture of 95% linear carbon
chain and 5% branched isomers with 98% C8 chain length and 2% C6.
PFOSA is a 99% C8 chain and is' a mixture of 70% linear and 30%
branched isomers. [N-ethyl-l-14C]Su1fluramid (14.1 mCi/mmol) was
also obtained from Griffin Corp.The stock solutions of these
compounds were prepared in ethanol and kept at -20°C. All other
reagents were obtained from Sigma Chemical Company, St. Louis,
MO.

Susceptible and field collected (C.I strain) German cockroach strains
were provided by S. C. Johnson & Son (Racine, WS). Insects were
maintained throughout the study under a photoperiod of 12:12 (L:D)
at 25-28°C and were supplied with dry dog chow (Purina) ad lib.

2. Mitochondrial Isolation:

For the 'isolation of well coupled mitochondria, water had to be
highly purified. Water was-prepared following the method described
by Toth et a1 (1986). Deionized water was glass distilled and then
redistilled from alkaline permanganate to remove all organic
contaminants. V

Liver mitochondria (RLM) were isolated from Sprague Dawley
rats of both sexes weighing 225-300 g by a method based on that of
Katyare et a1 (1971). Animals were sacrificed by decapitation and“ the
liver was quickly dissected and put in the homogenization medium
(containing 0.25 M sucrose, 4 mM Tris-HCI (pH 7.4) and 0.5 mM
EGTA). The liver was then minced and homogenized using a Potter-

Elvehjem-type homogenizer fitted with a Teflon pestle. The

74

homogenate was centrifuged first at 600g for 10 min then the
supernatant was centrifuged at 10,400g foranother 10 min to
sediment the mitochondria. The mitochondrial pellets were
resuspended and recentrifuged as before. The final pellets were
resuspended in the same medium to a final concentration of 1.0 - 2.0
mg protein/ml as determined by the method of Lowry et al. (1951)
using crystalline bovine serum albumin as a standard.

German cockroach thorax mitochondria were prepared from
the thoraces of 200 male Blattella germanica in 80 ml of isolation
medium (0.25 M sucrose, 4 mM Tris-HCI, 0.2 mM EDTA, 0.5 mM
EGTA and 0.1% BSA at pH 7.4). These were finely minced and
homogenized using a Potter-Elvehjem-type homogenizer fitted with a
Teflon pestle. The homogenate was filtered using 4 layers of ’
cheesecloth and centrifuged at 2,000g for 10 min. The supernatant
was then centrifuged at 18,000g for 10 min and pellet was
resuspended in the isolation medium and centrifuged for another 10
min at 18,000g. The final pellets were suspended to the desired

protein concentration as before.

3. Assay of mitochondrial respiration:

Oxygen utilization was measured polarographically using a Clark
oxygen electrode (Model 5300, Yellow Springs Instruments, Yellow
Springs, Ohio). The respiration medium for rat liver mitochondria
contained 0.1 M KCl, 5 mM K2HPO4, 1 mM EDTA, 20 mM Tris-HCl, 5
mM malic acid and 5 mM glutamic acid at pH 7.4. The mitochondrial
suspension (0.1 ml) was added to the reaction mixture to give a final

volume of 3 ml. State 3 respiration was initiated by the addition of 5

75

pl of ADP (0.4 umoles) using a 50 pl Hamilton syringe. The ADP:0
ratio, state 4 respiration and RCR (respiratory control ratio) were
calculated according to Estabrook (1967). Test compounds were

added in 5 ul ethanol and the effect on the above parameters of

coupling was calculated.

4. In vitro metabolism of sulfluramid

The metabolism of SULF was studied using microsomes and
mitochondria from rat liver. The S-lO fraction and microsomes were
isolated using the method described by Guengerich (1982) and the
mitochondria were isolated as described earlier. The reaction was
started by the addition of the tissue (mitochondria, microsomes and
S—10 supernatant) to the incubation medium (100. mM Tris-HCl and 4
mM MgC12) in the presence of 1 mM NADPH (or NADH) and 10'5 M
1‘lC-SULF at 37°C. Samples (10 ul) were taken at 0, 2, 4, 10, 20 and
40 min for TLC development on Whatman LKF6 plates (Whatman,
Maidstone, England) using acetone : toluene (25:75) as the solvent.
Zones corresponding to the radiolabelled SULF were visualized by
autoradiography, scraped and counted using a liquid scintillation

counter.

5. Artificial membrane studies with cytochrome aa3

To avoid complications due to possible metabolism of SULF during
mitochondrial incubations, Iiposomes containing purified cytochrome
aa3 (coupling site III) were prepared. These act as proton pumps and
consume oxygen but contain no cytochrome P450 or other xenobiotic-

metabolizing enzymes. The artificial membrane liposomes were

76

prepared from soybean phospholipids (Asolectin) and reconstituted
with purified cytochrome oxidase from bovine heart accordingito the
methods of Carroll (1977) and Casey (1979).

The respiration medium for the Iiposomes consisted of 10 mM HEPES,
41 mM KCI and 38 mM sucrose. In addition, 3.3 mM ascorbate, 0.2
mM TMPD, 30 pM cytochrome c and 0.6 pM valinomycin were

added. Oxygen uptake was measured with the Clark-type oxygen
electrode. Test compounds (5 pl) dissolved in ethanol were added
using Hamilton syringes. Compounds were tested in the presence and
the absence of valinomycin which acts as a K+ exporter to balance
charge distribution as H‘l‘ is imported into the vesicles. The
conventional uncoupler carbonylcyanide m-chlorophenylhydrazone

(CCCP) was included for comparison.

6. Mitochondrial Swelling

Mitochondrial swelling studies were conducted according to the
method of Chapel] and Croft (1966) and Brierley (1976) using a
Shimadzu UV. 265 spectrophotometer (Shimadzu Corp., Kyoto, Japan).
Rat liver mitochondria were prepared as described before in 330 mM
sucrose, 0.5 mM EGTA and 3.5 mM Tris-HCI at pH 7.4. The
mitochondrial swelling medium contained 30 mM K-acetate, 100 mM
sucrose, 5 mM Tris-HCl and 1 mM EGTA at pH 7.4. The final volume
was brought up to 1 ml. All test compounds used were at 0.12 mM as
a final concentration except for valinomycin (2 pM ) and succinate at
(0.4 mM). All additions were delivered in 2 pl volumes. Swelling was

monitored at 550 nm.

77

Results
Sulfluramid has only a low uncoupling effect even at higher
concentrations (100 pM) where low water solubility becomes an
obstacle (Figure 15). No uncoupling activity was evident at 10 pM.
However, as shown in this Figure, uncoupling at 100 pMslowly
increased during the 3-10 min incubation period. This is atypical of
most uncouplers which act essentially instantaneously e.g. see CCCP
in Figure 15. This delayed effect with SULF might be due to slow
resolubilization and redistribution of SULF which tends to
immediately precipitate from solution at 100 pM. Alternatively SULF
may be converted to an active uncoupler by metabolism in the
mitochondrial assays.
In contrast, the N-desethylated analog of SULF (NDES) showed a clear
and instantaneous uncoupling activity, even at 10 pM (Figure l6-A).
Typical of protonophoric uncouplers, its stimulation of respiration
was not inhibited by the FIFO-ATPase inhibitor, oligomycin (6
nmoles/mg protein,- Figure l6-B).
The relative uncoupling activities of SULF and its analogs are shown
in Figure 17. Only NDES showed strong activity. The. unfluorinated
analog of SULF (USULF) lacked even the small activity of SULF.
Similarly, MSULF and ISULF were essentially inactive at
concentrations up to 100 pM as were the sulfonic acid analogs, PFOSA
and OSA. While NDES showed appreciable uncoupling activity, it was
significantly less active (100-1000 fold) than such highly potent
uncoupler as CCCP and EL-499 (see Chapter 5).
To test the possibility that SULF is activated by mitochondria, its

metabolism by liver subfractions was assessed. SULF was rapidly

'73

Mitochondria
Substrate

V ADP

       

Uncoupler

v Sulfluramid
100 pM

J ’ .
Time
(min)

 

Figure 15: Uncoupling of rat liver mitochndria by sulfluramid
compared to the standard uncoupler, CCCP.

79

metabolized by rat liver microsomal and mitochondrial preparations
in the presence of NADPH but not NADH (Figure 18). About 80% was
destroyed by the S-10 fraction in the first two min of incubation. An
appreciable decline in SULF was also observed in mitochondrial
incubations, but only in the presence of NADPH.

To further examine whether sulfluramid was being activated through
metabolism by mitochondrial P450-linked MFO's, the effects of SULF,
NDES and PFOSA were studied on an artificial membrane system
(Figurel9) that was reconstituted with purified cytochrome oxidase
(aa3). Valinomycin increased the rate of oxygen consumption in both
control and uncoupler treatments (Figure 20) and was included in all
subsequent experiments. To test the response of this system to
uncouplers, CCCP (an established carbonylcyanide phenylhydazone
uncoupler) was added (10 nmoles). This increased the rate of 02
consumption several-fold (Figure 20). Maximum stimulation of
respiration of the Iiposomes (RR = 4) was achieved with 10'6 M CCCP
in the presence of valinomycin (Table 9). Under these conditions both
SULF and NDES increased the RR values at 105. NDES was more
active than SULF, increasing the RR values at 1 pM also. However,
NDES again was less active than CCCP which achieved maximal
stimulation (RR = 4) at 1 pM, while NDES showed an RR value of 2.2
at this concentration.

One of the possible metabolites of sulfluramid other than NDES is
perfluorooctanesulfonic acid (PFOSA). This compound was tested in
vivo on male German cockroaches and found to be significantly more
toxic than NDES. It also increased respiration in vivo rapidly and

potently (chapter 2). PFOSA may therefore be a toxic metabolite of

80

Figure 16: A schematic tracing of Clark oxygen electrode showing
the effects of the N-desethylated metabolite (NDES) in the
presence and absence of the ATPase specific inhibitor,
oligomycin, on rat liver mitochondria.

 

 

81

ADP

 
  
        

A) Glutamate

B) Glutamate Oligomycin

ADP

: A
33%
>a to
N :1
o v
rme
(min)

02=Zero

96 Stimulation of state 4

96 Stimulation at state 4

1200
5 —°— PFOSA
E I + OSA
'5
§
0 I 4 l I I U I I
I 4 I 1 I I I I 10- .0'3.0'2.0'1.00101102103
10' .0'3.0'2.0"I0°101 102103
1200 ' 1200
1000-
. —_'—‘ SULF
g °°°.—--— ISULF
a
" . —'A-—
-§ 600.—°- MSULF USULF
2 u
400-
zoo-M
l
O I I l I j 0] 4| 31 2| l T l
10'310‘210‘1100101 102103 10' .0‘ .0" .0".0°10‘ 102103

82

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 17: The uncoupling effects of sulfluramid (SULF), the
N-desethylated (NDES), N-methyl (MSULF), N-isopropyl
(ISULF), and unfluorinated (USULF) analogs of sulfluramid,
perfluorooctanesulfonic acid (PFOSA) and
octanesulfonic acid (OSA).

83

120
100 ‘ '

'1 _ —n— Micro (NADPH)
80 ( __, —v— S-10(NADPH)

 

-'-II- Mitoe (NADPH)
—9— Mitos (NADH)

 

% Remaining
M A
O O
l a I

 

 

0 ' I ' ———_— " .
o 1 0 20 so 40 50
Time (min) '

Figure 18: Metabolism of 14C-sulfluramid by rat liver subfractions
and the effect of both NADPH and NADH.

85

SULF. To determine its mode of action, the compound was applied in
vitro to mitochondria from rat liver and German cockroach thoraces
and was found to have a slight but not a significant stimulatory
effect on respiration at 100 pM concentration (Figure 21 and Table
10). Similarly it showed only low activity in the liposome
preparation.

