THE EFFECT OF INSECTICIDE SYNERGISTS ON PROPOXUR PHARMACOKINETICS IN THE GERMAN COCKROACH, Blattella germanica (L.)

By

HUSSEIN SANCHEZ ARROYO

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2000 For Diego and Maria Paula .

ACKNOWLEDGEMENTS

I would like to thank the following people who, in one way or another, contributed to the completion of this work:

Chuck Strong for teaching me laboratory techniques.

Debbie Hall for guiding me through the graduate school bureaucracy

Dr. Phil Koehler for serving as my committee chairman,

for his friendship, and for many opportunities.

Dr. Simon Yu for serving in my committee and for helpful discussions.

Dr. R. S. Patterson, R. J. Brenner, and O. N. Nesheim

for serving in my committee.

Finally I would like to express my profound gratitude

to Dr. Steven M. Valles for serving on my committee and for

his guidance during my research. His patience and

friendship were very important during this frustrating but

fulfilling period of my life.

iii TABLE OF CONTENTS

mas.

ACKNOWLEDGMENTS iii

ABSTRACT vi

INTRODUCTION 1

LITERATURE REVIEW 5

Life Cycle 5 Behavior/Ecology ^ Pest Status 7 Control Chemical Methods 10 Synergists 12 Non Chemical Methods 16 Insecticide Resistance 17 Insecticide Resistance Mechanisms 19 Decreased penetration 20 Enhanced metabolism 22 Target-site insensitivity 28 Behavioral resistance 32 Insecticide Pharmacokinetics in Insects 35 Insecticide Uptake 35 Cuticular Penetration 36 Translocation 37

MATERIALS AND METHODS 4 0

Insects 40 Chemicals 40 Insecticide Bioassays 41 Penetration Studies and In Vivo Metabolism Experiments . 43 Metabolic Studies 47 Preparation of Microsomes 47 Partial Purification of GSTs 48 In Vitro Oxidative Metabolism of [^^C [Propoxur ... 50

In Vitro Hydrolytic Metabolism of [^^C] Propoxur . . . 53

IV In Vitro Conjugation of [^^C] Propoxur by Glutathione S-Transf erases 54 Electrophoresis 54 Acetylcholinesterase Inhibition 56 Propoxur Sequestration 58 Statistical Analysis 59

RESULTS 50

Topical Bioassays 50 Penetration Studies and In Vivo Metabolism Experiments . 61 In Vitro Propoxur Metabolism 55 Propoxur Sequestration 58 Effect of Quinidine on Propoxur Penetration 69

DISCUSSION 89

REFERENCES CITED 98

BIOGRAPHICAL SKETCH 117

V Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

THE EFFECT OF INSECTICIDE SYNERGISTS ON PROPOXUR PHARMACOKINETICS IN THE GERMAN COCKROACH, Blattella germanica (L.)

By

Hussein Sanchez Arroyo

December 2000

Chairman: Dr. Philip G. Koehler Major Department: Entomology and Nematology

The role of piperonyl butoxide (PBO) and S, S, S-tributyl phosphorotrithioate (DEF) on propoxur toxicity, penetration

rate, and metabolism (in vitro and in vivo) were studied in

the Marietta strain of the German cockroach. Pretreatment

with 100 |ig/cockroach of the cytochrome P450 monooxygenase

inhibitor, PBO, or 30 |ig of the esterase inhibitor, DEF,

increased propoxur toxicity by 2 and 6.8-fold, respectively.

These data implicated hydrolysis as the most important

propoxur detoxification route. Conversely, in vitro studies

demonstrated that general esterases and glutatione

conjugation were not important components of propoxur

metabolism in this strain.

VI Propoxur was metabolized primarily by microsomal proteins fortified with NADPH. The major metabolites produced were W- hydroxymethyl propoxur, 2-hydroxyphenyl N- methyl carbamate and o-isopropoxyphenyl carbamate. Formation

of metabolites was NADPH - dependant ; no metabolism was detected with microsomal enzymes lacking NADPH.

Additionally, because PBO inhibited propoxur metabolism in a dose dependant fashion and the reaction required NADPH, the cytochrome P450 monooxygenase system was implicated as being responsible for the metabolism. Further studies showed that the addition of high concentrations of DEF (lO'^-lO'^ M) decreased the rate of propoxur metabolism by microsomal monooxygenases fortified with NADPH. Therefore, DEF

inhibition of cytochrome P450 monooxygenases is a plausible

explanation for the apparent discrepancy between in vivo

synergism of DEF and no metabolism of propoxur by esterases

in vitro.

Interestingly, pretreatment with synergists reduced the

rate of propoxur penetration significantly. PBO and DEF

reduced the penetration rate 2.9-fold and 2.7-fold at 2 hr,

respectively. The amount of metabolites produced in vivo was

reduced 5. 7- fold and 3.9-fold when PBO and DEF were added.

vii . .

INTRODUCTION

As a serious pest in the urban environment, the German cockroach, Blattella germanica (L.), has been exposed to

repeated applications of insecticides. The intense

insecticide pressure often results in resistance (Atkinson

are et al . 1991), and resistance ratios as small as 5-fold

considered significant enough to result in control failure

(Cochran 1989, Scott 1991) . Insects exhibit three

physiological insecticide resistance mechanisms, including

decreased insecticide penetration, enhanced metabolism, and

target site insensitivity (Roush 1991) . Behavioral

resistance, such as bait avoidance and habitat preference,

has been reported in some cases (Sparks et al . 1989, Valles

and Brenner 1999) In general, enhanced metabolism is the

most important mechanism of insecticide resistance in

cockroaches, and it is attributed to quantitative or

qualitative differences in the expression of detoxifying

enzymes. The primary detoxification enzyme systems involved

in insecticide resistance are cytochrome P450

monooxygenases, hydrolases, and glutathione S- transferases

(deBethizy and Hayes 1994)

1 . .

2

Piperonyl butoxide (PBO) and S, S, S-tributyl phosphorotrithioate (DEF) have been used traditionally as a specific synergists when studying the contribution of mixed

function oxidases and esterases, respectively, to

insecticide resistance. It is generally accepted that the addition of those synergists increases insecticide toxicity by blocking these enzymes, thus preventing insecticide

degradation and allowing the insecticide to exert its toxic

action unchallenged. However, it has been reported that

synergists do not always act as they were intended or

expected. For example, Valles and Yu (1996a) and Valles

(1998) reported that cockroaches treated with PBO were less

susceptible to permethrin or cypermethrin compared with

cockroaches not treated with PBO. However, Kennaugh et al

(1993) suggested that in other insects, the increased

toxicity was not due to a supposed increased enzymatic

level. Additionally, Valles et al . (1997) has shown that

micromolar concentrations of DEF can inhibit microsomal

oxidases in vitro, demonstrating that synergists are not

always enzyme specific.

Contact insecticides reach their target site

penetrating through the cuticle and into the hemolymph

where they are rapidly transported throughout the insect

body to the target organ (Matsumura 1985) . Gunning et al 3

(1995) reported that pretreatment with PBO accelerates insecticide penetration rate in esf envalerate resistant

strains of Helicoverpa armigera (Hiibner) . They concluded

that PBO might facilitate penetration of certain chemicals

through the cuticle of some insects acting in some way as a multi-metabolic inhibitor. Further, Bull and Pryor (1990)

reported that the absorption of topically applied PBO and

DBF slowed the rate of fenvalerate cuticular penetration in

houseflies, Musca domestica L. According to these reports,

pretreatment with synergists may accelerate or delay

insecticide penetration, and this alteration in insecticide

penetration rate could be insect specific.

The role that synergists play in modifying the rate

of insecticide cuticular penetration or in insecticide

metabolism has been poorly studied, and there currently are

no reports about propoxur pharmacokinetics in the German

cockroach. The objective of this research was to determine

the pharmacokinetics of propoxur in the insecticide

resistant Marietta strain of German cockroach, which is

moderately resistant to propoxur. The specific objectives

of these studies were to determine the role of the

synergists PBO and DBF on propoxur penetration and in vivo

and in vitro metabolism of this insecticide by cytochrome

P450 monooxygenases and hydrolases in the Marietta strain. 4

Additionally, the inhibitory effect of DEF on cytochrome

P450 monooxygenases and acetylcholinesterase was evaluated.

Finally, the possible sequestration of propoxur by proteins was determined. . . ,

LITERATURE REVIEW

Life Cycle

The German cockroach, Blattella germanica

life stages: egg, (Dictyoptera: Blattellidae) , has three nymph, and adult. The life cycle usually is completed

within 100 days (Archbold et al . 1987). Adult females form

an egg case 10-14 days after insemination (Cochran 1983)

and the interval between hatch of one egg case and

formation of a subsequent egg case varies from six to ten

days (Cochran 1983, Durbin and Cochran 1985).

Females carry their egg cases until they are ready to

hatch. The egg stage requires about 14 days and typically

each egg case contains between 30 and 40 eggs (Patterson

and Koehler 1992) The nymphal stage begins with egg hatch

and ends with the emergence of the adult and requires 60

days to be completed. The most frequently reported number

of molts is six (Ross and Mullins 1995)

Adult German cockroaches are 1.3 to 1.6 cm long, pale

brown or tan, and have two parallel dark stripes on the

pronotum; although fully winged, adults cannot fly (Ebeling

5 . . . .

6

1975) . A female may live for more than 200 days (Patterson and Koehler 1992)

Behavior /Ecology

German cockroach populations are characterized by

overlapping generations. German cockroaches are active primarily during dusk and night, and they remain in refuges

during the daytime. Adult male German cockroaches are more

active than nymphs and adult females. This activity

includes foraging for food, water, harborage, and searching

for mates. Drinking and eating coincide with peak times of

locomotory activity (Metzger 1995) Nymphs comprise a high

percentage of cockroach populations, often exceeding 80% of

field populations (Owens and Bennett 1983)

The German cockroach is gregarious and aggregation is

induced by pheromones which are the main elements

responsible for their spatial distribution (Ishii and

Kuwahara 1968) Harborages are marked with pheromones

contained in the feces. Aggregation behavior is most

pronounced in small nymphs followed by adults (Runstrom and

the Bennett 1990). Koehler et al . (1994) found that

harborage width preference increased with insect age.

Larger nymphs (3’^'^ and subsequent instars) are least apt to

aggregate, and this biological characteristic may be due to ., . .

7

the fact that large nymphs are more involved in migration when populations are overcrowded (Runstrom and Bennett

1990)

Pest Status

The German cockroach is considered the number one

it household pest in the United States (Brenner 1995) , and

is the most prevalent pest in low income apartments in the

United States (Koehler et al . 1987) . Cockroaches breed

within the home, sharing with man his food, water, and

shelter. They contaminate food, imparting an unpleasant

odor and taste, and because they frequent unsanitary areas,

their presence leads to suspicion of a threat to human

health by mechanical transmission of disease organisms

(Ebeling 1975, Benson and Zungoli 1997)

Medical implications of cockroach infestations are

associated with acute or chronic exposure to cockroaches

and their biotic associates (Brenner 1995) . They have been

reported as vectors of human pathogens (Alcamo and Frishman

1980) and producers of etiologic agents, such as defensive

and excretory secretions (Mullins and Cochran 1973) and

allergens (O'Connor and Gold 1999) The most common and

important human pathogens mechanically transmitted by

cockroaches are bacteria. These are acquired by walking .

8 over contaminated surfaces which are subsequently transferred to foodstuffs via normal foraging behavior

(Rueger and Olson 1969)

Recently, of more concern has been the severe allergic response in humans that is elicited by cockroaches. Allergy is an exaggerated response of the immune system to harmless antigens (O'Connor and Gold 1999). This response results in the release of several substances that damage tissues and eventually cause allergy, such as histamines (Brenner et al. 1991). Platts-Mills (1997) reported that asthma has been increasing in Western societies over the last 35 years, and this may be attributed to an increase in indoor activities. The risk of developing cockroach allergy is a function of several variables including (1) genetic predisposition, (2) the species of cockroaches, (3) the size of the infestation, (4) the duration of exposure, and

(5) the amount of allergen that becomes airborne in the context of ventilation and air flow patterns in buildings

(Koehler et al . 1990).

The amount and type of allergen necessary to sensitize genetically "at risk" children is unclear. The relationship between allergen exposure and asthma is also uncertain

reservoir (Sporik et al . 1999). However, the domestic

concentration of cockroach allergens is closely related to 9 the prevalence of sensitization in atopic (skin sensitive) children. Further, the prevalence of asthma had a limited relationship to these allergen measurements, suggesting that other factors play a part in determining which

allergic individuals develop asthma. Wilson et al . (1999) determined that allergy to cockroach and other indoor allergens might be a significant contributor to infantile asthma in rural settings.

Interestingly, one of the most potent allergens identified from the German cockroach was a glutathione S-

transferase (Arruda et al . 1997, Yu and Huang 2000). These proteins are important xenobiotic (including insecticides) detoxifying enzymes. They have been implicated as an important environmental factor that may aggravate asthma in sensitized persons and induce IgE antibody production

(Asturias et al . 1999, Arruda et al . 1997). Similarly, the

Cr-PI allergen from American cockroach induced lymphocyte proliferation and cytokine production in skin sensitive

patients (Jeng et al . 1996). In patients with cockroach allergy and asthma, multiple factors in addition to cockroach allergen appeared to aggravate the patient's asthma. A multimodality therapeutic regimen, which included medications as well as cleaning of the home, integrated pest management, and professional application of chemical . ,

10 controls, resulted in substantial clinical improvement

(O'Connor and Gold 1999).

Control

Chemical Methods

Being one of the most important pest in households,

the German cockroach has been exposed to almost all

insecticides available for urban insect control purposes.

To date, the most common insecticides used for controlling

cockroaches are broad spectrum insecticides which primarily

affect the nervous system, including organophosphorus

carbamate, and pyrethroid insecticides. Acephate,

and malathion are chlorpyrifos , diazinon, dichlorvos,

common organophosphorus insecticides, and bendiocarb and

propoxur are common carbamates recommended for cockroach

control (Matsumura 1985, Patterson and Koehler 1992). Among

the pyrethroids used for German cockroach control are

cyfluthrin, cypermethrin, deltamethrin, lamda-cyhalothrin,

and permethrin (Brannes 1997)

Recent concern over the toxicity of broad spectrum insecticides has resulted in the development of alternative, insect-specific chemicals with comparatively low mammalian toxicity. Among these new insecticide groups available for German cockroach control are . .

11

amidinohydrazones , f luoroaliphatic sulfonamides, chloronicotinyl nitroguanidines phenyl pyrazoles, and , macrocyclic lactones. These new insecticides interfere with different vital insect systems.

Hydramethylnon, an amidinohydrazone insecticide, acts by depressing respiration by inhibiting mitochondrial

electron transport (Hollingshaus 1987) . Sulfluramid, a f luoroaliphatic sulfonamide insecticide, also inhibits energy production by uncoupling oxidative phosphorylation

(Schnellmann 1990, Schnellman and Manning 1990)

Imidacloprid, a chloronicotinyl insecticide, causes a continuous activation of insect-specific nicotinic-

acetylcholine receptors (agonistic) , causing tetanic muscle contractions (Mehlhorn et al. 1999). Avermectins are macrocyclic lactones and fipronil, a phenyl pyrazole, activate the y-aminobutyric acid (GABA) -gated chloride channel which suppresses neuronal activity, resulting in ataxia, paralysis, and death (Bloomquist 1993, Cole et al

1993) .

Although these new active ingredients in baits are comparatively less toxic to humans, there are still environmental and public concerns, and the regulations imposed by the government have resulted in still further emphasis on decreasing insecticide use. Insecticide ,.

12 producers, researchers, and formulators have responded with bait technology. Bait formulations of hydramethylnon (Milio

et al . 1986, Appel 1990, Valles and Brenner 1999),

sulfluramid (Reid et al . 1990, Appel and Abd-Elghafar 1990,

imidacloprid (Appel and Tanley 2000) Reid and Klotz 1992) ,

abamectin (Cochran 1994), and fipronil (Kaakeh et al . 1997) are all very effective as stomach poisons. These baits provide excellent control when compared with conventional sprays and, most importantly, reduce insecticide use in urban environments significantly (Schal and Hamilton 1990)

Synergists

Synergists are chemicals that increase the toxicity of insecticides, though they are not toxic by themselves

(Brindey and Selim 1984) . These chemicals increase insecticide toxicity by inhibiting the enzymes that in turn detoxify the insecticides. Perhaps the most important

synergist developed was piperonyl butoxide (PBO) , which has been used widely in urban insect pest control. Pyrethrins, pyrethroids, and carbamates are the most common insecticides synergized (Showyin 1998); however, other insecticides are also improved with synergists. PBO improves pesticide efficacy by stabilizing certain formulations (e.g., pyrethrum) and interfering with .

13 detoxification of the insecticide which ultimately decreases the amount of insecticide required for control.

Casida (1970) classified synergists according to their structure, but from a physiological standpoint it is more useful to classify them according to the detoxification

mechanism they inhibit (Raffa and Priestler 1985) . The most numerous group inhibits cytochrome P450 monooxygenases.

Methyl enedioxyphenyl synergists, including sesamin, sesamolin, PBO, and sesamex, are among the most important cytochrome P450 monooxygenase inhibitors. Other cytochrome

P450 monooxygenase inhibitors include the W-alkyl compounds such as SFK-525A and MGK-264 (Raffa and Priestler 1985)

Although oxidative metabolism is the principal route of detoxification for many insecticides, others are metabolized hydrolytically or by conjugation with endogenous substances. S, S, S, -tributhylphosphorotrithioate

diisopropylf luorophosphate (TCP) synergists are (DEF) , and used to investigate hydrolytically-based resistance

mechanisms by researchers . Inhibitors of glutathione S- tranferase enzymes (which are very important in the metabolism of organophosphate insecticides) are poor

synergists in vivo (Soderlund and Bloomquist 1990) . Maleic acid diethyl ester has been used for providing limited .

14 evidence for the involvement of glutatione S-transf erase in

resistance to avermectins (Iqbal and Wright 1997) .

In some cases, insecticides can be synergized by other insecticides. For example, thiocyanates, such as Thanite, can synergize carbaryl more than 100 times by topical

application on houseflies (El-Sebae et al . 1964) . Liu and

Plapp Jr. (1990, 1992) have suggested that formamidines could be target site synergists of pyrethroids. Scharf et

suggested the use of mixtures of carbamates and al . (1997b) organophosphates as a way of synergizing the effect of each active ingredient for control of insecticide resistant

German cockroaches

Extensive investigation of synergists has led to a better understanding of the mechanisms of detoxification in insects, the basic biochemical processes involved in insecticide resistance, and the mode of action of

insecticides (Metcalf 1967, Bernard and Philogene 1993). In addition, Raffa and Priestler (1985) suggested the use of

synergists as research tools to define a compound's potential toxicity, to prevent the development of

insecticide resistance, or to make insecticide resistant populations more susceptible. A common method of attempting

to overcome insecticide resistance in field populations is

to introduce a synergist along with the insecticide (Casida . . .

15

1970) . However, synergists are only active in cases in which the resistance mechanism is attributable to an enzyme (s) that is inhibited by them (Scott 1990)

Unfortunately, some insects have developed resistance to synergists rendering them ineffective. For example. Hung and Sun (1989) reported that larvae of a diamondback moth population were resistant to piperonyl butoxide by an enhanced microsomal degradation mechanism of this synergist

Despite their usefulness in field and laboratory, synergists have been known to alter insecticide pharmacokinetics. For example, pretreatment with PBO accelerates the penetration rate of esf envalerate in insecticide resistant strains of Helicoverpa armigera

(Hiibner) ; however, in insecticide susceptible strains

insecticide penetration was unaffected (Gunning et al

1995) . In other experiments. Sun and Johnson (1972) reported that a variety of chemicals synergized the

toxicity of carbamates in the housefly {M. domestica) . They

found that the synergism in carbaryl-Thanite or SD9003-

Thanite mixtures by topical application was due largely to

the increase in the rate of penetration rather than to a

large decrease in the rate of detoxification.

Unquestionably, a certain degree of synergism in many .

16 insecticide- synergist combinations may be due to change in penetration rate, in addition to inhibition of detoxification. These authors proposed the use of the term quasi -synergism to describe this phenomenon (increased toxicity due almost exclusively to increased penetration rate, whereas the rate of detoxification is largely

unchanged) . According to their results. Sun and Johnson

(1972) speculated that quasi -synergism would be found more often in low-toxicity carbamates and only occasionally in organophosphorus or organochlorine insecticides.

Non Chemical Methods

Alternative strategies that have been suggested or attempted for controlling cockroaches include sanitation

(Robinson and Zungoli 1985) , structural modifications

(Runstrom and Bennett 1984) , temperature treatments (Forbes

and Ebeling 1987) , trapping (Moore and Granovsky 1983) , and enhancing insecticide efficacy by the addition of

aggregation pheromones (Miller et al . 1997) . The use of electronic devices for controlling cockroaches is widely suggested by manufacturers, but devices that produce ultrasonic pulses have proved unsuccessful for cockroach

control (Koehler et al . 1986a) .

17

Despite alternative strategies, insecticides remain the primary method of control for the German cockroach.

Unfortunately, insecticide use against isolated cockroach populations invariably results in the development of insecticide resistance. The current challenge to entomologists is the development of insecticide resistance management strategies for cockroaches to delay or prevent the development of insecticide resistance. Effective strategies can best be proposed when a complete understanding of the insecticide resistance phenomenon is achieved

Insecticide Resistance

Insecticide resistance is defined as the development of an ability in a population of some organism to tolerate doses of a toxicant which would prove lethal to the majority of individuals in a normal population of the same

species (Anonymous 1957) . The phenomenon of insecticide resistance has proliferated exponentially (Georghiou and

Mellon 1983) and its development is determined by a variety of genetic, biological or ecological, and operational

factors (Georghiou and Taylor 1986) . Genetic factors include a) number and frequency of resistant alleles, b) dominance of resistant alleles, c) penetrance. 18 expressivity, and interactions of resistant alleles, d) past selection with other chemicals, and e) extent of integration of resistant genome with fitness factors. The most important biological or ecological factors are a) generation turnover, b) offspring per generation, c) monogamy or polygamy, parthenogenesis, d) isolation, mobility, migration, e) monophagy or polyphagy, and f) fortuitous survival, refugia (Georghiou and Taylor 1977a).

Factors in the genetic and biological categories are inherent qualities of a population and are, therefore, beyond man's control. However, their assessment is essential in determining the risk for resistance of a target population (Georghiou 1983).

Operational factors in resistance are those related to the application of pesticides. These include a) the chemical nature of pesticides, b) the relationship to previously used chemicals, c) the persistence of residues and formulations, d) application and selection threshold,

e) the life stage selected, f) the mode of application, g)

space limiting selection, and h) alternating selection

(Georghiou and Taylor 1977b) . Operational factors are under man's control and their manipulation may help to delay

insecticide resistance. 19

Problems associated with insecticide resistance include increased insecticide applications, increased insecticide dosages, decreased crop yields, environmental damage from insecticide overuse, and outbreaks of human and animal diseases when vectors cannot be controlled (Scott

1996) . One of the hidden costs of resistance is the potential loss of all available insecticides as effective

control agents (Scott 1991) . Additionally, the knowledge of all the factors involved in insecticide resistance development will help to implement specific tactics to control insect pest populations.

Insecticide Resistance Mechanisms

Insects exhibit three physiological insecticide

resistance mechanisms, including decreased insecticide penetration, enhanced metabolism and target-site

insensitivity (Roush 1991) . Behavioral resistance, such as bait avoidance and habitat preference, has been reported in

some cases. However, physiologically-based insecticide

resistance remains the principal mode of resistance

exhibited by insects. All of these mechanisms have been

reported in the German cockroach, and in many instances the

resistance was attributable to multiple resistance . . .

20

mechanisms (Valles and Yu 1996a, Anspaugh et al . 1994,

Prabhakaran and Kamble 1994)

Decreased Penetration

To kill the insect, the insecticide must reach the target site in a lethal dose. If the mechanism of decreased penetration is present in the insect population, the insecticide is bound to components in the integument, preventing lethal accumulation of insecticide at the site

of action (Ahmad and McCaffery 1999) . Functionally, this can delay the penetration rate and provide the insect with additional time to detoxify the compound (Matsumura 1985)

Increased cuticular lipids also have been reported as a

factor responsible for resistance (Patil and Guthrie 1979)

The phenomenon of decreased penetration rate may be related to the presence of abnormal amounts of transport proteins (e.g., P-glycoproteins) in the integument, banning

reported four times more P-glycoprotein in et al . (1996a)

insecticide resistant larvae of tobacco budworm compared with susceptible larvae. Further experiments showed that

insecticide resistant budworm larvae treated with a P- glycoprotein inhibitor were more sensitive to thiodicarb.