Terada et al (1990) recently showed that two compounds which form
an ion-pair can cause uncoupling even though neither is active alone.
Extending this concept to PFOSA, a series of quaternary and tertiary
amines, polyamines and phospholipds as potential ion-pairing agent
were tested on both mitochondria and liposomes in the presence of
PFOSA. T ributylamine (TriBA) had the ability to potentiate the
uncoupling effect of PFOSA (Figure 2l-B) at a concentration at which
‘ TriBA alone showed only slight uncoupling activity (Figure 21-A).
The replacement of PFOSA by OSA (octanesulfonic acid) in the
presence of TriBA showed no potentiation (Figure 21-A) when
compared to PFOSA + TriBA (Figure 21-B).

A variety of other alkylamines were also tested in combination with
PFOSA. Lower alkylamines (e.g. TriBA, DiBA) were inactive in
combination with PFOSA. With longer alkyl chains (e.g. TriHA), the
amines themselves were active uncouplers. However, dihexylamine
(DiHA) gave an effect similar to that of TriBA (Table 10). Notably,
tetrabutyl ammonium ion (TetBA) was not active. The naturally
occurring amines (spermidine, cadaverine and phospholipidamines)
were also inactive. Studies with cytochrome oxidase Iiposomes gave
comparable results. All of these studies were conducted at a fixed

equimolar concentration of PFOSA and alkylamine. A study was then

86

Figure 20: A schematic tracing of Clark oxygen electrode showing
the effects of valinomycin and an uncoupler on an
artificial membrane reconstituted with cytochrome oxidase.

 

vesicles

Oxygen
(patom)

Time
(min)

 

valinomycin

ProtonOphore
(CCCP,10 umoles)

O 2: Zero

88

Table 9:The effects of sulfluramid and NDES on the respiration of
artificial Iiposomes reconstituted with the enzyme cytochrome
. oxidase.

 

 

Compound Concentration Valinomycin Respiratory
(M) Ratio (RR)**

Control Ethanol - 1.0

- + 1.5 (0.6)

CCCP 10‘6 + 4.0 (0.3)

Sulfluramid 10'7 + 1.8 (0.6)
10"5 + 1.6 (0.6)
10'5 + 2.6 (0.7)

NDES 10'7 + 1.7 (0.5)
10'6 + 2.2 (0.3)
10-5 + 3.2 (0.2)

 

(**) Respiratory Ratio = Rate of respiration after the addition of the uncoupler
divided by the rate of respiration before the addition of the uncoupler.
Values are 1 standard deviation of n = 3.

89

Figure 21: A schematic tracing of Clark oxygen electrode showing
the potentiation caused by the addition of tributylamine
(TriBA) to the perfluorooctanesulfonic acid (PFOS). The
effects of the unfluorinated analog, octanesulfonic acid
(OSA), was not changed by the addition of TriBA.

90

ADP

l osa
(0.1 mM) TriBA
(0.1 mM)

1

A)

     
 

ADP

B)

PFOS
(0.1 mM) TriB A

l “’“f‘”

ADP

Oxygen
(patom)

 

Time
(min).

02:

91

Table 10: The effect of monoamines, polyamines and phospholipids as
ion-pairing agents on the uncoupling activity of perfluorooctanesulfonic
acid (PFOSA) using rat liver mitochondria.

 

 

Additions" RCR % of control
respiration
Control 7.4 100
Solvent (ethanol) 6.7 100
SULF 3.9 194
NDES 1.0 833*
PFOSA 3.3 261
Tributylamine 3.5 222
Tetrabutylammonium Iodide 5.2 155
Dibutylamine ‘ 4.9 226
Diethylamine 5.0 186
Triethylamine 6.1 165
Dihexylamine 2.2 334
Trihexylamine l .0 677 *
Spermidine 5.6 173
Cadaverine 5.1 177
Phosphatidyl Choline 5.1 221
Phosphatidyl Ethanolamine 5.2 225
Ammonium Chloride 4.1 196
PFOSA + Tributylamine 1.0 833*
PFOSA + Tetrabutylammonium 1. 3,5 211
PFOSA + Dibutylamine 4.2 245
PFOSA + Diethylamine 5.0 186
PFOSA + Triethylamine 4.2 198
PFOSA + Dihexylamine 1.0 677*
PFOSA + Trihexylamine 1.0 677*
PFOSA + Spermidine 5.6 173
PFOSA + Cadaverine 5,1 177
PFOSA + Phosphatidyl Choline 5,1 221
PFOSA + Phosphatidyl 5.2 225
Ethanolamine
PFOSA + Ammonium Chloride 4,1 196

 

* Mitochondria are fully uncoupled and the addition of ADP has no effect
"All additions were at a final concentration of 10'4 M.

% Stimulation of state 4

respiration

92

 

 

 

 

 

2000 -

0 Varying TriBA

0 Varying PFOSA
1000 .1

___l____£ .
v H v
01 v 1 n"... 1 ..vv..., . . is"... . . infifw 1 "n... t . "..q—w-I-H-Irn
10" 10'3 10'2 10‘1 10° 101 102 103

Concentration (uM)

Figure 22: The effect of using varying rates of PFOSA and TriBA on
mitochondrial uncoupling.

93

conducted to find out which dose of ,PFOSA or TriBA would initiate
this potentiation effect. TriBA (0.1mM) was added to a series of
PFOSA concentrations (0.1 nM - 0.1 mM) and PFOSA (0.1 mM) was
added to a comparable series of TriBA concentrations. Their effects
were studiedon the respiration of rat liver mitochondria.
Potentiation in both cases started at 0.01 mM but was only notable at
0.1 mM (Figure 22).

To determine if the putative ion—pair is behaving like a true
uncoupler, a series of mitochondrial swelling studies were conducted
with SULF, NDES and PFOSA using RLM. Valinomycin alone caused
rapid swelling. Under these conditions SULF at 100 pM had little
effect. As shown in Figure 23-2, the presence of a true uncoupler
(CCCP, 1 pM) caused the mitochondria to shrink and the subsequent
effect of valinomycin was reversed. NDES (0.1 mM) behaved
similarly although with no initial shrinking before the addition of
valinomycin. A considerable valinomycin-like swelling was observed
after the addition of 0.1 mM PFOSA (Figure 23-3). This was
completely reversed by the addition of the same concentration of
TriBA (Figure 23-3-A). The addition of valinomycin did not cause
any additional effect in contrast to the cases of both CCCP and NDES.
These observations were repeated when TriBA was replaced by DiHA

but not with most of the other amines in Table 9, including TetBA.

Discussion
The results presented in this Study indicate that although
sulfluramid increased state 4 respiration at higher concentration, it is

not as potent an uncoupler as its N-desethylated metabolite or as the

94

Figure 23: Representatives recordings of swelling studies with rat
liver mitochondria.
l) the effects of sulfluramid (A) before the addition of
valinomycin and (B) after the addition of valinomycin.
2) the effects of (A) CCCP, and (B) NDES .
3) the effects of PFOSA and TriBA (A) before the
addition of valinomycin and (B) after the addition of
valinomycin .

A550

95

Succlnate
Rotenone

Succlnate 1
Rotenone B" ___f\
SULF

(0.1mM)

Valinomycin
\ (2 pM)

Time -
mln.

Valinomycin
(2 pM)

/

SULF

\/

Figure 23-1:

Shrlnk

Swell

96

 

     
     
 

fi—H
Valinomycini
(211M) \
i.
23:32:?
(luM)
a. l
Shrink
Succlnate
Rotenone
1 NDES
fl, __ (0.1mM), Swen
l Vanno
(211M)
O
in
1n
<(
“Tune
nun.

Figure 23-2:

97

    
 

 

Succlnate
Succinate
Rotenone
Rotenone
. PFOSA Valinomycin
A. (O . 1111M) _B__: (2 uM)
Valinomycin
(2 UM)! i
Shrink
TriBA
(0.1mM)
Swell
PFOSA
+
TriBA
(0.1mM)
0
L!)
m
4 \_/
Time
min.

Figure 23-3:

98

conventional uncoupler CCCP. The problems of solubility and binding
to glass made it inaccurate to further study SULF at high
concentrations. Insolubility may explain the unusual shape of SULF
02 tracing where resolubilization or redistribution after initial
precipitation may happen. Another reason for that shape could be
metabolic activation by mitochondria. Changing the lipophilicity of
sulfluramid did not improve its uncoupling potency. The N-methyl,
N-isopropyl and unfluorinated analogs of sulfluramid did not
increase state 4 respiration even at 0.1 mM with RLM. This disagrees
with Schnelmann and Manning (1990) and Schnelmann (1990) when
reporting the uncoupling activity of SULF at 10 pM on rabbit renal
cortical mitochondria.

The SULF metabolite, NDES, increased mitochondrial state 4
respiration, released oxygen consumption in the presence of the
FIFO-ATPase specific inhibitor oligomycin and caused mitochondria to
shrink in the presence of valinomycin at concentrations in the range
of l to 100 pM. This indicates it is acting as a true uncoupler and
raises the possibility that it could be one of the active metabolites
produced from sulfluramid. This metabolite is about 7-10 times more
potent than ‘sulfluramid when studied on rat liver mitochondria
which agrees with the findings of Schnellmann and Manning, 1990
who compared these compounds on the rabbit renal cortical
mitochondria.

Surprisingly since it stimulates respiration in vivo, PFOSA did not
have a significant uncoupling activity. The same conclusion was also
reached with its unfluorinated analog, OSA.The necessity for

fluorination of the alkane chain for biological activity in these

99

compounds is confirmed by the complete lack of activity of the
unfluorinated analog of PFOSA and of the unfluorinated analog of
sulfluramid in these studies. 1

One of the major objectives of testing SULF, NDES and PFOSA on
artificial membrane vesicles was to test the hypothesis that was
suggested by Schnellmann, 1990 and Schnellmann and Manning,
1990 that sulfluramid could be rapidly metabolized to NDES by
mitochondrial P450-linked MFO's. The fact that liposomcs contain no
dealkylating enzymes, makes the uncoupling ability of any
compound completely depend on its chemical-physical properties.
These studies show that both SULF & NDES can act as uncouplers and
the activity of SULF is low compared to that of NDES and particularly
(ID.

Schnellmann and Manning (1990) concluded that mitochondria
metabolize SULF. About 2% conversion was observed in 5 min.
Despite the low level of conversion, they felt that this explained the
observed uncoupling activity of SULF. In this study, mitochondria did
not metabolize SULF extensively unless NADPH was added and this is
not the case in typical mitochondrial incubations. Microsomal and S-
10 fractions rapidly degraded SULF, probably to NDES, while
mitochondrial fraction in the presence of NADH did not. These results
suggest that the effect of SULF on mitochondria is direct rather than
through metabolism to NDES though a low degree of conversion to
NDES may occur.Further, the calculated pKa valuues for NDES is
around 7.5 (based on an observed pKa of 9.5 in methanol) whereas
SULF has a calculated pKa of about 5.0 (A. Las, personal

_ communication). This difference is not so great that NDES would act

100

as a reasonably active uncoupler and SULF would be inactive.
However, in vivo NDES may be produced quite rapidly and NDES
could be a major factor in SULF toxicity. 1

Based on these results, the relatively high toxicity and ability of
PFOSA to stimulate respiration in cockroaches (Chapter 2) are not
explained. Certainly, PFOSA would not be expected to act as an
uncoupler because of its extremely acidic nature. Terada et al (1990)
recently showed that the alkylamine local anesthetic bupivacain's
activity changes from decoupling to uncoupling in the presence of the
anion ANS'(anilinonaphthalenesulfonic acid). This was attributed to
the formation of a lipophilic ion-pair between the two compounds.
This ion-pair was considered to have a relatively non-specific
disruptive effect on membrane integrity. To test this idea with
regard to PFOSA, a series of simple alkylamines were added with
PFOSA to the mitochondria. A strong potentiation of uncoupling was
seen with some amines. The optimum chain length for this effect
appeared to be a total of 12 carbon atoms (TriBA or DiHA). Any
potentiation by longer chain amines was concealed by their innate
uncoupling activity. The order of addition of PFOSA or TriBA to
mitochondria was not important. The data also show that
potentiation occurs when there is a 1:1 Stoichiometric ratio of the two
compounds.

To further understand the nature of this interaction, the effects of
SULF, NDES and PFOSA were studied on mitochondrial swelling in the
presence of the K+-ionophore, valinomycin. SULF had no significant
effect and NDES reversed the effect of valinomycin in a manner

typical of uncouplers. PFOSA alone gave an effect like that of

101

valinomycin. Upon the addition of TriBA the effect was reversed, as
in the combination of valinomycin and an uncoupler.