The LD 50 for thiodicarb was decreased by a factor of 12.5, 21 supporting the role of P-glycoprotein in pesticide

resistance (Lanning et al . 1996b).

It is generally agreed that the mechanism of reduced penetration does not provide large resistance levels to the

lethal effects of insecticides (<5-fold) . However, it may act as an intensifier for other resistance mechanisms

(Plapp and Hoyer 1968) . Ku and Bishop (1967) reported the first case of decreased insecticide penetration in an

insecticide resistant population of German cockroach. The susceptible strain absorbed a greater percentage of the applied dosage of carbaryl than did the resistant strain during the first 36 hr after treatment, but thereafter, penetration was about equal in the two strains. They concluded that despite exhibiting slower cuticular penetration and more rapid excretion of carbaryl, these

factors were only minor resistance contributors.

Valles et al . (2000) reported decreased cypermethrin penetration as an important resistance mechanism in the

insecticide resistant Aves strain of the German cockroach.

Similar results have been reported for other pyrethroids.

worked Anspaugh et al . (1994) and Bull and Patterson (1993) with permethrin in the same insecticide resistant strain

(Village Green) . These investigators reported that the

amount of permethrin that penetrated into resistant .

22 cockroaches was less than that of the susceptible strain

(Orlando) . The maximum difference in the insecticide rate of penetration was at 24 hr after treatment. Bull and

Patterson (1993) suggested that reduced penetration is a component of permethrin resistance in addition to kdr.

Additionally, Wu et al . (1998) reported decreased cuticular penetration in a fenvalerate insecticide resistant strain

(Munsyana) . The overall results for fenvalerate suggested that both metabolic and nonmetabolic mechanisms (decreased penetration and sodium channel insensitivity) may have been responsible for fenvalerate resistance in this strain.

Siegfried and Scott (1991) suggested that propoxur resistance in a Baygon resistant strain of the German cockroach was due to the combined effects of decreased penetration and metabolic detoxification. The involvement of a penetration barrier as a component of resistance was supported by loss of resistance when the insecticide was

injected as compared with topical application.

Enhanced Metabolism

Enhanced metabolism is the most important mechanism of

insecticide resistance in insects (Oppenoorth 1985)

Insecticide resistance due to enhanced metabolism is

attributed to quantitative and qualitative differences in ..

23 the expression of detoxification enzymes (Ahmad and Forgash

1976, Hemingway and Karunaratne 1998) . The primary detoxification enzyme systems involved in insecticide resistance are cytochrome P450 monooxygenases, hydrolases

glutatione S-transf erases (deBethizy and (esterases) , and

Hayes 1994)

Xenobiotic metabolism occurs in three distinct phases.

Phase I reactions introduce a hydrophilic functional group into the molecule providing an attachment point for subsequent reactions. Oxidations, reductions, and hydrolysis are typical Phase I reactions. Phase II reactions combine water-soluble endogenous agents which increase the molecule's water solubility. These conjugated products are, in general, more polar, less lipid soluble, and more readily eliminated from cells. Finally, phase III

is the transport of the manipulated xenobiotic out of the

cell (Ishikawa 1992) . In some cases the added chemical groups are recognized by specific carrier proteins involved

in facilitated diffusion or active transport (deBethizy and

Hayes 1994)

Oxidative metabolism is catalyzed by cytochrome P450

monooxygenases . These enzymes play an important role in

detoxification and/or activation of xenobiotics (Ohkawa et

The al . 1999) and endogenous compounds (Scott 1996). , .

24 cytochrome P450 monooxygenase system is located in the endoplasmic reticulum and is composed of cytochrome P450, and NADPH- cytochrome P450 reductase (Zhang and Scott 1994)

The most important oxidative reactions may be classified as desulfuration and ester cleavage, epoxidation and aromatic

hydroxylation, heterocyclic ring hydroxylation, N- 0- , and

S-dealkylation, sulphoxidation, dehydrogenation, and

dioxole ring cleavage (Kulkarni and Hodgson 1980) .

Cytochrome P450 monooxygenases oxidize their substrate by the introduction of oxygen to the substrate molecule. In this reaction, one atom of molecular oxygen is reduced to water while the other is incorporated into the substrate

(Hodgson 1983) . For the reaction to occur, oxygen, phosphatidyl choline, and a source of reducing equivalents

in the form of NADPH are required. The first electron is

transferred from NADPH to cytochrome P450 via NADPH

cytochrome P450 reductase (White and Coon 1980) . The second

electron can be accepted from NADPH -cytochrome P450

reductase as well as from cytochrome bs in some cases (Imai

1981, Zhang and Scott 1994) . Cytochrome bs appears to be

cytochrome P450 isoform specific.

Esterases have diverse functions in insects including

proteolysis, hormone metabolism, and xenobiotic metabolism

or sequestration (Aldridge 1993) . Most esterases have . .

25 general substrate specificity and they may have a broad biological function. The mechanism of hydrolysis of esters by many carboxylesterases has been established due to the reaction of organophosphorus compounds with their non-active catalytic center.

Esterases have been grouped into three categories based on their reactivity with organophosphorus compounds and by their substrate specificity. "A" esterases prefer carboxylesters with aryl groups bonded to the carbonyl carbon and can utilize organophosphate esters as substrates. "B" esterases prefer esters with alkyl groups bonded to the carbonyl carbon and are inhibited by organophosphate esters. Finally, "C" esterases prefer acetate esters and do not interact with organophosphates

Many insects have low levels of "A" esterases compared with mammals which often explains the selective toxicity of the organophosphorus insecticide malathion to insects

(deBethizi and Hayes 1994)

The overexpression of esterases in resistant insects often leads to enhanced metabolism of the insecticide.

However, they may also work by sequestering insecticides binding to and effectively preventing the insecticide from

reaching its intended target site. Many examples of

esterase overproduction have been reported for .

26 organophosphorus and carbamate insecticides (Vaughan et al

1998, Scharf et al . 1997a, Blackman et al . 1995, Field et

al . 1988, and Raymond et al . 1989) .

In German cockroaches esterase overproduction has been detected in many insecticide resistant strains. Elevated esterase activity was shown to correlate well with pyrethroid resistance in the German cockroach (Valles

1998) . Prabahakaran and Ramble (1995) reported that a higher quantity of esterase isozyme E6 from organophosphorus and carbamate resistant German cockroaches had 2-3 fold higher specific activity compared with the susceptible strain. However, the increased enzyme activity was due to the quantity of enzyme present rather than differences in structure.

Prabahakaran and Ramble (1995) conducted reactivation experiments that implicated the involvement of esterase E6 as a sequestrant in several resistant German cockroach strains. Further experiments performed by Prabahakaran and

Ramble (1996), and later by Scharf et al . (1997a) supported the hypothesis that the role of E6 and other esterases could be sequestration of toxic molecules (organophosphorus and carbamates) rather than hydrolysis.

Glutatione S- transferases are a large group of cytosolic enzymes that catalyze conjugation of organic . . , .

27 molecules possessing an electrophylic center with the tripeptide glutathione (Clark and Carroll 1989)

Glutathione conjugates are typically less lipophilic, more water soluble, and likely to be excreted (deBethizy and

Haynes 1994) . Glutathione S- transferases facilitate the nucleophilic attack of glutathione thiolate ion on the electron-deficient atom of the relatively hydrophobic electrophylic xenobiotic. In general, glutatione S- transf erase in invertebrates consists of two subunits, homodimers or heterodimers between 20-27 kDa (Clark and

Carroll 1989) . In susceptible German cockroaches, three glutathione S-transf erases have been isolated, all of which were homodimers with a subunit molecular weight of 25,550

(Yu and Huang 2000) However, in highly insecticide

resistant insect strains (e.g. houseflies) , higher molecular weights forms have been reported (Motoyama and

Dauterman 1977)

Each dimeric enzyme molecule possesses two

independent active sites, on each subunit. The active sites

consist of a glutathione binding site (G) and a hydrophobic site (H) for binding of the electrophylic

substrate. Catalysis is initiated by abstraction of the

glutathione thiol proton by a basic amino acid residue near

the active site. The enzyme strategically places the .

28 electrophylic center of the substrate in proximity to the active thiolate ion, which facilitates catalysis

(nucleophilic attack) . After conjugation, glutamate and glycine residues are cleaved from the conjugate leaving a water soluble mercapturic acid derivative (deBethizy and

Haynes 1994)

Organisms possess multiple glutathione S-transf erases with wide substrate specificity, and apparently, they may be selected to meet the needs of a particular population

(deBethizy and Haynes 1994) . Glutathione S-transf erases have been reported to be an important detoxification mechanism against organophosphorus insecticides (Lamoureux

and Rusness 1986) . However, although glutathione S- transferases are often overexpressed in insecticide resistant German cockroach populations, very little evidence exists concerning the contribution of these enzymes in metabolism.

Target-site Insensitivity

Decreased sensitivity of the target-site may result in insecticide resistance. This mechanism of resistance is often the most problematic and it may confer resistance to

an entire insecticide class (Scott and Dong 1994) . The most extensively studied example of target site insensitivity is . .

29 modified acetylcholinesterase, which confers resistance to organophosphorus and carbamate insecticide (Metcalf 1989,

Fournier and Mutero 1994, and Kanga and Plapp Jr. 1995)

These alterations result in two types of modifications

(quantitative and qualitative) that confer resistance to

insecticides (Fournier et al . 1992).

several Drosophila Fournier et al . (1992) constructed melanogaster strains that expressed quantitative increased amounts of enzyme. Qualitative changes in the enzyme, have been reported in Drosophila, even when only one gene (Ace) encodes acetylcholinesterase (Hall and Kanlcel 1976). Five point mutations in that gene were responsible for

resistance to various organophosphates and carbamates

(1992) (Mutero et al . 1994). Additionally, Brown and Bryson

concluded that the resistance in H. virescens was related

to the overall size or shape of the inhibitor molecule,

suggesting that the change in affinity responsible for

resistance was due to a change in the size of the active

site

Apparently, some species do not develop insecticide

resistance through modification of their

acetylcholinesterase (Fournier and Mutero 1994). In German

been suggested in a coclcroaches , decreased sensitivity has

Dubai strain (Hemingway et al . 1993); however, this 30 insecticide resistance mechanism has not been definitively documented despite extensive use of organophosphate and carbamates against the German cockroach for decades.

Siegfried and Scott (1992) suggested that the German cockroach lacks the genetic plasticity for modification of this enzyme.

Another important target site modification that results in insecticide resistance is the modification of the voltage-gated sodium channels of the nerve axon.

Mutation of the genes that code for these sodium channels results in resistance to pyrethroids and DDT and is known

as knockdown resistance {kdr ) . Pyrethroid insecticides and

DDT modify the sodium current of nerve cells by slowing down the inactivation of the sodium channel (Narahashi and

Lund 1980) . Therefore, sodium channel modification may

reduce insecticide binding and prevent its toxic action.

Knockdown resistance (kdr) to pyrethroid insecticides

due to reduced neuronal sensitivity to these compounds has

been studied extensively in cockroaches (Dong and Scott

1991, Scott and Dong 1994, and Dong et al . 1998).

Apparently, a single amino acid change (from Leu®®^ to

Phe^^^) in the para sodium channel protein is associated

with kdr to pyrethroid insecticides in German cockroach

(Dong 1997) . After working with several German cockroach , .

31

mutation strains, Dong et al . (1998) suggested that this was widespread in German cockroach populations in the

United States.

In D. melanogaster cyclodiene resistance has been shown to be associated with a single base pair substitution in the GABA receptor chloride ion channel gene Rdl

(f french-Constant et al . 1993). This substitution causes the replacement of an alanine with a serine in the second membrane spanning domain. The gene Rdl was cloned from a D. melanogaster mutant resistant to cyclodiene insecticide and

picrotoxin (f french-Constant et al . 1991), and apparently this gene manifests resistance through both the weakening of the insecticide binding to the antagonist -favored

(desensitized) conformation by a structural change at the insecticide binding site, and destabilizes the antagonist- favored conformation in an allosteric sense (Zhang et al

1994) . This mutation confers a high level of resistance to these insecticides in a wide range of insects (Stilwell et

al. 1995). Thompson et al . (1993) reported the same

substitution in American cockroach, suggesting that there are a limited number of mutations that can confer

resistance to cyclodienes.

The mechanism of decreased sensitivity to the microbial insecticide. Bacillus thuringiensis Berliner and .

32

Bacillus sphaericus crystal proteins, has been reported to occur in two different ways. The first is correlated with reduction of the affinity of the receptor on midgut brush-

border membranes to the toxic peptide (Rie et al . 1990,

Nielsen-Leroux et al . 1995). The second is correlated with differences in the midgut proteolytic activity (Oppert et

al . 1997, Forcada et al . 1996). In insecticide resistant diamondback moth, the B. thuringiensis crystal protein,

bind the brush border membrane of the CrylA(b) , did not to midgut epithelial cells of a field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule (Ferre et al

1991) . Apparently, the same mechanism occurs in Culex quinquefasciatus resistant to B. sphaericus Nielsen-

a resistant strain of Leroux et al . (1995) reported that this species had lost the functional receptor for B.

sphaericus toxin.

Behavioral Resistance

Behavioral resistance is defined as evolved behavior

that reduces as insect ' s exposure to toxic compounds or

that allow an insect to survive in what would otherwise be

a toxic and fatal environment (Sparks et al . 1989) . This behavioral resistance can be further divided into stimulus 33 dependant mechanisms, such as increased repellency and irritability which reduces contact with the insecticide, and stimulus -independent mechanisms such as exophily which do not require any contact with the insecticide (Georghiou

1972, Lockwood et al . 1984). Although behavioral resistance is still not well understood, many reports support its existence in both the presence and absence of physiological

and biochemical mechanisms (Sparks et al . 1989).

Behavioral resistance has been reported to baits used for German cockroach control. This behavioral resistance may be due to feeding deterrence to the active ingredient or an ingredient of the bait base. In German cockroaches, physiological resistance generally predominates over behavioral resistance in populations selected by

conventional means, such as dusts or sprays. However, with

toxic baits behavioral resistance may be an important mechanism because the cockroaches sometimes do not accept

it (Ross 1997) .

Experiments performed with a commercially available

chlorpyrif os-based bait. Raid Max, on a German cockroach

strain with low level of resistance to chlorpyrifos showed

that cockroaches preferred dog chow to the bait, although

cockroaches from a susceptible strain were attracted to the

bait (Ross 1997) . The strong attractiveness of insecticide 34 susceptible cockroaches to the bait was attributed to the attractiveness of the bait base and lack of repellence to the active ingredient. Subsequent experiments with treated dog chow showed that behavioral resistance to the bait was

caused by feeding deterrence of the active ingredient.

Interestingly, behavioral resistance to baits has been

found in German cockroach strains with at least some

resistance to pesticides (Ross 1998, Hostetler and Brenner

1994, and Valles and Brenner 1999) .

Silverman and Biemam (1993), and Silverman and Ross

(1994) demonstrated glucose aversion (glucose is an

ingredient of the bait base) as a behavioral resistance

mechanism, in a German cockroach strain with a history of

exposure to hydramethylnon bait. Strains that avoided

ingesting bait also displayed an aversion to glucose, which

was a component of the bait and was considered

phagostimulant . Substitution of fructose for glucose in

toxic baits significantly improved bait efficacy in field.

Rather than being learned, apparently this avoidance

behavior was inherited as an autosomal incompletely

dominant trait with heterozygotes ingesting intermediate

amounts of glucose. .

35

Insecticide Pharmacokinetics in Insects

Insecticide pharmacokinetics describe the factors that determine the quantitative relationships between administered dose and toxicant availability at the site of

action in insects (Welling 1977) . This phenomenon involves the process of penetration, internal distribution, metabolism, and excretion of insecticides. During penetration and distribution, some of the toxicant will be taken up by non-target tissues and perhaps become biologically unavailable. At any time during penetration and distribution, there may be opportunities for both enzymatic and non-enzymatic chemical conversions leading to more or less toxic molecules. The subsequent distribution of the insecticide or its metabolic products will also depend on its ability to cross semipermeable membranes separating the various biophases within the organism

(Brooks 1976)

Insecticide Uptake

In insects, the most generally accepted theory

describing how a contact insecticide reaches its target

site is penetration through the cuticle and into the

hemolymph where it is rapidly transported throughout the

insect body to the target organ (Matsumura 1985) . However, . .

36

Gerolt (1969) suggests that topically applied insecticide moves laterally through the integument and enters the target via the tracheal system. Regardless of which theory is correct, the fact that many insecticide resistant insect populations exhibit slower insecticide penetration across the cuticle and into the hemolymph support the importance of the integument in the dispersal of insecticide in insects

Cuticular Penetration

At least three factors play an important role in determining the amount of insecticide penetrating the insect body: lipid solubility, affinity for the cuticular components, and solubility in haemolymph (Matsumura 1985)

The eventual effect exerted by a certain quantity of toxic compound depends on the partitioning of the insecticide in

the insect (Sato 1992), its intrinsic reactivity with the

target, and its concentration as a function of time at the

target site. Moreover, the addition of various carriers to

insecticide solutions causes changes in penetration rate

(Welling 1977) . In some cases, insecticide penetration may

be inadvertently altered by such additions. An important

example of altered penetration rate is Icnown as quasi-

synergism, where the addition of some chemicals may

facilitate insecticide penetration, while the . .

37 detoxification rate remains unchanged. In this particular case, penetration may be more important than inhibition of detoxification of the insecticide (Sun and Johnson 1972)

synergism of Kennaugh et al . (1993) suggested that pyrethroid toxicity by piperonyl butoxide in Helicoverpa armigera Hiibner was not necessarily an indication of resistance due to increased detoxification by cytochrome

P450. They found that although the insecticide resistant population showed 20 -fold resistance toward pyrethroids and resistance was eliminated by piperonyl butoxide, no evidence for increased permethrin detoxification in the

insecticide resistant strain could be found. In this particular case, cytochrome P450 could be involved in the process of penetration of the insecticide through the

cuticle

Translocation

Once the insecticide gets inside the insect, it is

translocated to the tissue responsible for its degradation.

Various body tissues have been involved in xenobiotic

metabolism, such as fat body, gut, muscle, cuticle, and

haemolymph (Price and Ruhr 1969) . Cytochrome P450

monooxygenases are usually found in the endoplasmic

reticulum of numerous cell types. These enzymes have been 38 also found in some mitochondria where they are specialized

for the metabolism of steroid hormones (Hodgson 1983) . In

German cockroaches, cytochrome P450 monooxygenases have been demonstrated in the midgut, fat body, and Malpighian tubules of several different developmental stages (Valles

1998) with the midgut and fat body being generally the site of greatest activity. Comparatively, the Malphighian tubules exhibit the highest level of microsmal oxidation in

the American cockroach (Nakatsugawa and Dahm 1962) .

For other insects the tissues involved in metabolism are very similar. In houseflies these oxidative enzymes are

more active in the midgut (Tsukamoto and Casida 1967) . In

the European corn borer the greatest metabolic activity for methomyl is located in fat body tissues (Kuhr and Hesseney

methyl parathion 1977). Gunning et al . (1994) reported that

is equally distributed in different body tissues after 1.5

hours. After 4 hr the insecticide was concentrated in

excreta. Overall, the distribution in fat was very low over

the time. The most important esterase activity in German

cockroaches was located in the soluble fraction, and a

small percentage was located in the endoplasmic reticulum

(Scharf et al . 1997b). Prabhakaran and Kamble (1994)

reported that a small percentage of esterases are membrane

bound and are released into the soluble fraction. The 39

German cockroach is a major urban pest due to disease transmission and allergy-related health problems in human beings. The widespread and pervasive use of insecticides for cockroach control have resulted in many insecticide resistant German cockroach populations around the world.

For German cockroach the most important insecticide mechanism resistance are MFO, GST, esterases, decreased penetration and target site insensitivity. Physiological- based detoxification in conjunction with decreased penetration is an extremely important resistance mechanism

in German cockroach. Decreased penetration of insecticide,

a secondary resistance mechanism, may be modified by the

addition of additives or synergists. Understanding the way

additives alter penetration may help to better understand

insecticide pharmacokinetics and eventually lead to more

effective insecticide formulations. . , . ,

MATERIALS AND METHODS

Insects

German cockroaches used throughout this study were originally collected from an infested institutional kitchen and cafeteria in Marietta, GA, in 1992 (Valles and Yu,

5001 1996a) . Cockroaches were fed laboratory rodent diet #

(PMI Feeds, St. Louis, MO). The colony was reared as described by Koehler and Patterson (1986b) at 26°C, 50% RH,

and a photoperiod of 12:12 (L:D) h.

Chemicals

Technical grade propoxur and S, S, S- tributyl

phosphorotrithioate (DEF) were purchased from ChemService

(West Chester, PA) Glucose-6-phosphate, glucose-6-

phosphate dehydrogenase, nicotinamide adenine dinucleotide

acid (EDTA) phosphate (NADP) , ethylenediaminetetraacetic

dithiothreitol (DTT) and phenylmethylsulf onylf luoride

(PMSF) were purchased from Sigma (St. Louis, MO). Piperonyl

butoxide (PBO) and 1 -phenyl -2 -thiourea (PTU) were obtained

from Aldrich (Milwaukee, WI)

40 . .

41

[^‘‘C] Propoxur with radiocarbon in the ring position

(specific activity 23.5 mCi mmol'^) was purified using two- dimensional thin-layer chromatography (TLC) plates (silica

gel 60; Merck, Darmstadt, Germany) using hexane : ethyl

acetate : ethanol (35:6:5 by vol) for the first dimension chromatogram. After drying, the plates were developed in the second dimension using benzene: ethyl acetate (6:1 by vol) The plates were exposed to Biomax MR-2 film (Eastman

Kodak, Rochester, NY) for three days at -80°C. Propoxur was scraped and subsequently eluted from the silica gel with acetone. Purity was verified by TLC using propoxur standard

(20 |ig) . N- hydroxymethyl propoxur, o-isopropoxyphenyl carbamate, 2 -hydroxyphenyl N-methyl carbamate, and 2- isopropoxyphenol were supplied by Bayer, Inc. (Kansas City,

MO) All other chemicals were of analytical quality and procured from commercial suppliers.

Insecticide Bioassays

For assessing insecticide toxicity and for developing a general understanding about insecticide resistance mechanisms expressed in the Marietta strain, adult male

German cockroaches (7-14 days old) were anesthetized with

CO 2 and treated topically with propoxur and synergists dissolved in acetone. The propoxur solution (1 ^1) was . .

42 applied to the first abdominal sternite. At least five solutions of different concentrations that caused > 0 % and

<100% mortality were used. At least three replications containing 10 cockroaches per dose were conducted. PBO

(100 |ig per cockroach) or DBF (30 |ig per cockroach) was

applied to the first abdominal sternite 1 h prior to propoxur application as described by Atkinson et al

were (1991) . The synergist dosages used in the experiments

the most commonly reported for German cockroaches when

insecticide resistance mechanisms are studied; no mortality

was observed when cockroaches were treated at these

synergist concentrations. Mortality was recorded 24 h after

treatment and cockroaches were considered dead if unable to

right themselves in 15 seconds after being turned onto

their dorsum. LD 50 values were determined by probit analysis

using mortality as the dependant variable (Finney 1971)

To assess the effect of synergist pretreatment on the

ability of propoxur to knockdown cockroaches, time

bioassays were conducted. Cockroaches were treated with DBF

or PBO as described for topical bioassays. One hour after

synergist treatment, 8.25 |o,g of propoxur (LD 90 ) was applied

to each cockroach on the first abdominal sternite. Insects

unable to right themselves after being turned onto their

dorsum were considered knocked down. Knockdown was assessed 43

at 5 -min intervals. LT 50 values were determined by probit analysis using knockdown time as the dependant variable

(Finney 1971) .