A possible explanation for these observations is offered in Figure 24.
It is assumed that PFOSA acts as a transporter of potassium, like
valinomycin. A limitation on its activity would be the energy barrier
of movement of‘ the anion with its highly localized charge across the
lipophilic environment of the membrane. Trialkylamines with
sufficient lipophilicity act as weak uncouplers. Their action also is
limited by the presence of a localized charge in the protonated form
and also by their high pKa value which does not favor dissociation of
the protonated form at the inner interface of the membrane. The
combination of PFOSA and alkyamine could form an exchange system
whereby one potassium is extruded from the potassium-rich matrix
in exchange for the import of one proton from the proton-rich
external environment. Further, by forming a lipophilc ion-pair within
the membrane, the limiting factors in the diffusion of the charged
species is removed. This model also explains the complete lack of
interaction between tetrabutylammonium ion and PFOSA. Although
this combination could form a lipophilic ion-pair, TetBA as a
quaternary amine cannot act as a protonophore in that model.
Further testing of this model is needed, especially for the hypothesis
that PFOSA is a K+-transporter. It is not clear whether a similar ion-
pairing effect occurs in vivo.

A further factor that may be involved in the activity of these
perfluorinated compounds on mitochondria, and particularly that of
PFOSA, is their strong capacity to act as surface-active agents

(Hoffmann and Ulbrich, 1977; and Shinoda and Nomura, 1980).

a 102

ea 9 ‘

  
   
 
  

//////////////////

   
    
    
    

 

 

PFOSA' — — —- —>PFOSA'

Haunt _. — — —» arr-1+

 

 

   

Outside

. ////////////////// .nslde

Inner Mitochondrial
Membrane

Figure 24: Suggested model explaining the mechanism bywhich
ion-pairs migt uncouple oxidative phosphorylation.
R3NH“: alkylamine cation.

103

Surfactants at sufficient concentration can disrupt the integrity of the
mitochondrial membrane leading to ion leakage. Fully fluorinated
alkylsulfonates are about 10-fold more active as surfactants than
their unfluorinated analogs and can reduce the surface tension of
water at concentrations below 1 mM. It is significant that in this
study, the unfluorinated analogs of SULF and PFOSA were completely
inactive on mitochondria at highest concentrations tested (0.1 mM)
and the unfluorinated analog of SULF shows virtually no insecticidal
action (Vander Meer et al 1990). Surfactant activity is also strongly
dependent on the counter-ion present with PFOSA. The A
tetraethylammonium ion makes a particularly active combination
with PFOS (Shinoda et 'al 1972). The ability to form micelles in water
is well developed in such molecules and could also be involved in the
disruption of mitochondrial functions. However, the critical micelle
concentration (cmc) for PFOSA-tetraethylammonium is about 1 mM
(Hoffmann and Ulbrich, 1977). This concentration seems to be too
high to explain the uncouplinggeffects of PFOSA on mitochondria
when combined with alkylamines, which occurs wi high activity at
0.1 mM. However, specific information on the cmc for the PFOSA-
alkylamines most active as uncouplers (combinations with TriBA or
DiHA) is lacking and judgement on the significance of micelles in this
regard must be reserved.

The relative roles of PFOSA and NDES in the toxicity of sulfluramid
also remain to be determined. It can best be. assessed by metabolic
studies with the appropriate radiolabeled version of sulfluramid (in
the fluorinated chain) which was not available forthis study. Our

results suggest that metabolic activation by microsomal mixed

104

function oxygenases is a critical feature for the toxicity of

sulfluramid (Chapter 2).

105

References

Guengerich, F. P. 1982. Microsomal enzymes involved in toxicology
analysis and separation. In Principals and methods of Toxicology.
pp. 609-634. (ed. A. Wallace Hayes) Raven press, New York.

Guenthner, R. A. and Victor, M. L.1962. Surface active materials

from fluorocarboxylic and fluorosulfonic acids. I & EC Prod. Res.
Dev. 1:165-169.

Hoffmann, H. and Ulbrich, W. 1977. Kinetische und
thermodynamische messungen zur aggregation von perfluorirten
tensiden. Ziet. Phys. Chem. 106: 167-184.

Olorunsogo, O. O. and Malomo, S. O. 1985. Sensitivity of oligomycin-
inhibited respiration of isolated rat liver mitochondria to
Perfluidone, a fluorinated arylalkyl sulfonamide. Toxicology 35:
231-240. '

Schnellmann, R. G. 1990. The cellular effects of a unique pesticide
sulfluramid (N-ethylperfluorooctane sulfonamide) on rabbit renal
proximal tubules. Toxic. in vitro 4: 71-74.

Schnellmann, R. G. and Manning, R. O. 1990. Flourooctane
sulfonamide: A structurally novel uncoupler of oxidative
phosphorylation. Biochem et Biophys. Acta 1016: 344-348.

Shinoda, K.; Hat, M. and Hayashi, T. 1972. The physicochemical
properties of aqueous solutions of fluorinated surfactants. J. Phys.
Chem. 76: 909-914.

Shinoda, K. and Nomura, T.1980. Miscibility of fluorocarbon and
hydrocarbon surfactants in micelles and liquid mixtures. Basic
studies of oil repellent and fire extinguishing agents. J. Phys.

Chem. 84: 365-369. .

106

Terada, H.; Shima, O.; Yoshida, K. and Shinohara, Y.l990. Effects of
the local anesthetic Bupivacaine on oxidative phosphrylation in
mitochondria. Change from decoupling to uncoupling by the
formation of a leakage type ion pathway specific for H+ in
cooperation with hydrophobic anions. J. Biol. Chem. 265: 7837-
7842.

Toth, P. P.; Ferguson-Miller, S. M. and Suelter, C. H. 1986. Isolation of
highly coupled heart mitochondria in high yield using a bacterial
collagenase. Meth. Enzymol. 125: 16-27.

Vander Meer, R. K., Lofgren, C. S.and Williams, D. F. 1985.
Fluoroaliphatic sulfones: A new class of delayed-action

insecticides for control of Solenopsis invecta (Hymenoptera:
Formicidae). J. Econ. Entomol. 78: 1190-1197.

Vander Meer, R. K., C. S. Lofgren, and D. F. Williams, 1990. Methods
for control of insects. United States Patent # 4921696.

Williams. D. F.; Lofgren, C. S. and Vander Meer, R. K. 1987. J. Agr.
Entomol. 4: 41-47.

CHAPTER 4

Inhibition of Mitochondrial Respiration by the
Quinazolinamine Pesticide, Fenazaquin (EL-436).

108

INTRODUCTION

Quinazolines are a new class of pesticides with activity especially
against mites, some insects and fungi. One of these compounds, 4-[2-
[4-(1,1-dimethylethyl)phenyil]ethoxy]quinazoline (Figure 5-B)
(fenazaquin; EL-436) is now being developed primarily as a miticide.
This compound has an oral LD50 between 500-5000 mg/kg in rats
(Elanco Technical Report, 1989). Major toxicity symptoms in
mammals are hypoactivity, ataxia, lethargy and coma. In our initial
studies, when the compound was fed in bait formulation to male
German cockroaches (Blattella germanica), no death was observed at
doses up to 1.0% over a period of two weeks. On the other hand,
when the compound was injected into the haemocoel, the insects
showed hypoactivity and became prostrate shortly after injection.
These observations led us to hypothesize that the compound might
be acting upon the respiratory system. This study describes the
effects of fenazaquin on the mitochondrial electron transport as a

suggested site of action.

MATERIALS AND METHODS
Chemicals:
Fenazaquin (EL-436) and its analogs were provided by DowElanco
Research Laboratories, Greenfield, IN. Stock solutions of these
compounds were prepared in ethanol and kept at -20°C. All other
reagents were obtained from Sigma Chemical Company (St. Louis,

MO). Rotenone was recrystallized from hexane before use.

109

Mitochondrial Isolation:

Liver mitochondria (RLM) were isolated from Sprague Dawley rats of
both sexes weighing 225-300 g. The mitochondria were isolated by a
method based on that of Katyare et al. (1971). Animals were
sacrificed by decapitation and the liver was quickly dissected and
put in the cold homogenization medium (containing 0.25 M sucrose, 4
mM Tris-HCl (pH 7.4) and 0.5 mM EGTA). The liver was minced and
homogenized using a Potter—Elvehjem-type homogenizer fitted with a
Teflon pestle. The homogenate was centrifuged first at 600g for 10
min then the supernatant was centrifuged at 10,400g for 10 min to
sediment the mitochondria. The mitochondrial pellets were
resuspended and recentrifuged as before. The pellets were
resuspended to a final concentration of 1.0 - 2.0 mg protein/ml as
determined by the method of Lowry et al. (1951) using crystalline

bovine serum albumin as a standard.

Assay of mitochondrial respiration:

'Oxygen uptake was measured polarographically using a Clark type
oxygen electrode (Model 5300; Yellow Springs Instruments, Yellow
Springs, Ohio). The respiration medium for rat liver mitochondria
contained 0.1 M KCl, 5 mM K2HPO4, 1 mM EDTA, 20 mM Tris-HCl, 5
mM malic acid and 5 mM glutamic acid at pH 7.4. Glutamate and
malate were replaced by 5 mM succinate in some assays. The
mitochondrial suspension (0.1 ml) was added to the reaction mixture
to give a final volume of 3 ml. State 3 respiration was initiated by
the addition of 5 pl of ADP (0.4 pmoles) using a 50 pl Hamilton

syringe. State 4 respiration rates and respiratory control ratios (RCR)

110

were calculated according to Estabrook (1967). The inhibitors were.
tested on mitochondria that were fully uncoupled by the addition of
CCCP at 10'6M. Inhibitors were added in 5 p1 ethanol and the percent

inhibition of respiration was calculated.

Sf-9 cell respiration:

Cellular studies were conducted on the Sf-9 cell line derived from the
. pupal ovary of Spodoptera frugiperda. Initial cultures were obtained
from ATCC (American Type Cell Culture, Rockville, MD). Subculturing
and maintenance were carried out according to the method of
Summers and Smith (1987). Cells were cultured in Ex-Cell 400 low
protein (10 pg/ml) nutrient medium (J.R. Scientific Inc., Woodland
CA). Cultures were maintained in a 27°C incubator until they become
confluent. Confluent cultures were harvested using a cell scraper and
the suspended cells were sedimented at 2,500 rpm for 5 min. The
medium was gently decanted and the cells of a single flask (75 cm2
containing 20 ml media as a final volume) were resuspended in 2 m1
of medium. This suspension contained 0.5 - 1.0 mg protein/ml. The
oxygen level was monitored using the Clark oxygen electrode. A 1 ml
volume of cell suspension was brought up to 3 ml by the addition of
2 ml of the plain Ex-Cell medium to start the reaction. A strongly
uncoupling concentration of CCCP (0.01 mM) was added followed by
the inhibitor in 5 pl ethanol. The percent inhibition of respiration
was calculated compared with control incubations containing ethanol

only.

111

(Cockroach respiration in vivo:

Male cockroaches of a susceptible strain originally supplied by S. C.
Johnson & Son (Racine, WI) were slowed down by cooling for 3-5 min
in a freezer before injection. Injection was subcuticular between the
first and second abdominal segments using a finely drawn 10 pl glass
pipett. The injection volume was 1 pl delivered slowly to prevent
excessive bleeding. Fenazaquin and rotenone were dissolved in DMSO
(200 pl) and added to water (800 pl) containing Tween-20 (1%) as an
emulsifier just before injection. Controls were injected with 1 pl of
this solvent. Insects were tested in groups of four confined in a small
' piece of glass tubing (12 x 50 mm) with both ends sealed with
muslin. Insects were then monitored in a 1 liter chamber of a Li-
6200 C02 analyzer (LiCor Inc., Lincoln, NE). The machine was .
calibrated daily using a known mixture of C02 in air (507 ppm).
Injected insects were left for 10 min to recover before beginning
respiration monitoring. Carbon dioxide levels in the chamber were

then measured every 5 min for 50 min.