Ppnptration Studies and In Vivo Metabolism Experiments

Propoxur penetration studies were assessed to determine how synergists might affect the rate of propoxur penetration. Insecticide penetration was determined by a

Cockroaches method modified from Argentine et al . (1994).

were treated topically with radioactive propoxur for

varying periods of time. To assess penetration, propoxur

and its metabolites were quantified from the surface of the

excreta (excreted) and cockroach (external) , in the

continued within the cockroach (internal) . A sublethal dose

of [^^C] propoxur (10,000 dpm, 0.04 |ag) was applied to the

first abdominal sternite of adult males (7-14 days old) in

1 |il of acetone. The cockroaches were dried briefly with

gentle fanning, then placed individually into 20 ml glass

scintillation vials for varying periods of time (15, 30, 60

two and 120 min) . After the specified period of time,

treated cockroaches were placed together into 5 ml of

acetone and swirled gently for 15 seconds. This extraction

process was repeated and the acetone extractions were

combined. For the 0 time point, treated cockroaches were 44

extracted immediately after insecticide application by placement directly into a scintillation vial containing 5 ml of acetone.

For metabolite identification and quantification, an

aliquot of 2.5 ml of the extraction solution was

transferred to a 15 ml conical centrifuge tube and taken to

dryness under a gently stream of nitrogen. The sample was

resuspended in 100 pi of acetone, vortexed for a few

seconds, and spotted onto a fluorescent 5 x 20-cm silica

gel TLC plate (250 pm, Merck, Darmstandt, Germany) . The

tubes were rinsed three additional times with 100 pi of

acetone, which was also spotted onto the TLC plate. N-

hydroxymethyl propoxur, o- isopropoxyphenyl carbamate, 2-

hydroxyphenyl N-methyl carbamate, and 2-isopropoxyphenol

(40 pg) were spotted along with the extracts for metabolite

verification by short wave UV light. The plates were

developed twice in hexane: ethyl acetate : ethanol (35:6:5 by

. vol) as described by Shrivastava et al . (1969) After

drying (4 h) the plates were exposed to Biomax MR-2 film

(Eastman Kodak, Rochester, NY) for three days at -80°C.

Identification of metabolites and propoxur were made by

comparing the positions of unknown autoradiographic bands

with known metabolites under UV light (254 nm) . Propoxur .

45 was quantified by scraping the zone of radioactivity from the plate and counting by liquid scintillation spectrometry. All metabolites were also quantified by scraping the entire TLC plate (less the propoxur) and counting. The total radioactivity was determined by summing the radioactivity present on the TLC plate and in the remaining extract. The liquid scintillation counter was a

Packard Model 4530 (Downers Grove, IL) with 95% efficiency

for ^'^C. Metabolism was expressed as a percentage of the total recovery of radioisotope. A quench curve was established with an extended series of quenched samples.

Internalized propoxur was determined by homogenizing

the solvent-rinsed cockroaches with a motor-driven Teflon pestle and glass mortar in 4 ml of acetone. The homogenate was then centrifuged at 500g for 3 min. The supernatant was

removed by serological pipette and placed into a 20 ml

scintillation vial. The extraction procedure was repeated

two additional times and the 12 ml supernatants combined.

Metabolism was quantified as described for the external

wash

Production of metabolites from cockroach excreta was

determined by adding 8 ml of acetone to the holding vials

and spotting an aliquot of the extract on TLC plates.

Metabolism was quantified as described for the external . .

46 wash. Three replicates of each time interval were conducted

Decreased insecticide penetration may be due to the presence of P-glycoprotein in the integument of resistant

insects as reported previously (banning et al . 1996a,

1996b) . P-glycoproteins are transport proteins that can be

inhibited by compounds such as quinidine. To assess the possible effect of P-glycoproteins on insecticide penetration in the Marietta German cockroach strain, 1 |il of

quinidine (0.01 mg/ml acetone) was applied topically to 20

adult cockroaches on the first abdominal sternite (banning

et al . 1996a) . This treatment was repeated every 12 hours

for 48 hours (4 total applications) . After the last

quinidine application, and after CO 2 recovery, six

cockroaches were topically treated with 1 |^1 of

[^^C] propoxur (10,000 dpm, 0.01 pg) on the abdominal

sternite. Penetration rate was compared with an acetone

treated control by determining the radioactivity washed

from the external surface, extracted from inside the

cockroach, and in the excreta. Three time points were used:

0, 30 and 120 min using the procedure described previously

for insecticide penetration. The control was treated with

acetone .

47

Metabolic Studies

The objective of the in vitro metabolic studies in the

Marietta German cockroach was to determine which enzymes and tissues were responsible for propoxur metabolism.

Additionally, PBO and DBF were used to inhibit oxidative

and hydrolytic enzyme systems, respectively. For metabolic

studies, enzymes present in the microsomal fraction and the

105,000Max supernatant were evaluated.

Preparation of Microsomes

For microsomes preparation, adult males were

decapitated, then cut with scissors to remove the last

three abdominal segments. The entire alimentary canal was

gently removed from the posterior opening and placed into

iced-cold 1.15% KCl . The contents of the foregut and midgut

were removed by teasing open the tissues longitudinally

with fine forceps to minimize detoxification enzyme

inhibition by liberated digestive proteins (Valles and Yu

1996b) . The tissues were recombined (less the head) and

homogenized for 30 seconds in a protected buffer (0.1 M

sodium phosphate, pH 7.5, containing 10% glycerol, 0 . 1 mM

DTT, 1 mM EDTA, 1 mM PMSF, and 1 mM PTU) using a motor-

driven Teflon pestle and glass mortar (Siegfried and Scott

1992) .

48

The homogenate was filtered through two layers of cheesecloth, then centrifuged at 10,000gMax- The supernatant was filtered through glass wool and further centrifuged at

105,000gMax for 1 h. The resulting pellet (microsomes) was rinsed and suspended in 0.1 M sodium phosphate buffer, pH

7.5. The above procedures were performed at 0-4°C. Protein determinations were made by the method of Bradford (1976) using bovine serum albumin as the standard. All enzyme assays were conducted at least three times. For determining tissue-specific activity, microsomes derived from the alimentary canal (foregut, midgut, hindgut, and Malpighian

and carcass were obtained as described tubules) , fat body, before

Partial Purification of GSTs

Preparation of glutathione S- transferases (GSTs) was accomplished with an affinity column (Pharmacia,

Pascataway, NJ) . Three hundred cockroaches (with the head removed) were homogenized in sodium phosphate buffer (SPB)

(0.1 M, pH 6.5) and soluble fraction prepared by differential centrifugation, as described previously, with the exception of not using protected buffer during tissue preparation. The 105,000gMax supernatant was used as source of GSTs. The soluble fraction was filtered through a 0.45 pm . .

49 filter and then partially purified using a prepacked glutathione sepharose 4B affinity column (Pharmacia

Pascataway, NJ) by following the manufacturer's instructions. The column was washed with 10 ml of phosphate

buffered saline (PBS) (150 mN NaCl, 16 mM Na 2 HP04 , 4 mM

P and then equilibrated with 6 ml PBS + 1 % NaH2 04 , pH 7.3)

Triton X-100. Aliquots (40 ml, approximately 100 mg) of the soluble fraction were loaded onto the column and washed with 20 ml of PBS. The bound material was eluted with 15 ml of 5 mM glutathione in 50 mM Tris-HCl, pH 8.0, and collected in 1.5 ml fractions.

Glutathione S-transf erase activity was measured with

4 l-chloro-2 , -dinitrobenzene (CDNB) as substrate using the method of Habig and Jakoby (1981) as modified by Yu (1982)

The 3 -ml reaction mixture contained 1 ml of 15 mM glutathione, and 100 |al of GST source in 1.9 ml of 0.1 M

sodium phosphate buffer (pH 6.5) Reference samples were devoid of an enzyme source. Samples were shaken briefly and

then incubated for three minutes at 25°C, then 2 0 |il of CDNB

(150 mM in methyl cellosolve) was added and mixed. The

change in absorbance at 340 nm was measured for two minutes

on a Varian Model 3 Bio uv/vis spectrophotometer equipped with a diffuse reflectance accessory. Enzyme activity was

expressed as nmol CDNB conjugated per min per mg protein 50 using the molar extinction coefficient of 9.6 mM-1 cm-1 for

the product, S- (2 , 4-dinitrophenyl) glutathione.

In Vitro Oxidative Metabolism of Propoxur

In vitro metabolism of propoxur was studied using a

method described by Valles et al . (2000) using microsomal enzymes derived from the alimentary canal (foregut, midgut, hindgut, and Malpighian tubules), fat body, carcass and whole body (excluding the head) of adult males (7-14 days old) of the insecticide-resistant Marietta strain of German cockroach. The 2 -ml incubation mixture contained 1 mg of microsomal protein suspended in 0.1 M sodium phosphate buffer, pH 7.5, and 0.04 ml methyl cellosolve containing

0.128 i^g [^^C] propoxur (32,000 dpm) . The reaction mixture was fortified with an NADPH-generating system composed of

1.8 pmol NADP, 1.8 |imol glucose-6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase. When DBF was used, 20 ^il of an acetone solution was first added to a reaction vessel and allowed to dry. Reactions were initiated by the

addition of the enzyme source. Duplicate incubations were

carried out at 3 0°C in a water bath with shaking for 2 hr.

The reaction was terminated by the addition of 0.2 ml of 4

M HCl followed by plunging the reaction vessel in ice. For

the control, the enzyme source was boiled in a water bath . .

51 for 15 min before use. When microsomes derived from specific tissue sources (alimentary canal, fat body, and carcass) were evaluated, 0.25 mg of microsomal protein was used for each assay. Reactions were repeated in the presence and absence of an NADPH source to verify oxidation by microsomal monooxygenases

Propoxur and its metabolites were extracted by adding

4 ml of diethyl ether to the incubate and shaking for 20 min on a rotary mixer. The shaken incubate was centrifuged for 3 min at 500g and the ether phase was removed by pipette. The extraction process was repeated three additional times. The combined ether extract was taken to dryness under a gentle stream of nitrogen and resuspended

in 100 |j,l of acetone. The entire extract was spotted onto a

5 X 20 cm TLC plate along with known propoxur metabolites.

The plates were developed twice in hexane: ethyl

acetate : ethanol (35:6:5 by vol) as described previously

(Shrivastava et al . 1969, Valles et al . 1996b). The plates were allowed to dry for 24 h at which time they were exposed to Biomax MR-2 film (Eastman Kodak, Rochester, NY) for ten days at -80°C. Metabolite determinations were made by comparing the positions of unknown autoradiographic bands with known metabolites under UV light (254 nm) The zone of radioactivity corresponding to the metabolites and :

52 propoxur were scraped separately from the plates and counted in a liquid scintillation counter. In all instances boiled microsomal tissue was used as a blank to correct for nonenzymatic propoxur degradation.

Propoxur metabolism was quantified according to the following formula:

MTcorrected ~ I'^Tact ive MTboiled

where

MTcorrected = Corrected radioactivity from metabolites

(dpm)

MTactive = Metabolite radioactivity from active enzyme

source (dpm)

MTboiied = Metabolite radioactivity from boiled enzyme

source (dpm)

Propoxur metabolism was expressed as pmol/2hr/mg protein. Total recovery of radioactivity was determined by

summing all radioactivity from the TLC plates and from the aqueous fraction.

To determine if propoxur metabolism by NADPH fortified microsomes was catalyzed by cytochrome P450 monooxygenases,

an experiment was performed using increasing concentrations

of PBO and microsomes as enzyme source. The 2 ml reaction vessel contained 0.3 ml of NADPH generating system, 20 ^il of methyl cellosolve containing 0.08 pg of [^^C] propoxur 53

mg of protein and |il of PBO in methyl (20,000 dpm) , 0.5 20 cellosolve. Final concentrations of PBO in the incubation

tubes were 10"^, 10'^, and 10'® M. Two replicates were

conducted for each PBO concentration. Samples were

extracted, taken to dryness, spotted onto TLC plates,

developed, counted, and quantified as described.

In Vitro Hydrolytic Metabolism of Propoxur

The 105,000grMax supernatant (soluble fraction) and pellet (microsomes) were used as sources of hydrolytic

enzymes. Enzyme preparation was accomplished as described

under the in vitro oxidative metabolism section with the

exception of not using protected buffer during tissue

preparation. To evaluate propoxur metabolism catalyzed by

the microsomal and soluble esterase-containing fractions,

the 2 -ml incubation mixture contained 1 mg of microsomal or

soluble fraction protein suspended in 0.1 M sodium

phosphate buffer, pH 7.5, and 0.04 ml methyl cellosolve

containing 0.128 |^g [^^C] propoxur (32,000 dpm). Duplicate

incubations were carried out at 30°C in a water bath with

shaking for 2 hr. The reaction was terminated by the

addition of 0.2 ml of 4 M HCl followed by plunging the

reaction vessel in ice. For the control, the enzyme source

was boiled in a water bath for 15 min before use. ,

54

Extraction and metabolite determination were accomplished as described under oxidative metabolism.

In Vitro Conjugation of Propoxur by Glutathione S-

Transf erases

To assess the possible contribution of glutathione conjugation as a component of propoxur metabolism, GSTs were partially purified by affinity chromatography and evaluated in vitro against [^^C] propoxur hydroxymethyl propoxur, [^^C] 2 -hydroxyphenyl N-methyl carbamate, and [^^C] o-isopropoxyphenyl carbamate. A total volume of 2 ml containing 1 unit of enzyme source (1 unit catalyzed the conjugation of 10,000 nmol CDNB per minute),

1 ml of 15 mM glutathione, 0.04 ml of methyl cellosolve

containing 0.1 |^g [^'^C] propoxur (25,000 dpm) , in 0.1 M sodium phosphate buffer, pH 6.5. Incubations were carried out at 30°C in a water bath with shaking for 2 hr. The reaction was terminated by the addition of 4 ml of diethyl ether. The extraction of propoxur and its metabolites was performed as described for oxidative metabolism.

Electrophoresis

Native polyacrylamide gel electrophoresis (PAGE) was conducted on a mini trans blot cell (Bio Rad) using a 5% stacking gel (0.75 mm thick) and 7.5% separating gel (non- .

55 denaturing) to assess the purity of the affinity purified

GSTs (Bollag and Edelstein 1991) . The GST sample (0.88 mg ml) was diluted 1:3, 1:5, 1:10, and 1:20 using 0.1 M sodium phosphate, pH 6.5. Samples were further diluted 1:3 with native sample buffer (312.5 mM Tris-HCl, pH 6.8, 50% glycerol, 0.05 bromophenol blue, 10% sucrose) and 2 0 p,! was loaded into each well. Separation was conducted with electrophoresis buffer (25 mM Tris, 192 mM glycine, pH 8.8) at 50V for 15 minutes, then 200V until the marker dye ran within 1 cm of the gel base. All the experiments were performed at 0-4°C.

After electrophoresis, gels were rinsed with deionized water and then placed in a container with 50 ml of staining buffer capable of detecting glutathione S- transferase activity (0.1 M sodium phosphate buffer, pH 8.0 containing

4 . 5 mM GSH, 1 mM CDNB, and 1 mM nitroblue tetrazolium

[NBT] ) . Incubations were performed at room temperature under gentle agitation. After 10 min, gels were washed with deionized water and incubated at room temperature for 10 min in 50 ml of 0.1 M tris-HCl buffer, pH 9.6, containing 3

mM phenazine methosulfate (PMS) (Ricci et al . 1984) . The gel was dried on Promega (Madison, WI) drying film. Blue insoluble formazan appears on the gel surface, except in the glutathione transferase area (negative staining) 56

Acetylcholinesterase Inhibition

Based on the bioassay results with DBF, hydrolysis of propoxur was implicated as a major detoxification route in the Marietta cockroach strain. However, in vitro metabolic data showed that very little esterase-based detoxification occurred. In fact, oxidation of propoxur catalyzed by cytochrome P450 was the principal route of metabolism in vitro. A plausible hypothesis for this apparent discrepancy was that the greater synergism observed with DBF pretreatment may have occurred as a result of enhanced inhibition of acetylcholinesterase in the presence of DBF

(an organophosphorus pesticide) . To test this hypothesis comparative experiments were conducted in which in vitro acetylcholinesterase activity was inhibited with propoxur and a mixture of propoxur and DBF. To examine the possible synergistic effect of DBF on acetylcholinesterase

inhibition, the I 50 for propoxur was used in the presence of increasing DBF concentrations. Acetylcholinesterase inhibition was accomplished using the method of Bllman et

al . (1961) with slight modifications. Thirty adult males were decapitated and the heads were homogenized for 30 seconds in 10 ml of ice cold 0.1 M sodium phosphate buffer, pH 8.0, using a motor-driven Teflon pestle and glass mortar. The homogenate was filtered through two layers of , 1 . ,

57 cheesecloth. The filtrate was centrifugated for 15 min at l,000g. The supernatant was filtered through glass wool and this filtrate was used as the enzyme source. The 3.2 ml reaction contained 2.7 ml of buffer (0.1 M sodium

phosphate, pH 8.0), 0 . ml of dithiobisnitrobenzoic acid

(0.1 M) 0.1 ml of acetylthiocholine iodide (0.03 M) and

0.3 ml of enzyme source was added to each tube to start the reaction. When insecticide inhibitors were used, a 0.3 ml solution of the insecticide (propoxur or propoxur + DBF) was added to 0.6 ml of enzyme source and incubated at room temperature for 10 min. Immediately after incubation, 0.3 ml of the inhibitor/enzyme mixture was added to the enzyme reagents and analyzed. Enzyme progression was followed by the production of 5-thio-2-nitrobenzoic acid and analyzed at 412 nm for 2 min. The initial concentration of inhibitor was prepared in methyl cellosolve and then serially diluted with buffer (0.1 M sodium phosphate buffer, pH 8.0) A complete reaction mixture, less acethylthiocholine iodide, was used as reference for correcting nonenzymatic hydrolysis. Three replications were conducted for propoxur and DBF inhibition.

Acetylcholinesterase activity was calculated using a molar extinction coefficient of 13.6 mM'^ cm'^ for 5-thio-2- nitrobenzoic acid. I 50 (the molar concentration of inhibitor 58 causing a 50% inhibition of enzyme activity) was calculated by plotting percentage of inhibition versus log inhibitor concentration (O'Brien, 1976). Three replicates were conducted.

Propoxur Sequestration

Another possible hypothesis accounting for the discrepancy between the in vivo synergism of propoxur by

DEF, and no hydrolytic metabolism in vitro is sequestration. Specifically, if esterases in the cockroach bind to propoxur and prevent it from reaching the target site, then DEF could tie up these esterases, effectively resulting in synergism by virtue of mass action.

Sequestration has been reported as a important mechanisms of resistance in German cockroaches (Prabhakaran and Kamble

1995, Scharf et al . 1997b) To evaluate the hypothesis of propoxur sequestration by enzymes, soluble and microsomal fractions were prepared by differential centrifugation and used as the sources of possible sequestering proteins.

Propoxur (5,0 00 dpm, 0.005 |ig) was added to an incubation tube and taken to dryness, 0.25 mg of microsomal or soluble fraction protein was added in 750 pi of sodium phosphate buffer (SPB, 0.1 M, pH 6.5) and incubated for 15 minutes at room temperature. An aliquot of this sample (500 pi) was . .

59

loaded onto a HiTrap desalting gel filtration column (5 ml;

Pharmacia Pascataway, NJ) . The bound sample was eluted with

6 ml of 0.1 M sodium phosphate buffer, pH 7.5. Fractions

(0.5 ml) were collected in 20 ml scintillation vials. An aliquot was taken from these samples and protein content was determined by the method of Bradford (1976)

Radioactivity was quantified in the remaining portion of each fraction by liquid scintillation spectrometry (Packard

Model 4530; Downers Grove, IL) . The rationale was that if propoxur bound the enzymes, then a large portion of the radioactivity would elute from the column with the protein fractions (first off the column)

Statistical Analysis

Insecticide bioassay data were analyzed by probit

analysis (Finney 1971) . Significant differences for median lethal data were determined by nonoverlapping fiducial limits. Data obtained from different treatments were analyzed by Students 's t-test or by analysis of variance

(ANOVA) followed by Duncan's test when appropriate (SAS

Institute 1990) . RESULTS

Topical Bioassavs

Table 1 summarizes the results obtained from studies of insecticide toxicity of propoxur and synergists on adult

male Marietta cockroaches. The LD 50 obtained by topical bioassay for propoxur was significantly different compared with cockroaches treated with synergist + insecticide.

Pretreatment with the cytochrome P450 monooxygenase inhibitor, PBO, or the esterase inhibitor, DEF, increased

propoxur toxicity by 2 and 6 . 8 -fold, respectively. Partial suppression of resistance levels by PBO and DEF suggested the involvement of cytochrome P450 monooxygenases and hydrolytic enzymes, respectively. Apparently hydrolysis is the most important resistance mechanism as greater synergism occurred with DEF. However, it has been reported that propoxur is detoxified principally in the German cockroach by microsomal monooxygenases with hydrolases playing a minor role in the detoxification process

(Siegfried and Scott 1991) . Resistance to propoxur was not completely eliminated by the addition of either synergist

60 . .

61 suggesting that additional mechanisms may be involved in propoxur resistance.

Table 2 shows the effect of synergist pretreatment on the ability of propoxur to knockdown Marietta German

cockroaches. Determination of LT 50 showed that the addition of DBF killed cockroaches more quickly, compared with propoxur alone and propoxur + PBO. Interestingly, no synergism occurred with PBO treatment.

Penetration Studies and In Vivo Metabolism Experiments

The effects of PBO and DBF on the penetration rate and metabolism of propoxur were assessed. Propoxur treatment alone was used as a control for comparison and total recovery was compared with the zero time point (i.e.

immediately after treatment)

Radiolabel recovery is summarized in Table 3. Recovery declined over time in all treatments and was the same for propoxur and synergist treatments 15 and 30 minutes after

treatment. However, significantly more propoxur was

recovered from DBF and PBO treated cockroaches at 60 and

120 minutes when compared with cockroaches treated with propoxur alone

Table 4 summarizes the recovery of [^^C] propoxur from

external washes, from inside the cockroaches and in the 62

excreta over a 2 h time period. Recovery of external

[^^C] propoxur from cockroaches treated with propoxur and propoxur + DBF or PBO declined progressively with time.

After 2 hours only 24.3% was recovered from external washes of cockroaches treated with propoxur alone. Conversely, recovery in PBO and DBF-treated cockroaches was significantly greater compared with those not treated with a synergist, regardless of the time after treatment. This data indicated that PBO and DBF retarded propoxur penetration into the German cockroach. Indeed, this notion is supported by decreased quantities of parent propoxur within the cockroaches at all time points when compared

with non- synergist treated cockroaches (Table 4) . As anticipated, the recovery of propoxur was initially low within the cockroaches for all treatments and increased throughout the 2 h treatment period. Propoxur recovery was

significantly lower among synergist treated cockroaches at all time points, compared with the control. This difference was obviously due to low propoxur penetration when the

insects were pretreated with synergists. Interestingly, despite reduced insecticide penetration caused by PBO and

DBF, these synergists still caused a significant increase

in propoxur toxicity in bioassay experiments, indicating

that they were effectively penetrating the cuticle. In .

63 fact, verification that the synergists were inhibiting detoxification is evidenced by a significantly lower quantity of propoxur metabolites inside the cockroaches

(Table 5) . These data further support the notion that PBO and DBF pretreatment decrease the rate of propoxur penetration into German cockroaches.

In the absence of PBO or DBF, propoxur penetration was very rapid. Greater than 75% of the applied dose had penetrated the cuticle within 2 hours (based on external

cuticular washes) . However, when the cockroaches were pretreated with DBF or PBO, the rate of propoxur penetration was reduced significantly. Among PBO and DBF- pretreated cockroaches, only 28 and 32% of the applied propoxur dose penetrated the cuticle after 2 hours, respectively (based on external washes)

Regarding metabolite production in vivo, metabolites were quantified in external rinses, within the cockroach, and in the excreta. There were no significant differences in metabolite quantities from external rinses in all

treatments and at all time points (Table 5) . Less than 1% of the applied amount was recovered as metabolites in external washes among all treatments.

Internal metabolite determination showed a very different situation. At 15 minutes the quantity of propoxur .

64 metabolites was low for all treatments. However, after two hours of incubation, propoxur metabolite recovery in the absence of synergists, was 21.7% of the applied dose (Table

5) . The addition of PBO or DBF decreased the production of metabolites to less than 6%. Metabolite production among all treatments increased through time. These results showed that the addition of PBO or DBF significantly slowed propoxur detoxification in the German cockroach; 91.9% of the total internalized propoxur was recovered as metabolites in the control two hr after treatment, while only 42.5% and 43.2% were recovered as metabolites in the

PBO and DBF treatments, respectively.