Mite toxicity assays:

Two week old squash seedlings were used in this assay. Seedling
leaves were infested with 075-100 adult two spotted spider mites
(Tetranychus urticae). Compounds were dissolved and the leaves
were sprayed to run off and left to dry. Mite counts was made after

24 h.

112

RESULTS
German cockroach males that were injected with fenazaquin showed
an obvious hypoactivity that was followed by prostration at a rate
that was dose-dependant. No evidence for excitation was seen except
for the first few minutes after injection which would be attributed to
the effects of handling and injection. With fenazaquin, mortality was
first seen at 0.5 pg/insect and 1.5 pg/insect caused 50% mortality
within 1 h. With rotenone, mortality was first seen at 0.05 pg/insect
while 0.08 pg/insect caused a 50% mortality within 1 h. When C02
production was monitored, a decrease in output was observed with
both compounds and was proportional to the concentration injected
(Figure 25).. Injecting the solvent or lower fenazaquin concentrations
usually caused an elevation in C02 production compared to untreated
controls, but this elevation changed to inhibition as the concentration
was raised. Fenazaquin was about 10-fold less active than rotenone
in this test: A single high concentration of KCN (20 pg/insect) was
also injected to determine C02 production in the complete absence of
mitochondrial respiration. With KCN, C02 production was reduced by
70% compared to the controls, but not eliminated. Percent inhibition
of respiration was calculated from:
% inhibition = 100- [(A/B)(100)]
A = C02 production for treatment - KCN rate
B = CO2 production for control - KCN rate

IC50 values (concentrations needed to inhibit respiration by 50%)
were calculated graphically from plots of % inhibition versus log

concentration of the inhibitor.

113

30

 

        

Rotenone Fenazaquin

 

(ppm/g/min)

10-

002 Concentration

 

0 KCN

 

 

o I I IIIIII' I I I IIIII" I I I IIIII' I I IIIIII'

.001 . .01 .1 1 10

Concentration
(uglinsect)

Figure 25: The effect of fenazaquin and rotenone on 002

IIIIIII

production by German cockroach males. The effect of

single dose of KCN is also shown.

100

114

At the whole cell level, the addition of CCCP to the respiring Sf-9 cells
clearly increased the oxygen consumption as expected (Figure 26).
The addition of fenazaquin (3 pM) reduced uncoupler-stimulated
respiration almost to zero, while KCN at 100 pM completely abolished
oxygen consumption. The leo for EL-436 was 54 pmoles/mg protein
compared to 21 pmoles/mg protein for rotenone.
The same approach was taken in testing the compound at the
mitochondrial level. Using glutamate plus malate as a substrate,
respiring mitochondria were tested in the presence and absence of
CCCP (Figure 27). The normal response to the addition of a limited
amount of ADP is seen in Figure 27-A with a transition from state 4
(phosphorylation acceptor limited) to state 3 and a return to state 4
as ADP is depleted. The addition of fenazaquin (3 «M) slightly
decreased state 4 respiration and completely eliminated the response
to subsequent additions of ADP. In the presence of 1 pM CCCP, where
mitochondria were completely uncoupled and Oz consumption was
maximized, fenazaquin was also able to inhibit mitochondrial oxygen
consumption although this oxygen consumption is not coupled to ATP
synthesis.
With succinate as a substrate, the response to fenazaquin was quite
different (Figure 28). The addition of fenazaquin to the succinate- '
respiring mitochondria did not affect their ability to utilize oxygen
when stimulated either with ADP (Figure 28-A) or with CCCP (Figure
28-B). However, KCN was still capable of bringing respiration to a
halt (Figure 28-B).

Based on the inhibition of mitochondrial and cellular respiration, a

limited structure- activity study was carried out with fenazaquin

115

Figure 26: Representative polarographic traces showing the
effect of CCCP, KCN and fenazaquin on respiration of
the Sf-9 cell line.

116

CCCP
(Fl) (10 ilM)

 

Solvent

 

(B)

(C)

   

Fenazaquin

(3 llM)

 

 

KCN
(0.1mM)

Oxygen .
(patom)

 

 

 

kl

117

Figure 27: Representative polarographic traces showing the effect
of fenazaquin on rat liver mitochondria in the presence of
glutamate. The effect is shown with and without the
addition of an uncoupler (CCCP).

118

ADP

Glutamate (A)

Fenazaquin
(3 11M)

    
     

Glutamate CCCP
(B)
Fenaz uln
(3 pM

: A l

a E

e :‘e

0

Time

(min)

 

119

Figure 28: Representative polarographic traces showing the effect
of fenazaquin on rat liver mitochondria in the presence of
succinate. The effect is shown with and without the
addition of an uncoupler (CCCP).

 

 

120

         
   
    

Fenazaquin

(3:14) (A)

Succinate

Succinate

Fenazaquin
(3 1M)

1

Oxygen
(patom)

Ime
(min)

 

02 =Zero

121

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analogs differing in the para substituent in the phenyl ring, as shown
in Table 11. Several compounds were more active as inhibitors than
fenazaquin (V). The most active compound was the 4-phenoxy analog
(1) which was 5-10 fold better than fenazaquin and 2-3 fold more
active than rotenone. The unsubstituted analog (IX) was 6-fold less
active than fenazaquin and 35-100 fold less' active than the 4-
phenoxy compound (I). The compounds were also tested for toxicity
against Tetranychus urticae . The most active compounds were

numbers III and V (fenazaquin) with LC50 of about 4 ppm.

DISCUSSION
The injection of fenazaquin into German cockroach caused
hypoactivity and a gradual decrease in mobility without prolonged
excitation which suggested respiration as a possible site of action.
The symptoms were similar to those reported by Harvey and Brown
(1951) for‘rotenone poisoning in Blattidae. If the compound inhibits
mitochondrial respiration it should decrease the rate of Oz
consumption and increase COz production. Monitoring C02 production
following the injection of fenazaquin revealed a strong inhibition of
C02 production. The effect of fenazaquin in this regard was
comparable to that of rotenone although fenazaquin was less potent.
However, the concentrations to inhibit respiration and cause
mortality after injection into cockroaches are well correlated for both
fenazaquin and rotenone. Neither compound was capable of reducing
COz production to the level seen after the injection of a large dose of
KCN indicating that some fenazaquin-insensitive, cyanide-sensitive

pathways of CO2 production are operating. The inhibition of

124

respiration seen in vivo was confirmed by the results with Sf-9 cells
and mitochondria in vitro. Fenazaquin was a strong inhibitor of
oxygen consumption by Sf-9 cells whether the respiration was basal
or stimulated with an uncoupler. Rotenone gave a comparable
response but was 2-3 fold more active than fenazaquin. Similarly
with rat liver mitochondria utilizing glutamate-malate as a substrate,
inhibition of oxygen consumption was rapid and complete. The
potencies of fenazaquin and rotenone were virtually identical on the
insectan Sf-9 cells and on the mammalian mitochondria. The
inhibition of uncoupler-stimulated respiration showed that inhibition
occurs in the respiratory chain and not at the level of the F1Fo-
ATPase as seen with oligomycin or aryltins (Corbett et al 1984).

In order to determine the site of inhibition by fenazaquin in the
respiratory chain, mitochondria respiring with succinate were
studied. Fenazaquin, like rotenone (Ragan, 1987), failed to inhibit this
system. This establishes that fenazaquin inhibits respiration in the
region of complex-I (NADH-coenzyme Q oxidoreductase) and no
effect was observed on the subsequent elements of the respiratory
chain. Further studies using Complex‘l isolated from beef heart
(Ahamadsahib et al, unpublished data) confirms this as a site of
potent inhibition by fenazaquin.

In order to understand some aspects of the structural requirements
for inhibition and toxicity of quinazolinamines, a series of analogs
with differing substitutions in the phenyl group of the side chain was
examined. Considerable variations in inhibitory potencies were
observed. Several compounds, particularly those with bulky,

lipophilic groups in the para-position of the ring were better

 

125

inhibitors than fenazaquin. The most potent analog (with a 4-
phenoxy substituent) was 2-3 times more active than rotenone (as a
respiratory inhibitor with an ICso value less than 10 nM for insect
cells and rat liver mitochondria. In general, the inhibitory potency
within these analogs was similar for the two in vitro systems,
indicating that little selectivity can be expected between insects and
vertebrates at the level of the target site.This potent inhibition can
reasonably explain the poisoning symptoms observed in cockroaches
and vertebrates.

A reasonable agreement exists between inhibitory activity against
rat liver mitochondria and the Sf—9 cells.

In general, the most active inhibitors were also the most active
acaricides. However, within this group (compounds I-V), the
correlation of in vitro activity to mite toxicity was poor. Compounds
with relatively low inhibitory activity (compounds VIII-XII) showed
either low or no toxicity to mites. Poor inhibitors (compounds VIII-
XII) were uniformly if low activity. Differences in acaricidal activity
within this group depend not only on target site activity but also on
rates of uptake, metabolism and non-specific binding which may
tend to confound correlations with in vitro, inhibitory activity.
Consequently, we believe that the sum of the evidence indicates that
the primary toxic'mechanism for these quinazoline ethers is as
inhibitors of respiration at Complex 1. As such they represent a

structurally novel group of inhibitors at this site.

126

References

Corbett J. R., Wright K. and Baillie A. C. (1984) The biochemical mode
of action of pesticides. 2nd ed. Academic Press. London.

Ernster L., Dallner G. and Azzone G. F. (1963) Differential effects of
rotenone and amytal on mitochondrial electron and energy
transfer. J. Biol. Chem 208: 1124-1131

Estabrook R.W. (1967) Mitochondrial respiratory control and the
polarographic measurement of ADP:O ratios. Methods Enzymol
X: 41-47

Harvey GT. and Brown A.W.A. (1951) The effect of insecticides on
the rate of oxygen consumption in Blattidae. Canad. J. 2001.29: 42

Horgan DJ. and Singer (1968) Studies on the respiring chain-linked
reduced nicotinamide adenine dinucleotide dehydrogenase. XIII.
Binding sited of rotenone, piericidin A and Amytal in the
respiratory chain. J. Biol. Chem. 243: 834

Ilivicky J. and Casida J. (1967) Uncoupling action of 2,4-
dinitrophenols and 2-trifluoromethylbenzimidazoles and
certain other pesticide chemicals upon mitochondria from
different sources and its relation to toxicity. Biochem Pharmacol.
18:1389-1401

Jeng M., Hall C., Crane F. L., Takahashi N., Tamura s. and Folkers K.
(1968) Inhibition of mitochondrial electron transport by
piericidin A and related compounds. Biochemistry 7: 1311- 1322

Katyare S. S., Fatterpaker P. and Sreenivasan A. (1971) Effects of
2,4-dinitrophenol (DNP) on oxidative phosphrylation in rat liver
mitochondria. Archs. Biochem. Biophys.144: 209-215

127

Lindahl P. E. and Oberg K. E. (1961) The effect of rotenone on
respiration and its point of attack. Exptl Cell Res. 23: 228-237

Ragan C. I. (9187) Structure of NADH-ubiquinone reductase

(complex-I). Current topics in bioenergetics (ed. C. P. Lee) 15:
l -3 6

Story B. T. (1981) Inhibition of energy-coupling Site-I of the
mitochondrial respiratory chain. "Inhibitors of Mitochondrial
F unctions" (ed M. Erecinska and D. F. Wilson) pp. 101-108
Pergamon Press, Oxford. '

Summers M. D. and Smith G. E. (1987) A manual of methods for
baculovirus vectors and insect cell culture procedures. Texas
Agric. Exper. Station Bulletin No.1555: 1-56.

Chapter 5

The Mitochondrial uncoupling activity of

the new polyfluorocarboxanilide insecticide,
EL-499.

129

INTRODUCTION

The polyfluorocarboxyanilides particularly EL-499 (2'-bromo-4'-
nitro-l,2,2,3,3,4,4,5,5,6,6-undecafluoro-cyclohexane carboxanilide)
are a group of .new compounds being studied by DowElanco Co.
(Indianapolis, IN) as soil or household insecticides. The Structure of
these compounds indicated that they may act as uncouplers of
mitochondrial oxidative phosphorylation because they are highly
lipophilic weak acids containing an aromatic moiety. EL-499 has an
acute oral LD50 of 65.5 mg/kg in rats and 230 mg/kg in mice,
indicating a degree of safety greater than with some other potent
uncouplers.