The appearance of absorbed radioactivity in excreta

was very low among all treatments (Table 5) . There were no differences among all treatments and time points, except for the propoxur + DBF treatment at 60 minutes, perhaps due to experimental error. The amount of metabolites recovered was less than 4% in all cases. Metabolite recovery in the excreta was consistently lower among synergist treatments compared with the control .

65

In Vitro Propoxur Metabolism

In vitro metabolic studies were performed in the

Marietta German cockroach to determine which enzymes and tissues were responsible for propoxur metabolism. In a first set of in vitro experiments, propoxur metabolism was determined using the whole insect as a tissue source. For subsequent experiments the tissue sources were the alimentary canal (comprised of the foregut, midgut,

hindgut, and Malpighian tubules) , fat body and carcass

(integument, muscle, nervous tissue).

Table 6 summarizes the metabolism of propoxur by microsomes of adult male German cockroaches. When

[^^C] propoxur was incubated with microsomal proteins

with fortified NADPH (monooxygenases) , fat body-derived microsomes metabolized propoxur at the highest rate, followed by the whole insect- and carcass-derived microsomes. The lowest detoxification rate occurred in microsomes prepared from the alimentary canal

The major metabolites produced were i'?- hydroxymethyl propoxur, 2 -hydroxyphenyl N-methyl carbamate, and o-

isopropoxyphenyl carbamate (Figs. 1 and 2) . In some experiments, additional unknown metabolites were produced in smaller quantities. Formation of metabolites was NADPH-

dependant ; no significant metabolism was detected with .

66 microsomal enzymes lacking NADPH. Additionally, PBO inhibited propoxur metabolism in a dose dependant fashion

monooxygenases as (Table 7) , implicating cytochrome P450 being responsible for the metabolism. 2-Isopropoxy phenol was produced with denatured (boiled) microsomal preparations, active microsomal preparations without added

NADPH and a complete incubation mixture without microsomes indicating that it was a non-enzymatic product.

Representative autoradiograms of [^'*C] propoxur and metabolites following incubation with either resuspended microsomes fortified with NADPH or NADPH + DBF are shown in

Figs. 1 and 2. Tests performed with glucose-6-phosphate dehydrogenase before and after experiments with microsomes showed that the NADPH generating system worked throughout the 2 h incubation time (Fig. 3)

The addition of DBF (10'^ or 10'^ M) did not significantly decrease the metabolite production in fat

body and carcass-derived microsomes (Table 6) . However, a significant difference was observed in metabolism using whole insect -derived microsomes, even at the lowest DBF concentration (10'^ M) which decreased the quantity of metabolites produced by half. Metabolism by alimentary canal-derived microsomes was inhibited only at 10*^ M DBF.

Overall, there was a clear indication that the addition of . . .

67

DEF at 10"^ M decreased propoxur metabolism. These results support the hypothesis that when applied at high

(>10"* inhibit to some degree, concentrations M) , DEF may microsomal monooxygenases (Scott 1991)

[^^C] Propoxur metabolism by soluble enzyme (cytosol) preparations or microsomal preparations (in the absence of

NADPH) were not significantly different from boiled blanks.

These results indicated that propoxur hydrolysis was not an

important detoxification route in this strain (Table 6)

Furthermore, despite negligible in vitro hydrolytic

metabolism of propoxur, DEF treatment in the bioassays

nonetheless caused an apparent increase in propoxur

toxicity. This apparent discrepancy may possibly be

explained by enhanced inhibition of acetylcholinesterase in

the presence of DEF. In order to test this hypothesis,

acetylcholinesterase activity in the German cockroach was

10'’ inhibited by 5 x M propoxur (I 50 ; Table 8) and then

subsequently treated with increasing concentrations of DEF

to examine possible effects. The results showed that DEF

did not affect the acetylcholinesterase activity at any

concentration tested (Table 9)

To assess the possible contribution of glutathione

conjugation as a component of propoxur metabolism,

glutathione S- transferases were partially purified and 68 later visualized on a Native PAGE gel by using Ricci's

(1984) technique (Fig. 4) . The partially purified enzymes were incubated in vitro with propoxur and propoxur microsomal metabolites (W- hydroxymethyl propoxur, 2- hydroxyphenyl N-methyl carbamate, and o-isopropoxyphenyl

carbamate) . No conjugates were produced after a 2 h incubation. Lack of propoxur metabolism by the soluble fraction and glutathione S-transf erases suggested no involvement of these enzymes in propoxur metabolism in the

Marietta strain.

Propoxur Sequestration

Sequestration experiments were performed in an attempt to explain the discrepancy between the in vivo synergism of propoxur by DEF and yet no hydrolytic metabolism in vitro.

Assays performed with the HiTrap desalting gel filtration column indicated that propoxur sequestration did not occur by proteins present in the soluble and microsomal fractions

(Figs. 5 and 6) . When microsomal and soluble fraction preparations were incubated with [^“^C] propoxur for 2 h, the majority of the propoxur eluted after the proteins

indicating that excessive binding was not occurring. Almost all proteins eluted in the first four 0.5 ml collected

samples, while propoxur eluted later. These results 69 suggested that propoxur sequestration by proteins present in soluble and/or microsomal fractions was not occurring.

Effect of Ouinidine on Propoxur Penetration

Low propoxur penetration rate did not change when quinidine was added, indicating that P-glycoprotein may not be involved in propoxur cuticular penetration in this

strain (Fig. 7) . Other mechanisms may be changing the penetration rate of propoxur through the cockroach cuticle when in the presence of PBO or DEF. 1' 1

70

German

1 cn 'Xt

J3 1 0 • • 04 \ to CO 1 CN X) 0 0 0 in CO CN q H rH rH synergists

r- if) 00 + iH rH rH •

Cn

propoxur 0) cn U) a iH CT» CN • ro CQ ro CN 0 • 0 + and P d CN cn r-- CN b o\o X in rH 0 0 o —cn •«w' d propoxur 0 cr» ro IT) S CO CN d P • • P rH 0 0 p p ro CD applied ro LO b w • X CO 0 0 0 o d +1 o +1 +1 +1 QJ d d ro r- 0 cn • 1— topically Q CO CN ro rH P males. O •H of 0 d 4J (T5 adult m w 50 d p 4-) 4J LD + + u m p 0) -H 1. CQ d U CQ cn H ;:3 d d c cn X X X -H 0) cockroach q 0 0 0 \ a (D a d d cn >i Table d 0 0 0 u d d a cn CO d d oJ X) '

71

0 XI cn KO Oi • • German CO 1 0 rH 0 0 0 0 0 rH to q CN CN rH

synergists LO C' cn KD

• • • tH D- cn

,— + CN CN 4J CO • • rH 00 m cn CN • rH (N cn 10 propoxur rH '— cn (U P C ft >1 LO • m o\o rH rH 00 LD — •— ««_r- + cn and — rH r- b 0 CN CO • • • Lfl X H r- 00 0 0 P rH rH rH ft 0 propoxur

ft o Q1/1 m LD r- O' applied ft 0 0 0 b CO X o +1 +1 +1 +1 ft o

0 ft ft CD r' 0 0 CN rH o ir> • • • topically p CO CO (O cn p

males.

o of *H 0 Cn 4J adult PQ a b 50 ft Q LT + H- 4J p CQ 2. CQ Sh b CD •H H b b OJ cn cn X X X J-) Q) cockroach q 0 0 0 b (U ft ft 0^ a a Table b 0 0 0 •H I>0 b b Jh e CO CO ft ft Ch 10 XI . 1

72

males

propoxur 4J X! b b to CD CN 0) adult r- I— CO 4J 4-) CN CN rH C b [^'*C] c rH 0)

treatment Td e 01 rH ro (U o (U B 5 CO B - *H +1 +1 +1 *H o 4-) b

CO ' m and O 4J • • • u (0 O CN CN b CD cn cn cn X 0) b topical >1 Q b 01 (propoxur > b b b 0 CN CO CN ^ o tj r- CN — o 4-) U OJ • • LT) after b rH CN n g o o u o\o +1 +1 +1 fC3 O tu 4-1 crc 00 cn V

cn w -Sc. O Ui strain ro 00 cn 00 -b 4J +1 4-) radioactivity H b b b b CD CN 00 to CN n 0) 4-1 0) b cockroach rH rH g OJ ‘W S -H of +1 +1 +1 4-i n

CO O 00 CO (U ^ Ln to to to Cn O r-i CTi cn cn H (C3 b 4-1 b German recovery m u b -H H 44 6 -H b b O ft a hi m ft 4-) -H Total ft Q O CO Marietta dj

QJ + + (0 44 b 3. b b CO m (D b b b (U 6 X X X b tj S4 4-1 the 0 0 0 t—H -H (U

I 4-4 b ft ft ft 05 —I 4-) Table (U 0 0 0 a b b b > o of b b H ft ft ft r) r-| 1 1

73

0)

4J U m rQ 03 b 8b r4 m C-' in . 0 rH (N rH CN rH

-51 -51 51 -51 -51 ± CD Q) 8 CO (X) V£) o . I—I o • • • • • (d CN CN t> 00 CTl 12 B m r4 CN >X) CN QJ 4J :5 M U 03 03 0.3b n3 H b b 4J CD V£) ro rH rH a £ a 0) I— rH CN •H H o 6 QJ ± 4J 6 -51 -51 -51 -51 -51 (t3 •H 55 05 4J CO CTl CN rH • • • • • X S-i O 10.2 0 4J 4J '43 KO LTI CN Oj (0 CO CN 0 S-I 5-1 QJ a 4-) 54 44 u QJ rO > u 03 b 03 u 1.1b 0 (N in O' CQ O • • • • • QJ QJ CN CN m rH rH 4J 6 54 a *H -51 -51 -51 -51 -51 ± 0) 4J o\o 5-1 CN rH rH 00 rH 7.6 n3 CD O • • • • • a sn n 'X) O O CN 00 •H CTl 00 rH CQ nJ 54 m b > b 05 b 03 03 03 u 0.6b 5^ 4J CN 'S' o in VO (U (C > CN CN 00 rH o 0 m u QJ -51 -51 -51 -51 -51 ± QJ b u CT\ 00 (Tl in 5.3 • • • • • u •H 0 4-) u u (T5 a O a a o 0 (0 0) m w 0 CQ DEF 6 b (0 a Q •H a b 54 ct 55 4J -5 -5 -5 (T5 QJ (0 H 03 p5 O Pi N 4-1 54 54 54 -H 54 54 . (C >1 c 1— :5 55 55 rH 55 55 4-) 4J 54 QJ «3 X X X 03 X X 4J O 0 B a 0 0 0 a 0 0 —(U QJ <0 0) 4-) 54 a a a u a a t 1 H 54 0) 03 0) 0 0 0 0) 0 0 b 54 4J 4J QJ 4-) 54 54 54 4J 54 54 rO fO X (fl 54 X a a a a a a s H O 4J H H 1 i

74

•

4J 5-1 (0

-t-l +1 +1 4J CN C O 4-) 0) CN n VO CQ rH O OJ 4-) u 4-1 d O d X X r-\ 'd a 4J (minutes) LO 00 m n3 rH o o 5^ m X! -H -H +1 >1 4J (U •H XI O vf 'S' rH to time VO ro to LD r! S rH -d 0) d u % d m at O 6 (D CQ d X rQ ' o CN VO ro CQ M-l d 4J d rH o rH d u

recovered 0) d -H -H -H E „ d 5 n to VO m sh . , ^ O 0) o ro rH a u -d o\o rH X QJ -- t3 Qj d ° • 0) d d X d o '4' V o 4J

+1 +1 +1 - as

i 1 \ 1 4-J in , w d 4J rH ro CN CO u d d -t-l d X d CQ u 4H QJ d d 4H d d d •rH d O td (U T3 •fH m M 6 4-) ts 04 Q d d d 0) •H r-H 0 d + + d dl 4J u d 4-1 d 4-) d d d rH d . Jd d d d d CQ o 4J d d d X X X d •rH O 0 B 4J 0 0 0 d 4-1 0) d D) 4J 0) a ft ft r— CQ •rH 1— d (U d d 0 0 0 d d d X 4-> 4J 0) O d d d > d cn d X d 5h X CU Ot ft d •fH H O 4J u n! s CQ 1 1 1 1