The purpose of this project was to investigate the uncoupling effect
of EL-499, on mitochondria from different sources. The effects of this
compound on insects in vivo were also tested to explor the
hypothesis that this compound exerts its toxic action primarily

through uncoupling.

Materials and Methods
1. Chemicals
EL-499 (2'-bromo-4'-nitro-l,2,2,3,3,4,4,5,5,6,6-undecafluoro-
cyclohexane carboxanilide) was provided by DowElanco Laboratories
(Greenfield, IN). Four other analogs were also included and they are
analog-I: perfluoro-t-butyl analog of EL-499, analog-II: perfluoro-l-
methylpentyl analog, analog-III: 4'-nitro-1,2,2,3,3,4,4,5,5,6,6-

undecafluorocyclohexane carboxanilide and analog-IV:

130

no2

'1' / (CH2)5-n
CH3-C=C 0(0) . o c\
H (CH2)n

CF3\
/C8 NJ}. N02
CF3 H

N02

Figure 29: The chemical structure of pesticides that showed
uncoupling selectivity. A) karathane (Dinocap, Pesticide
Manual, 1979). B) arylhydrazone (Holan and Smith, 1986).

131

Br F2 F2
0
NO N g F
2 H F 2
F F2
EH22
Br Br
I? 91:3 I? F
N02 N 43-9-05; N02 :11 -C'G-CF2-CF2-CF3
H 01:3 CF3
inning-I . Ariana-11
F2 F2 F2 F2
0 0
II II
N02 N -c F2 N -c F2
H F H F
l:2 F2 l:2 F2
Analog-III Analog-1!

Figure 30: The chemical structure of EL-499‘ with the four analogs
tested in the structureactivity relationship.

132

30). The other reagents were purchased from Sigma Co. (St. Louis,

MO).

2. Mitochondrial Isolation:

Rat liver mitochondria (RLM) were isolated from Sprague
Dawley rats of both sexes weighing 225-300 g. The mitochondria
were isolated by a method based on that of Katyare et al. (1971).
Animals were sacrificed by decapitation and liver was quickly
dissected and put in the isolation medium containing 0.25 M sucrose,
4 mM Tris-HCl (pH 7.4) and 0.5 mM' EGTA. The liver was _
homogenized using a Potter-Elvehjem-type homogenizer fitted with a
Teflon pestle. The homogenate was centrifuged first at 600g for 10
min then the supernatant was centrifuged at 10,400g for 10 min to
sediment the mitochondria. The mitochondrial pellets were
resuspended and recentrifuged as before. The final pellets were
resuspended to a final concentration of 1.0-2.0 mg protein/ml as
determined by the method of Lowry et al. (1951) using crystalline
bovine serum albumin as a standard.

Tobacco hornworm midgut mitochondria (THMM) were
prepared using the method of Mandel et al, 1980. Twenty last-instar
larvae of Manduca sexta ( Carolina Biological Supply Co., Burlington,
NC) were dissected and the midguts were placed on ice . The
Malpighian tubules were removed along with the contents of the
midgut including the peritrophic membrane. All dissected midguts
were washed at least two times in the isolation medium (225 mM
manitol, 75 mM sucrose, 0.2 mM EDTA and 1% BSA at pH 7.4) and

then homogenized in a tapered ground glass homogenizer fitted with

133

glass pestle. The homogenate was centrifuged at 2,000g for 10 min.
The supernatant was then centrifuged at 18,000g for 10 min. To
maximize the yield, the pellets of the .low speed spin were
rehomogenized and the supernatants were combined for a second
spin at 18,000g for 10 min. Pellets were then resuspended in the
same medium to the desired protein concentration.
Etiolated corn shoots mitochondria (CSM): Using the method of
Day and Hanson (1977), corn seedlings that were grown in the dark
were taken before the first leaf opened (approximately 6-7 days
old). The harvested seedlings were left in ice-cold water for one
hour, dried and then weighed. The grinding solution (2 ml/g corn)
was 0.4 M sucrose, 50 mM TES, 5 mM EGTA and 0.1% BSA at pH 7.6.
The shoots were quickly ground in a cold mortar and pestle then
strained through 4 layers of cheesecloth. The homogenate was
centrifuged at 1000 g for 10 min and the supernatant was
recentrifuged at 1000 g for 10 min. This supernatant was then
centrifuged at 12,000 g for 10 min. The mitochondrial pellet was
suspended in the grinding solution and layered on top of 15 ml 0.6 M
sucrose and centrifuged once more at 12000 g for 20 min. The final
pellet was then washed and suspended at a concentration of 10-15
mg protein/ml.-
3. Assay of mitochondrial respiration

Oxygen uptake was measured polarographically using a Clark
oxygen electrode (Model 5300, Yellow Springs Instruments, Yellow
Springs, Ohio). The respiration medium for rat liver mitochondria
contained 0.1 M KCl, 5 mM K2HPO4, 1 mM EDTA, 20 mM T ris-HCl, 5

mM malic acid and 5 mM glutamic acid at pH 7.4. The respiration

134

medium for the tobacco hornworm midgut mitochondria contained
225 mM manitol, 75 mM sucrose, 0.2 mM EDTA, 11 mM Tris-HCl, 11
mM KCl, 22 mM Na-phosphate, 5 mM glutamate and 5 mM malate at
pH 7.4. For plant mitochondria, the respiration medium contained
250 mM sucrose, 10 mM TES, 5 mM KH2PO4, 5 mM MgC12, 1 mM
EGTA, 5 mM glutamate, 5 mM malate and 1% BSA at pH 7.2. In each
case the mitochondrial suspension (0.1 ml) was added to the reaction
mixture to give a final volume of 3 ml. State 3 respiration was
initiated by the addition of 5 ml of ADP (0.4 mmoles) using a 50 ml
Hamilton syringe. The ADP:0 ratio, state 4 respiration and RCR were
calculated according to Estabrook (1967). Test compounds were
added in 5 ml ethanol.

4. ATPase activity

Rat liver mitochondria were prepared as described earlier but in a
slightly different homogenizarion buffer that contained 275 mM
sucrose, 4 mM HEPES and 0.5 mM EGTA.

The reaction was carried out in 16x100 mm glass test tubes by
adding 1 ml of a reaction mixture containing 222 mM KCl, 100 mM
sucrose, 150 mM HEPES and 1.5 mM EGTA at pH 7.4, 1 m1 substrate
containing 120 mM MgSO4 and 18 mM ATP, 10 ml of test compounds
in ethanol and 200 ml mitochondrial suspension. The volume was
brought to 3 ml with water. Tubes were incubated at 37°C for 15 min
and the reaction was terminated by the addition of 7 ml of chilled
70% perchloric acid. Protein was pelleted at 3000 g for 10 min and
0.1 ml aliquots of the supernatant were taken to assay for the
liberated Pi using the colorimetric method described by Myers and

Slater (1957).

135

5. Cockroache respiration in vivo:

Male cockroaches of a susceptible strain (S. C. Johnson Co.,
Racine, WS) were sedated by cooling for 3-5 min in a freezer before
injection. Subcuticular injections were made between the first and
second abdominal segments using a syringe tipped with a finely
drawn glass needle. The injection volume was 1 ml delivered slowly
to prevent excessive bleeding. EL-499 was dissolved in DMSO (200
ml) and added to water (800 ml) containing Tween-20 (1%) as an
emulsifier just before injection.

Insects were tested in groups of four confined in a small piece of
plastic tubing with both ends sealed with muslin. Insects were then
monitored in a 1 liter chamber of a Li-6200 C02 analyzer (LiCor Inc.,
Lincoln, NE). The instrument was calibrated daily using a known
mixture of C02 in air (507 ppm). Injected insects were left for 10 min
to recover before beginning respiration monitoring. Carbon dioxide

levels in the chamber were then measured every 5 min for 50 min.

Results and Discussion

This research was started as a part of a projected study of the
selectivity of uncouplers in vitro. Uncouplers sthat are more potent
on insects rather than on mammals or plants would be valuable . One
of the compounds that was reported to have this uncoupling
selectivity is karathane (Dinocap, Figure 29-A) (Pesticide Manual,
1979) another group is the arylhydrazones (Figure 29-B) (Holan and
Smith, 1986). Previous work by Iliviky and Casida (1969) has also
suggested that uncoupling may vary in potency between

mitochondria from different sources by a factor of lO-fold or more.

136

 

 

 

6001 _
—o— RLM T
‘ —-— Tl-lMM
500.. —I— CSM H
= u
.2 400
3 .
h
*0—
O a.
It
3 g 300-
a
2
0" ‘
5
0
-- 200-
*2
a:
100-
. ___'____.4.
o __.__J
10'2 10'1 100 101 102 103 104 105

Concentration (nM)

Figure 31: The uncoupling activity of EL-499 on Manduca sexta
midgut mitochondria (THMM), rat liver mitochondria
(RLM) and etiolated corn shoot mitochondria (CSM).

[Pijliberated
(pmolelis minlmg protein)

137

  
  
  
 

20-
—-— EL-499
—fi— Analog-I
. —O— Analog-ll
—o— Analog-Ill “
10 " ‘r _

 
 

 

o .4 III... /‘

r .
I .0 I .0. O

10'2 10'1 100 1o1 102 103 104 10s 106

Concentration (nM)

Figure 32: The The effect EL-499 and three of its analogs on the

ATPase activity of rat liver mitochondria .

138

' —O— 0.03 ug/msect
—t— 0.15 ug/msect
—o—- 0.5 ug/msect

a

:5 --—- Control (solvent)
0

U)

.E

E

E 2

E

Q

8

N

O

o 1

 

Time (h)

Figure 33: The in vivo effect of injected EL-499 on respiration in
German cockroach males.

139

EL-499 was tested on mitochondria from rat liver. Manduca sexta
midgut and etiolated corn shoots. Results observed were typical of
protonophoric uncouplers (Figure 31). State 4 respiration was
strongly stimulated and the ADP:O ratio fell to zero. Respiratory
stimulation was not affected by oligomycin. The uncoupling potency
of this compound was extremely high and did not differ significantly
between RLM and THMM. CSM were about a 50-fold less sensitive.
The estimated UCso's (uncoupling concentration that causes 50%
activity) supported this conclusion also. The values for THMM. RLM
and CSM were-5.0 pmoles/mg protein, 10.0 pmoles/mg protein and
250 pmoles/mg protein, respectively. This places EL-499 among the
most active uncouplers yet described.

Uncouplers are also known to cause ATP depletion by reversing the
HT-translocating ATP synthase to hydrolyze ATP. The ability of EL-
499 to stimulate ATPase activity at low concentration is clearly
shown in Figure 32. Using the same approach, the structure-activity
relationships were breifly studied with four further‘analogs of BL-
499. The parent compound (EL-499) was the most active followed by
analog-II which has a five-carbon perfluorinated chain, then analog-
1 which has only a four-carbon chain. EL-499 was much more active
than analog-III that does not have the bromo substituent in the
phenyl group and analog-IV, lacking phenyl ring substituents, was
inactive at 0.1 mM. Uncoupling activity increases with carbon chain
length in the perfluorinated moiety. At the same time, both the
bromo and the nitro groups are significantly essential for high

activity. This probably relates primarily to the need to maintain the

140

pKa within a optimum range e.g. for poysubstituted diphenylamines
this pKopt is approximately 7.5 (Nizamani, 1983).

The monitoring of carbon dioxide production in male cockroaches
injected with EL-499 also showed the potency of this uncoupler in
vivo (Figure 33). At 0.5 and 0.15 mg/insect, EL-499 was able to kill
the insects after about two hours preceded by a stimulation in C02
production to maximum that was about 5-fold higher than control
rates. At 0.03 mg/insect (about 0.6 mg/g), the C02 respiration was
approximately doubled compared to controls. Though it caused a
more modest degree of stimulation, this dose was still lethal after
about 8 h. I

In conclusion, EL-499 is a new very potent uncoupler (more than
1000-fold more potent than NDES, Chapter 3). The presence of both
the bromo and the nitro groups is essential for the potency of BL-
499. EL-499 also maximized the production of C02 of the German
cockroach males and was highly toxic to them. The idea of having
different degrees of potency on mitochondria from different sources
(selectivity) is possible since a clear difference was observed
between animal and plant mitochondria in sensitivity to EL-499. This
could not be attributed to the presence of BSA, a known binder of
uncouplers (Weinback and Garbus 1969) in the plant mitochondria

since BSA was also present in the insect preparation.

141

References

Day, D. A. and Hanson, J. B. (1977) On methods for the isolation of
mitochondria from etiolated corn shoots. Plant Sci. Lett 11: 99-
104.

Holan, G. and Smith, D. R. J. (1986) A new selective insecticidal
uncoupler of oxidative phosphorylation. Experientia 42: 558-560.

Ilivicky J. and Casida J. (1967) Uncoupling action of 2,4-
dinitrOphenols and 2-trifluoromethylbenzimidazoles and
certain other pesticide chemicals upon mitochondria from
different sources and its relation to toxicity. Biochem. Pharmacol.

18: 1389-1401.

Katyare S. S., Fatterpaker P. and Sreenivasan A. (1971) Effects of
2,4-dinitrophenol (DNP) on oxidative phosphorylation in rat liver
mitochondria. Arch. Biochem. Biophys. 144: 209-215

Mandel, L. J.; Moffett, o. F.; Riddle, r. G. and Grafton, M. M. (1980)
Coupling between oxidative phosphorylation and active transport
in the midgut of tobacco hornworm. Amer. J. Physiol.: Cell Physiol.
7: C1-C9.

Myers, D. K. and Slater, E. C. (1957) The enzymatic hydrolysis of
adenosine triphosphate by liver mitochondria. Biochem. J. 67 :
558- 572.

Nizamani, S. M. (1983) The toxicity and mode of action of
diphenylamine pesticides. A thesis, Purdue University.

Weinback, E. C. and Garbus, J. (1969) mechanism of action of
reagents that uncouple oxidative phosphorylation. Nature 221:
1016-1018. '

Summary and Conclusions

143

Summary and Conclusions

several new pesticides with novel structures and unknown modes
of action have been studied. The first one is an insecticide named
sulfluramid (N-ethyl perfluorooctane sulfonamide; Finitronm) that is
marketed for use against ants and household insects especially
cockroaches. In this study, different approaches have been taken in
studying the effects of this new compound on the German cockroach,
Blattella germanica (L.). Sulfluramid (SULF), N-desethyl' sulfluramid
(NDES) and perfluorooctanesulfonic acid (PFOSA) have been fed to

m~mm

adult cockroaches (susceptible and field strains) in diet pellets.
Mortality was delayed, even at high dosesWith PFOSA acting most
rapidly. The field strain showed lower sensitivity. The effect of
piperonyl butoxide (PBO) as an MFO inhibitor was also studied by
incorporating it at a single concentration in pellets with the three
compounds. Piperonyl butoxide antagonized the toxicity of
sulfluramid. The same case was also noticed for the susceptible strain
with NDES. No effect of PBO on PFOSA toxicity was observed.
Sulfluramid metabolism was studied in vivo, and the susceptible
strain was found to eliminate SULF faster than the field one.
Monitoring carbon dioxide (C02) for cockroaches that were injected
with SULF, NDES and PFOSA revealed that both NDES and PFOSA were
able to immediately increase the insects C02 production. SULF did
not show this activity initially with doses up to 10 pg/insect but ,

considerable respiratory stimulation occured after several hours.

144

This results suggests that SULF acts indirectly, being metabolically
converted to NDES and/or PFOSA which stimulate respiration.
Another approach was to study the mode of action of sulfluramid
and related metabolites in vitro. Sulfluramid acted as a very poor
uncoupler against mitochondria from rat liver, German cockroach
flight muscles, Manduca sexta midgut and cytochrome c oxidase-
containing reconstituted membrane vesicles. The uncoupling activity
was 7-10 times higher when NDES was used. Despite its ability to
stimulate respiration in vivo, PFOSA did not have any direct
uncoupling activity but when mixed with some alkylamines, e.g.
tributylamine, it was able to exert a strong uncoupling effect. This
phenomenon was further studied using different alkylamines,
poylamines and phospholipids. Only tributylamine and dihexylamine
were active. Typical of uncouplers, NDES caused mitochondria to
shrink in the presence of K-acetate but PFOSA alone resembled
valinomycin (causing mitochondrial swelling). When tributylamine
was added to PFOSA to initiate uncoupling, mitochondria shrunk. A
possible explanation for these results is that PFOSA alone acts as an
ionophore for K-t- and in combination with a lipophilic amine enhances
protonophoric activity through ion-pair formation.

A second compound studied is the novel acaricide (DowElanco named
EL-436) fenazaquin, (4-[(4-(l,l-dimethylethyl)phenyl)ethoxy]
quinazoline). The mechanism of action of this compound was studied
at the the mitochondrial (rat liver), cellular (Sf-9 cell line) and whole
organism (German cockroach) levels. Both mitochondrial and cellular
studies proved that the compound acts as a powerful inhibitor of the

mitochondrial electron transport process at complex I of the

145

respiratory chain. In this respect, its effects are similar to those of
rotenone. The inhibitory activities of fenazaquin and its more potent
analogs were comparable to that of rotenone with 150 values in the
10-50 pM range. This action was confirmed in vivo by studying
respiratory inhibition caused by fenazaquin in cockroaches.

The third compound, EL-499 (2-bromo-4-nitro-
perfluorocarboxanilide) is an experimental insecticide. This
compound acts as an extremely powerful uncoupler of oxidative
phosphorylation with activity on insect and vertebrate mitochondria
I at concentrations below 10 nM. Plant mitochondria are about 50-fold
less sensitive. Respiratory stimulation and mortality in cockroaches

Occur at doses below 1 pg/g.

S 'E' l'

1- Sulfluramid and NDES act as uncouplers of mitochondrial
respiration with NDES more active. They increase in vivo respiration
in cockroaches. Sulfluramid does so after a delay. PFOSA also
increases in vivo respiration but does not uncouple mitochondrial
respiration. The mechanism of PFOSA stimulation of respiration
remains to be established.

2- The above evidence and studies with PBO indicate that
sulfluramid is metabolically activated in cockroaches to NDES and/or
PFOSA.

3- Tolerance to sulfluramid occurs in field strain. This may be
explained by a lower rate of activation but other differences also
contribute to tolerance which is also observed with possible active

metabolites, NDES and PFOSA. ‘

146

4- The acaricide/insecticide fenazaquin (EL-436) inhibits insect
respiration in vivo and act as a potent inhibitor of the mitochondrial
electron transport system at complex I. This is a reasonable
explanation of its pesticidal properties.

5- The insecticide EL-499 is an extremely strong uncoupler of

oxidative phosphorylation.

Appendices

Appendix 1

148

APPENDIX 1

Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in
the named museum(s) as samples of those species or other taxa which were
used in this research. Voucher recognition labels bearing the Voucher
No. have been attached or included in fluid-preserved specimens.

Voucher No.: 1992-01

 

Title of thesis or dissertation (or other research projects):

Novel Pesticides Affecting Mitochondrial Functions

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums: None

Investigator's Name (3) (typed)

 

Gadelhak Gabg; Gadelhak

 

 

Date 7/28/1992

 

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in
Nbrth America. Bull. Entomol. Soc. Amer. 24:141-42.

Deposit as follows:

Original: Include as Appendix 1 in ribbon copy of thesis or
dissertation.

Copies: Included as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator,
Michigan State University Entomology mseum.

149

APPENDIX 1.1

Voucher Specimen Data

Pages

1 of 1

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Appendix 2

The role of prostaglandins in insect reproduction
and the actions of formamidines as reproductive
toxicants.

151

Abstract

The role of prostaglandins in insect reproduction and the
actions of formamidines as reproductive toxicants.

The effects of formamidineinsecticides on insect reproduction
were studied. Chlordimeform (CDM) and demethylchlordimeform
(DCDM) were given to adult tobacco budworm Heliothis virescens in
the food source. Egg production was monitored and a reduction in the
number of eggs laid per female was observed. The same approach
was taken to study the effect of both CDM and DCDM on the
reproduction of the onion fly Delia Antiqua. As the dose of both
compounds increased a reduction in the total eggs/female was also
seen. Delia was considerably less sensitive than Heliothis in this
response. Both compounds were studied in vitro as inhibitors of
prostaglandin synthetase. A complete inhibition of the enzyme
System was reached at 1 mM of both CDM and DCDM. However, levels
of prostaglandin synthetase were very low in the insects observed
and inhibition of the insect enzyme was not studied. In addition to
inhibition of prostaglandin synthetase, the formamidines also mimic
the effects of the insect neurotransmitter, octopamine. The effect of
octopamine and a prostaglandin (PGan) on the contractility of the
onion fly oviduct was investigated in an attempt to find a
relationship between octopamine, octopamine agonists (CDM & DCDM)

and prostaglandins in their effect on the egg laying process.

152

INTRODUCTION

Reproduction is of fundamental importance for all organisms.
In insects, reproduction is a complex process that involves multiple
behavioral and physiological mechanisms.

There are many possible means of insect control through which an
interference with these mechanisms could lead to unsuccessful
reproduction. Reproductive impairment can arise through the use of
natural products (Kubo. 1987 and Grazzini, 1991) or the use of
synthetic pesticides (Hollingworth and Lund, 1982; Uchida et a1
1986).

One of the biochemical aspects of reproduction in both
mammals and insects is the involvement of prostaglandins (PG’s).
The prostaglandins' best known function in insects is the release of
egg-laying behavior as observed in crickets by Brady (1982). Many
compounds were found to inhibit the enzyme, prostaglandin
synthetase such as -the antiinflamatory agents aspirin and
indomethacin. One group of compounds that was found to inhibit this
enzyme in mammals are formamidine insecticides (Yim et al, 1978).
In addition, formamidines were proved to mimic the action of
octopamine (Hollingworth and Murdock, 1980). Octopamine acts at
the neuromuscular junction in locusts controlling the contraction of
oviducts (Orchard and Lange, 1984). Both of these actions could lead
to decreased reproduction in insects. Since the effect of formamidines
as reproductive toxicants has not been well studied in insects, one of
the main goals of this study is to shed some light on their potency

and mechanism as reproductive toxicants in insects.

153

Short Literature Review

Following Bergstrom's isolation of prostaglandins from mammalian
seminal vesicles (1962), a widespread interest arose in PG's and
related compounds of arachidonic acid metabolism in mammals. This
interest intensified when it was discovered that aspirin and other
non-steroidal antiinflamatory agents act by inhibiting PG synthesis.
Prostaglandins are biologically active metabolites of arachidonic acid,
a 20-carbon polyunsaturated fatty acid (PUFA). Related PUFA
metabolites include prostacyclins, thromboxanes and leukotrienes,

which together with PG's are called eicosanoids (Corey et a1 1980).

II E [25' . . | l °|es

The presence of PG's in insects was first reported by Destephano and
Brady (1974) in the house cricket Acheta domestica. The presence of
prostaglandins in the reproductive tract and their role in oviposition
were described in this and subsequent work (Brady and

Destephano 1977; Yamaja and Ramaiah 1980; Hagan and Brady
1982). PG's studies in insects have been reviewed in detail by Brady
(1983). Only a few types of PG's have been reported in insects i.e.
PGE (E1 & 152) and PGF (F1, F2, F211). The amounts of PG's present in
insects tissues differ between authors. Murtaugh and Denlinger
(1982) using an RIA method analyzed PG's in the head and thorax of
seven insect species. Galleria mellonella had the lowest level (1
pg/insect) and Periplaneta americana had the highest level (547

pg/insect). Comparing the levels in mated and non-mated females,

154

Destephano and Brady (1977) reported 589 pg/mated insect and 5
pg/virgin insect in the house cricket, while Hagan and Brady (1982)
reported 71 pg/ mated insect compared to 24 pg/virgin insect in
Tricoplusia ni. Other eicosanoids have not been studied in insects.
Since PG's behave as tissue or local hormones, they are not stored,
but are generated when a stimulus activates phospholipase A2 to
free arachidonic acid from esterified phospholipids. The enzyme fatty
acid cyclooxygenase initiates the reaction on arachidonic acid
followed by the other enzymes of PG biosynthesis (Figure 34). After
release, PG's, like many other local hormones are rapidly removed by
metabolism or diffusion (McGiff, 1981). Other authors have reported
the presence of PG's in many other insects belonging to different

orders. Table 12 summarizes some of their work.

155

Table 12: Prostaglandins in insects and their effect in stimulating

 

 

 

 

oviposition.
Order Insect FTC: presence Effect on
oviposition
Orthoptera Locusta migratoria +1- -
Telogryllus commodus ++ ++
Acheta domestica ++ 1+
Periplaneta americana 1+ ND
“91501319” Bombyx mori ++ ++
Trichoplusia ni ++~ -
Galleria mellonella ++ ND
Heliothis virescens +1- ND
Manduca sexta 1» ND
Saturniid moths ND ND
Dlptera Musca domestica + ND
Drosphila sp. ND ND
Sarcophaga crassipalpis +1- NI)
Culex pipiens + ND
Hemlptera Oncopeltus fasciatus + ND
9019091978 Tenebrio molitor + ND
Tribolium castaneum + ND
Hymenoptera Apis mellifera + ND
Thysanura Thermobia domestica + ND!
Acarlna Tetranychus sp. ND ND
:Absent

+ :Low level
+1- :High level
ND :not determined

Sources: Brady (1983), Murtaugh and Denlinger (1982), Yamaja and
Ramaiah (1980), Howard et a1 (1986), Sasaki et al (1984), Wakayama
et a1 (1986), Ragab et al (1987) and Lange (1984).

156

II E 1' [EG' . . ts

In mammals, PG's are ubiquitous, (Gilman et a1 1985) with
regulatory functions in many biological processes and tissues . In
particular. they play several important roles in reproduction, e.g. in
steroidogenesis, ovulation, luteolysis and other reproductive aspects
in females. In males they are involved in spermatogenesis and sperm
maturation. The metabolites of arachidonic acid appear to exert their
actions by a variety of receptor-mediated mechanisms. For example,
PGE2 stimulates formation. of steroid in the adrenal cortex by
activating adenylate cyclase, suppresses epinephrine-induced
lipolysis by inhibiting adenylate cyclase, and stimulates uterine
contraction by increasing the intracellular concentration of free
calcium.

The physiological function of PG's in insects have not been studied
except for their effect on reproduction. In some insects they are
potent in stimulating oviposition. This action has been demonstrated
in the house cricket (A. domestica), the Australian field cricket
(Telogryllus commodus) and the silk moth (Bombyx mori). There is a
contrast in the site of synthesis of PG's between crickets and silk
moths. While the male cricket transports only the enzyme PG
synthetase to the female which provides the arachidonic acid
precursors, the males of the silk moths synthesize PG's themselves
and deliver them within the spermatophores to the females. The
physiological mechanism by which PG's stimulate oviposition is not
know. In addition to the above species, Uchida et al (1986) showed

that injection of PG's‘into females of the Brown planthopper,

157 '

Nilaparvata lugens, increased oviposition. On the other hand,
oviposition in some other insects showed no response to PG's i.e.
Manduca sexta (Sasaki et a1 1984), Trichoplusia ni (Hagen and Brady
1982) and Leptinotarsa decemliniata (Pferoen et al 1981).

Given the widespread presence of PG's in insects and their multiple
functions in other organisms, it could be expected that there are
other functions for them in insects that have not yet been revealed.-
Some of these possible functions have been superficially studied. The
effect of PG's on hatching and fertility of insect eggs was studied by
Datta and Banerjee (1978) who found that topical-

treatment of the eggs of the red cotton bug (Dysdercus sp.) with
PGA1 and A2 reduced hatching. Another aspect relating to
development and growth was studied by Singh and Datta (1980)
where exogenous PGE1 given to B. mori larvae was found to increase
cocoon and pupal weights. Murtaugh and Denlinger (1982) and
Stanley-Samuelson (1980) reported that PG's have been identified in
nervous and muscle tissues in many insects belonging to different
orders. Nanda and Ghosal (1978) reported that PGEza drastically
depletes neurosecretory cells of the pars intercerrbralis of
Periplaneta americana but information on any function of PG's in the
insect nervous system appears to be lacking.

The well-established role of PG's in thermoregulation and fever in
vertebrates has led a few investigators to study their effects on
thermoregulation in invertebrates (Brady, 1983). PG's caused a
number of arthropods ( scorpions and lobsters) to seek conditions
that elevated their body temperature ("behavioral fever"). This effect

was not studied in insects.

158

Eff | [25' ll . .1.” . . ts

Most inhibitors of PG biosynthesis work on the fatty acid cyclo-
oxgenase step in arachidonic acid metabolism (Figure 1). Examples of
these inhibitors are the non-steroidal antiinflammatory agents like
aspirin and indomethacin. Both these inhibitors wereactive in
decreasing oviposition in B. mori (Yamaja and Ramaiah, 1980), and
the related compound N-acetyl-p-aminophenol lowered the egg
counts in A. domestica (Destephano and Brady, 1977). However,
indomethacin had no inhibitory effect on cricket PG synthetase
(Destephano and Brady, 1976). Recently, Tribolium castanium , the
red flour beetle, has been shown to secrete phenylketones into the
surrounding flour. Phenylketones inhibit the conversion of
arachidonic acid into PGE2 in both mammals and insects (Howard et
al 1986). The production of PG synthesis inhibitors (salicylates,
phenylketones) by plants and other insects suggests that they may
be useful as sterilants for pests or competitors. This idea is
strengthened by the observation of Kubo (1987) that several natural
plant products, particularly anacardic acid, act as insect sterilants
and are potent inhibitors of PG biosynthesis (Grazzini et al 1991).
The effects of pesticides on PG biosynthesis are not well studied
either in mammals or in insects. The only report in which PG's are
involved in the physiological effect of a pesticide was by Yim et a1
(1978) who observed that two formamidines (chlordimeform and
amitraz) inhibited PG biosynthesis and had both antipyretic and anti-
inflammatory activity in rats. Chlordimeform was also found to
decrease PGE2 and PGF2 levels in brain tissue of treated mice (Brady

et al, unpublished data). Formamidines have been shown to decrease ‘

159

reproduction and to inhibit oviposition in several species of insects
and ticks (Hollingworth and Lund, 1982). More recently, the new
insecticide, buprofezin (2-t-butylimino-3-isopropyl-5-phenyl
perhydro-l,3,5-thiadiazin-4-one) which works as an insect growth
regulator, and aspirin were shown to block PG synthesis, inhibit
oviposition and decrease population levels in the plant hopper
Nilaparavata lugens and the lady beetle Henosepilachna
vigintiotopunvtata (Uchida et al 1986). Since this effect was reversed
by B-ecdysone, Uchida et 01 suggested that buprofezin acts
indirectly, inhibiting PG biosynthesis through a decrease in ecdysone
levels. However, the link between ecdysone and PG biosynthesis in

insects remains to be established.

Materials and Methods
l-lobamhudmmfldifis

Rearing; Tobacco budworm (Heliothis virescens) pupae were obtained
from the USDA laboratories in Stoneville, MS. Pupae were allowed to
hatch and adults were fed a 10% honey solution.

Larvae were reared on a semi-artificial diet composed of:

22.5 g agar

300 g . ground pinto beans

75 g brewers yeast

20 g alphacel

9 g ascorbic acid

6 g methyl paraben

3 . g sorbic acid

26 g Vanderzant vitamin supplement

160

250 mg tetracycline
5 ml formaldehyde (37%)

Larvae were reared individually in 2 02 cups with enough diet to
carry larva through pupation. Pupae were collected from cups and
differentiated by sex. They were used for bioassays or to maintain
the colony.

Bioassays: To assess the reproductive toxicity of both formamidines
and nonsteroidal antiinflamatory agents on tobacco budworm, a
simple assay was developed. A cage was made from two 200 ml cups
joined together at their openings with adhesive tape. On the inside,
two strips of cheese cloth were taped to the top one container and
allowed to hang for egg deposition. A virgin male and a virgin female
were placed in the cage with the treatment applied in the food
source (10% honey solution). Eggs were collected and counted every
day. The food source was changed every other day and if necessary,

the whole cage was replaced.

2- QILiQn_f_ly_su1di_es

Mm Onion flies were collected from Grant, Michigan, reared
and maintained by the Insect Behavior Laboratory at Michigan State
University. The bioassay used the method described by Havokkala
and Miller (1987) where a green surrogate onion stem was used to
induce females to lay eggs. Refrigerated pupae of the onion fly Delia
antiqua were placed in a hatching cage to ensure using flies of the
same age. After emergence, flies (males and females) were left
together in these cages for 7 days to ensure mating. Special cages

with food (powdered milk, powdered sugar, brewers yeast, soy

161

flakes and yeast hydrolysate at 10:10:1:1:20, respectively), water
and artificial oviposition sites were prepared for the bioassay. Ten
pairs of insects were placed in each cage. On the third day of
oviposition (tenth day postemergence), test compounds (5, 50 and
500 ppm of both CDM and DCDM) were introduced in the water
source. Eggs were collected and counted every day using the
floatation method.

Oviduct contractility: Oviducts of active onion fly females were
surgically removed and mounted in a contractility measuring
apparatus. The method and a detailed description of that apparatus

were reported by Mowry et al (1987).

3_ I v' ' E5 1 . 1
Bovine seminal vesicles (BSV) were used as the source for PG
synthetase. Microsomes from BSV were prepared and lyophlized. PG
synthetase activity was determined following the method described
by Flowers et al (1973) and Yim et al (1977). Microsomes were used
to start the reaction. The assay buffer (100 mM Tris-HCl, 5mM
epinephrine, 5 mM glutathione, luM 3H-arachidonic acid) was at pH
8.2. The different PG's generated were separated by TLC using Iodine
to visualize them. They were then scraped and counted. Unlabeled PG
standards were used to locate zones of interest.

Limited studies on insect PG synthetase were conducted on several
insect tissues. Reproductive tracts of both male and female A.
domestica, and nerve cord and muscle fibers of P. americana were
tested for PG synthetase activity using the procedure of Destephano

and Brady (1974). A single concentration (0.1 mM) of both CDM and

162

 

H \\\H R’ Arachidonic acid

 

 

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__ R'
l°~-‘
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Cycle-oxygenase
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. I (1);: PGGsiendoperoxide)
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Figure 34: Mechanism of prostaglandin biosynthesis ( after

Samuelsson, 1987)

163

DCDM was also tested in inhibition studies on the male reproductive

system of A. domestica.

Results
The feeding of an antiinflamatory drugs like aspirin clearly affected
the number of egg laid by females of tobacco budworm causing a
60% reduction at 1000 ppm in the adult diet (Figure 35). When the
same approach was taken to test two formamidines (CDM and DCDM),
the same effect was noticed with the formamidine being about a
100-fold more potent than aspirin.
Using the same idea, both CDM and DCDM were introduced through
the water source in three different concentrations to onion fly
females. A dose related effect on the number of eggs laid by females
was also apparent as shown in Figure 36. However, this insect
seemed to be much less sensitive to the effect of the formamidines
than H. virescens.
The effects of formamidines on the prostaglandin synthetase were
studied on bovine seminal vesicles. The inhibition of this system
was also related to the dose applied (Figure 37) with DCDM (approx.
ICso = 3 pM) being more potent than CDM (approx. ICso = 30 pM). In
studying PG synthetase activity in insects, male reproductive tracts
of A. domestica were found to be the most active source (E2 and F22
were 2-3 fold over the background) when compared to female
reproductive tract of the same insect, central nerve cord of P.
americana and muscle fibers of P. americana. DCDM was also more

potent than CDM (60% inhibition for CDM compared to 100% for

164

 
  
   
  

800 - CONTROL (10% honey)
—0— COM (10 ppm)
600 . —I— Asprin (1000 ppm)

DCDM (10 ppm)

Comuiailve number of
eggs/femalelday

 

 

0 2 4 6 a 10 12
Days after hatching

Figure 35 : The effect of aspirin , chlordimeform (CDM) and N-
demethylchlordimeform (DCDM) on the number of eggs
laid by females of the tobacco budworm Heliothis
virescens. Numbers are means of n=8.

165

eggs/female/day

 

 

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3-1
C
:5 -——I-—' COM
2 2‘
. "-'-' DCDM
1-—I-—-I-I-H-I-nf—F-I-I-rrvn1 sssnwlkswrrvd‘
.1 1 10 100 1000

Concentration (ppm)

Figure 36 : The effect of (CDM)and (DCDM) on the number of eggs
laid by females of the onion fly Delia antiqua. Numbers
are means :t SE of n=20

166

    

 

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.\ .0.
q
--fiI-- (film
20'
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.0001. .001 .01 .1 1 10 100 1000

Concentration (uM)

Figure 37 : The inhibition of prostaglandin synthetase (PG
synthetase) by formamidines (CDM & DCDM). Numbers

are means i SE, n=4.

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171

DCDM at 0.1 mM) in inhibiting the PG synthetase activity (PGE2) of
the male reproductive tract of A. domestica.

These observations led us to further investigate the effect of
prostaglandins on the egg laying process. The onion fly oviduct was a
good model since the methodology was carefully studied by Mowry
et al, 1987 at the Pesticide Research Center of Michigan State
University. The oviduct contractility seemed to differ greatly
between preparations as shown in the controls of figures 38-A and
39-A. The addition of octopamine typically initiated rhythmic
contractions (Figures 38-B and 339-3). Following 0A treatment,
contractions lasted longer than the spontaneous contractions in
controls (Figure 38-B). The subsequent addition of a prostaglandin
(PGan ) attenuated the OA-induced contractions (Figure 39-C). To
confirm this result another addition of octopamine followed by an
addition of PGan (Figures 39-D & 39-E) were administered on the
same preparation. The second addition of PGF2a decreased
considerably but did not completely abolish the octopaminergic

contractions of the oviduct.

Discussion
The presence of prostaglandins (PGE2 &PGF2a) in insects was studied
on the reproductive systems of the house cricket males and females
in addition to the ventral nerve cord and the thoracic muscles of the
American cockroaches. The only tissue that showed any
prostaglandin synthesis activity was the the reproductive tract of the

male of the house crickets. No activity was detected in females

.- hl‘hAAHKCH‘A-L— _

172

of the same insects or both tissues of the American cockroach, and
this agrees with the findings of Murtaugh and Denlinger, 1982 where
they found up to 39 pg/mg protein of PGE2 and 9.7 pg/mg protein of
PGF2a in the males of the cricket and no prostaglandins were
detected in American cockroaches, Milkweed bug, Mealworms and
honey bees.

The effect of prostaglandin synthesis inhibitors on reproduction was
studied on the tobacco budworm. A significant decrease in the
number of eggs/female was caused by the presence of aspirin in the
food source. The same results were reported by Yamaja Setya and
Ramaiah (1980) on their studies on the silkmoth B. mori. Both
aspirin- and indomethacin-injected males produced a significantly
lower number of eggs when mated with untreated females. Treating
females with prostaglandin reverses this effect.

The effects of pesticides (as prostaglandin synthesis inhibitors) on
egg production was studied using formamidines on both H. virescens
and D. antiqua. Both CDM and DCDM were found to reduce the
number of egg/female in tobacco budworm moths and the onion
flies. The only insecticide that was studied previously as an inhibitor
of the prostaglandin system was buprofezin by Uchida et al (1986)
and Izawa et a1 (1986). In both cases, buprofezin was found to
reduce the number of eggs laid by the planthopper Nilaparvata
lugens and the ladybird Henosiplachna vigintioctpunctata.
Formamidines both inhibit PG biosynthesis and mimic octopamine,
both of which actions could block oviposition. Thus, a limited study
on the effect of prostaglandin and octOpamine on the contractility of

the onion fly oviduct was initiated to test the effect of octopamine

173

agonists (formamidines) and prostaglandins on this oviduct
contractility. The observations of these studies indicated that
octopamine triggered oviduct contractions. The addition of PGE2
antagonized this elevated rate of contraction .

In conclusion, prostaglandin synthetase from both mammalian and
insect sources was inhibited by both CDM and DCDM with reasonable
potency. Both formamidines reduced the number of eggs/females
when applied on two insects from two different orders (Lepidoptera
and Diptera). The relationship between the actions of both
octopamine and its agonists (formamidines) in decreasing the
number of eggs produced, increasing the frequency of oviduct
contractions and inhibiting PG synthetase activity is yet to be
explored. More studies will also be needed to confirm the in vitro
effects offormamidines on oviduct contractility and to crystalize
the relationship between prostaglandins and octopamine in
reproduction. The in vivo effects of formamidines on insect

reproduction also needs to be investigated in more details.

_‘ ‘ 7": 12mm

174

References

Bergstorm, S.; R. Ryhage; B. Samuelsson and J. Sjovall 1962b. The
structure of prostaglandin E, F1 and F2. Acta Chem. Scan. 16: 501-
502.

Bergstrom, S.; F. Dressler, C. Karbisch, R. Ryhage and J. Sjovall
1962a. The isolation and structure of a smooth muscle
stimulating factor in normal sheep and pig lungs. Ark. Kern. 20:
63-66.

Brady, U. E. 1983. Prostaglandins in insects. Insect Biochem. 13:
443-451.

Collins, E. and T. Miller 1977. Studies on the actions of biogenic
amines on the cockroach heart, J. Exp. Biol. 67 : 1-15

Corey, E. J.; H. Niwa; J. R. Falck C. Mioskowski; Y. Arai and A. Marfat
1980. Recent studies on the chemical synthesis of eicosanoids.
Adv. Prostaglandin Thromboxane Research. 6: 19-25.

Datta, S. and P. Banerjee 1978. Prostaglandins, cyclic AMP, U-7118
and acetic acid as insect growth regulators and sterilants. Indian
J. Exp. Biol. 16: 872-875

Destephano, D. B. and U. E. Brady 1974. Synthesis of prostaglandin by
reproductive tissue of the male house cricket Acheta domestica.
Prostaglandins 6: 7l-79.

Destephano, D. B. and U. E. Brady 1977. Prostaglandin and
prostaglandin synthetase in the cricket Acheta domestica. J.
Insect Physiol.23: 905-911.

Destephano, D. B.; U. E. Brady and L. B. Woodalll976. Partial
characterization of prostaglandin synthetase in the reproductive

 

175

tract of the male house cricket Acheta domestica. Prostaglandins
11: 261-273.

Flower, R. J.; H. S. Cheung and D. W. Cushman 1973. Quantitative
determination of prostaglandins and malondialdehyde formed by
the arachedonate oxygenase (prostaglandin synthetase) system in
bovine seminal vesicle. Prostaglandins 4: 325-341

Grazzini, R., D. Hesk, E. Heininger, G. Hildenbrandt, C. C. Reddy, D. Cox-
Foster, J. Medford, R. Craig and R. O. Mumma 1991. Inhibition of
lipoxygenase and prostaglandin endoperoxide synthase by

anacadic acids. Bioch. Bioph. Res. Comm. 176: 775-780.

Hagan, D. V. and U. E. Brady 1982. Prostaglandins in the cabbage
looper Trichoplusia ni. J. Insect Physiol. 28: 761-765.

Havukkala, I. J. and J. R. Miller 1987. Daily periodicity in the
ovipositional behavior of the onion fly, Delia antiqua (diptera:
Anthomyiidae). Environ. Entomo.l6: 41-44. '

Hollingworth, R. M. and A. E. Lund 1982. Biological and neurotoxic
effects of amidine pesticides. Insecticides Mode of action.
Academic press, pp. 189-227.

Hollingworth, R. M. and L. L. Murdock 1980. Formamidine pesticides:
Octopamine like actions in a firefly. Science 208: 74-76.

Howard, R. W.; R. L. Jurenka and G. J. Blomquist 1986. Prostaglandine
synthetase inhibitors in the defensive secretions of the red flour

beetle Tribolium castaneum (Herbst.) ( Coleoptera: Tenebrionidae).
Insect Biochem. 16: 757-760.

Izawa, Y., M. Uchida and M. Yasui 1986. Mode of action of buprofezin
on the twenty-eight-spotted ladybird, Henosepilacnha
vigintioctopunctata Fabricius. Agric. Biol. Chem. 50: 1369-1371.

176

Kubo, I. K. M., N. K. Komatsu, S. Yamagira, Y. Ohashi, K. Sakamoti, Y.
Hirakawa and T. Kamikawa 1987. Chem. Lett. 1101-1104

Lange, A. B. 1984. The transfer of prostaglandin-synthesizing
activity during mating in Locusta migratoria. Insect Biochem. 14:
551-556.

McGiff, J. C. 1981. prostaglandins, prostacyclin and thromboxanes. A
Rev. Pharmac. Toxicol. 21: 479-509.

Mowry, T. M.; T. Adams and J. Miller 1987. Mechanoptical transducer
for quantifying activity in small insect muscles. J. Insect Physiol.
33: 867-873.

Murtaugh, M. P. and D. L. Denlinger 1982. Prostaglandins E and F2a in
the house cricket and other insects. Insect Biochem.12: 599-603.

N anda, D. K. and M. S. Ghosal 1978. Effects of prostaglandin PGF2a on
the cerebral neurosecretory cells of Periplaneta americana
(Blattodea). Vest. C31. 2001. Spol. XLII: 139-142

Orchard, 1. and A. B. Lange 1984. Cyclic AMP in locust fat body:
Correlation with octopamine and adipokinetic hormones during
flight. J. Insect Physiol. 30: 901-904.

Peferoen, M.; R. Huybrechts and A. DeLoof 1981. Longevity and
fecundity in the Colorado potato beetle, Leptinotarsa
decemlineata. Ent. exp. appl.29: 321-329

Platt, N. and S. E. Reynolds 1986. The pharmacology of the heart of a
caterpillar, the tobacco hornworm, Manduca Sexta.J. Insect
Physiol32: 221-230.

Ragab, A. C. Bitsch, J. M. Thomas, J. Bitsch and H. Chap 1987.
Lipooxygenase conversion of arachidonic acid in 'males and

177

inseminated females of the firebrat, T hermobia domestica
(Thysanura). Insect Biochem. 17: 863-870.

Samuelsson, B. 1987. An elucidation of the arachidonic acid-cascade.
Discovery of prostaglandin, thromboxane and leukotrienes. Drugs
33: 2-9.

Sasaki, M. and L. Riddiford 1984. Regulation of reproductive behavior
and egg maturation in the tobacco hawkmoth Manduca sexta.
Physiological Entomology 9: 315- 327.

Singh, B. D. and S. Datta 1980. Effect of cyclic AMP (c-AMP) and
prostaglandin-El on postembryonic developmentof silkmoth,
Bombyx mori L. Indian J. Entomol. 42: 197-204.

Stanley-Samuelson, D. W. 1980. Long-chain polyunsaturated fatty
acids in whole-animal and specific-tissue extracts in insects.
Am. Zool. 20: 577.

Uchida, M.; Y. Izawa and T. Sugimoto 1986. Antagonistic effect on the
20-hydroxyecdysone to an insect growth regulator, Buprofezin, in
Nilaparavata lugens Stal. Agric. Biol. Chem. 50: 1913-1915.

Wakayama, E. J.; J. W. Dillwith and G. J. Blomquist 1986.
Characterization of prostaglandin synthesis in the housefly,
Musca domestica (L.). Insect Biochem. 16: 903-909.

Yamaja Settya, B. N. and T. R. Rmaiah 1980. Effect of prostaglandin
and inhibitors of prostaglandin biosynthesis on oviposition in the
silkmoth Bombyx mori. Indian J. Exp. Biol. 18: 539-541.

Yim, G. K. W.; M. P. Holsapple; W. R. Pfister and R. M. Hollingworth
1987. Prostaglandin synthesis inhibited by formamidine
pesticides. Life Sci. 23: 2509-2516

 

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