75

of m to to to h! 0.27b iH rH LT) CO rH O rH CN

O o o CN O males +1 +1 +1 +1 +1 ±

rH rH in O n o O O rH > 00 5.53 CN • • • • • adult CO rH O rH O rH 00 (U CN 4-) b in g (d to to to h! 0.14b •H rH CTi CN cr> B o CN O rH 00

treatment. o O O rH o QJ 6 +1 +1 +1 +1 +1 ± *H metabolites r) r' O cn ID 00 O o ro rH l> ID 3.60 4-) • • • • •

after n3 o o O CN CN rH u (D > (0 to to times O 00 (T\ in to h! 0.22b propoxur u n O CN O 0) • • rH 00 g o o o • • rH o o\o +1 +1 +1 +1 +1 ± [^^C] 00 00 00 varying rH r- ID O m 2.7 ro • • • • • o O o ID rH

as at

to to to

o I— to XI 0.4a rH CM CN 00 CN recovered O O O o O cockroaches ± +1 +1 +1 +1 +1

og ID CN in m m 1.8 T— o• o• O• m• o•

German Radioactivity O ft g O 0) OP ft 0 PQ DBF IQ ft Q H ft T3 g 4J a H + + Id + + fO cm N 4J Sh g Jh H g iH Marietta 5. >1 g 1— b b b t— b b b 4J k 0) Id X X X Id X X X O 0 B g o 0 0 g 0 0 o (d 0) 4J g ft ft ft u ft ft d M 0) n3 0) o 0 0 0) 0 0 o 4J 4J ^H iH J-l Table 4J 4J 0) g g g the X (d g X ft ft ft g ft ft pa 0 4J pa H 1 1 1 I

76

• 0) m s-i C (0 (0 fO fO 0 rH CN CD CT\ CN 0) 0 0 0 4J 4.) +1 +1 +1 (U

in 0 in in rH nj 0 4-1 • • • —. (N C CQ rH rH 4.) n n (U (U CQ 4-1 (U (U 4J 4-1 (0 nJ 43 44 -H m CN CO -H C in U3 CN o £ r) 0 0 0 Oj 4-) dJ e +1 +1 +1 •rl

4-) cn T— 0 -g >, a 0 cn CN 4J JQ (U 4J l£> • • • H m m n CN rH Ti C 4::! (U rO ^4 U 5 (U 0) (0 O g > n3 (t3 m d) I— 0 0 CN rH CQ - CJ in CN 0 q; • • • CQ 44 d 0 0 0 44 to d u o\o +1 +1 +1 (U d d B C d in in -H d Q 0 0 rH d rH n • • • OJ 0 ro CN rH ft u 43 X (U 'O in (U d 0 (T3 fO m CO 0) 00 0 . C T) QJ •H rH 0 c + + d 43 44 u rC d 44 d 4-) U d d H (0 . t>i d d d d CQ u in 4) h to cn m X ta d X ft ft ft QJ -H Eh u 0 44 H fO s CQ 1 1 1 1 1

77

of

microsomes

ied

NADPH-fortif w CO

(0 fd fd -l-l rO b b U3 ro rH • • fd CD o (N • 6 B • • • CN CN CN CQ UD o ro rH in rH b CN by b iH CN +1 +1 +1 X 0 \ +1 +1 +1 0 Xi (— rH rH strain. a b 0 in (X) l> • • • 0 -U B ja • • • ro rH VO S-l 0) a Q ro C^ H P in ro a g V) CN CN S rH rH rH

(pmol/2h/mg)

cockroach

§

ro ro

metabolism German w o o o o o O O O Q rH rH rH rH

Marietta

propoxur

X a the Q [^^C] < 1 + + + 1 H- + + of

vitro

tissues

4J 4-) 4J 4J In CJ CJ U u QJ 0) 0) QJ 6. CQ CQ CQ CQ b b b b l>i *H •H -H •H P P P P 0) 0 0 0 0

different b 0) (D (U 0) b b b b

1 1 Table CQ t— — I— — CQ o 0 0 0 4J 4J 4J 4J H b b b b cO fd fd fd H IS IS IS a a Ph a 1 1 1 1 1 1 1 1

78

CQ Cl o H Q) 4J Cl fO (0 d •H Cl B dJ Cl JJ 4J CQ M 4J QJ QJ CO 1 4J Cl QJ — -H 0 4J 03 n5 05 03 05 4-1 d lO U) rH H r^ l> m O ^ QJ d 0 • • . . . • QJ •H B 6 CQ CQ CO o rH (N M 03 Cl 4J Cl -d rH d QJ 03 d 1— CN 4-1 Cl X 0 \ +1 +1 +1 +1 H-l +1 S --I 4-1 03 0 (— -H ^ •H a 03 CD rH CO in CO ro QJ a 0 Cl ^ X! 0 4-) B n • • • • . . CQ M/m Cl QJ —a Q in rH rH P m rH ro O. 03 a 6 in r- 12: CN rH Q'xi d 4-) >1 (0 QJ •H Xi OJ •> QJ ^ ^ X)-r. QJ Q) CQ Cl s - 5 o d ^ OJ (0 B u O o d 4-1 0 Cl Q W o w B ^ Q U) d Xi rH +1 o ^ U Oin d (U Xi § ^ d O 4.) (0 (U V 4J Q| ICI ^ d m o QJ C4 -fl B 4J 4-) (U cn 03 d 1 -1- -1- -1- 1 + + 1 + (0 QJ QJ 4J Cl Cl 4J QJ 4-1 X! 0) o QJ 5 ,d 4-1

1 1 1 d I— — — — 4J U d 03 03 03 03 03 X3 -H d d d d 4J 0,-g ^X3 03 03 03 03 0) d u U u U 03 d (U H 4J 0 Cl o u >1 >1 03 QJ Cl Cl Cl Cl 4J 4-> 4J • 03 03 03 03 CQ d QJ CD CQ CQ CQ CQ 4J 4J 4J 4.) 0) QJ QJ XJ CQ CQ CQ CQ d d d d B I—* d QJ 03 03 4.) CO 03 03 QJ QJ QJ QJ pH -O 1— U U CJ U B B B B 03 CO (15 S, o XI Cl Cl Cl Cl •H •H •H •H QJ 03 tJI • rH > g 03 Ph 03 03 03 03 rH rH rH rH Cl •H H U u u u < c < < 4J CQ J3 9

79

Table 7. Effect of PBO on propoxur metabolism produced by microsomes prepared from adult German cockroaches of the Marietta strain. The whole body was used as tissue source.

PBO pmol/2hr/mg ± SE Concentration (M)

0 51.4 ± 2.5a^

10-05 50.2 ± 3 . Oa

o O 13 . ± 0.9b

10'°^ 6 . 1 ± 0.6b

^ Values with different letters are significantly different from the control by Students t-test. (P < 0.05). Three replications were conducted. 31 . 61 . 59

80

Table 8. Acetylcholinesterase inhibition by propoxur in Marietta adult males. In vitro enzyme inhibition to determine the I50 value

Propoxur Activity ± SE Cone. (M) (% of control)

Control 100

10'°'^ 1 . X 68 . ± 4.4

-°" 3 . X 10 49.4 ± 3 .

10-06 1 . 1 X 38.4 + 3 .

3.3 X 10-06 32.7 ± 5 8

10-05 1 . 1 X 26 . ± 1.9

^ Propoxur I 50 = 5 x 10 M Specific activity of the control = 21.56 nmol/min/mg .

81

Table 9. Effect of DEF on acetylcholinesterase activity in 10’^ the presence of 5 x M propoxur (I 50 ) in Marietta adult males

Propoxur DEF Specific I50 Concentration activity ± SE (M) (nmol/min/mg protein)

0 0 21 . 56 ± 1.69a

10''^ 10''^ 5 X M 36 . 86 + 0.19b

10''^ 10'® 5 X M 39 . 11 ± 2.25b

10''^ 10'® 5 X M 44.26 ± 8 . 87b

5 X lO"'^ M 10'^ 42.35 ± 3.23b

Values are specific activity of means ± SE from two experiments. Means were not different (P < 0.05) by Duncan's multiple mean separation test. 82

A B

propoxur (Rf=0.47)

*4 o-isopropoxyphenyl carbamate(Rf=0.34)

A^-hydroxymethyl propoxur (Rf=0.31)

2-hydroxyphenyl N-methyl carbamate (Rf=0.29) Unknown A Unknown B Unknown C

Fig. 1. Autoradiogram of TLC separation of [^'^C] propoxur metabolites produced by microsomes of adult males of the Marietta German cockroach fortified with NADPH. A, boil. B, active. 2-isopropoxyphenol (Rf=0.95) 83

A B

propoxur (Rf=0.47)

o-isopropoxyphenyl carbamate (Rf=0.34)

^ hydroxymethyl propoxur (Rf=0.31)

- 2-hydroxyphenyl N-methyl carbamate (Rf=0.29)

Unknown A

Fig. 2. Autoradiogram of TLC separation of [^“^C] propoxur metabolites produced by microsomal fraction of adult males of the Marietta German cockroach fortified with NADPH + DEF. A, boil. B, active. 2 - isopropoxyphenol

(Rf = 0 . 95) . .

84

Fig 3. Ability of G6PDH to convert G6P to 6- phospho-glucono-5- lactone and reduce NADP to NADPH at time 0, and after being incubated in a shaking water bath at 30°C for 2 hr. The experiment showed that G6PDH is capable of producing NADPH throughout the experiment period 85

Fig. 4. Glutathione S- transferase partially purified. Ricci's Method. 86

30.00

25.00 TJ 0)

0> 20.00 > o u o 15.00 3 X o Q. 10.00 O

5.00

0.00

Fraction

Fig. 5. Amount of protein and propoxur obtained in serial fractions from microsomal esterases using the HiTrap desalting gel filtration column in the Marietta strain. 87

35.00

30.00 T3 0) k_ 25.00 O > o u 20.00 0> 3 X 15.00 o Q. o 10.00

5.00

0.00

1 2 3 4 5 6 7 8 9 10 11 12

Fraction

Fig. 6. Amount of protein and propoxur obtained in serial fractions from soluble esterases using the HiTrap desalting gel filtration column in the Marietta strain. 88

Time (min)

Fig. 7 Effect of quinidine on propoxur recovery from cuticle, internal tissues, and excreta from Marietta strain German cockroach. A = Control, B = Quinidine. DISCUSSION

Topical bioassay data from the Marietta strain revealed that the addition of synergists increased the

toxicity of propoxur significantly (Table 1) . The level of propoxur tolerance was reduced 1.8 -fold when cockroaches were pretreated with PBO (a cytochrome monooxygenase inhibitor) and 6.7-fold when pretreated with DBF (an

esterase inhibitor) . These data suggest that both oxidative and hydrolytic enzymes participate in the detoxification of propoxur in vivo in the Marietta strain of German cockroach. Furthermore, hydrolysis appears to be the primary detoxification route of propoxur in the German cockroach; synergism was over 3 -fold greater with DBF compared with PBO.

In vitro metabolic experiments with radiolabeled propoxur were conducted to confirm the bioassay results which indicated that the major detoxification route of propoxur was hydrolytic. Surprisingly, experiments with cytosolic and non-NADPH fortified microsomes (i.e. esterases) showed no propoxur metabolism regardless of the

tissue source (Table 6, Figs. 1 and 2) . However, propoxur

89 .

90 was readily metabolized by microsomal enzymes fortified with NADPH. These data strongly suggest that propoxur is detoxified by cytochrome P450 monooxygenases. Indeed, this notion was confirmed when the experiments were repeated (in vitro metabolism with NADPH fortified microsomes) in the

presence of PBO . Under these conditions, metabolism was reduced in a dose -dependent manner. The highest concentration of PBO (10'^ M) reduced the in vitro propoxur metabolic rate by 89% (Table 7)

Interestingly, cytosolic and microsomal esterases did not metabolize propoxur as was expected, based on the topical bioassay data. A similar example was reported by

resistance to Siegfried et al . (1990). They found that chlorpyrifos was partially suppressed with DBF, suggesting the involvement of hydrolytic enzymes. However in vitro metabolism of [^^C] chlorpyrifos with cytosolic fractions was not detected. High levels of hydrolysis were detected when

[^^C] chlorpyrifos oxon was used as substrate. These results may imply that hydrolytic enzymes also metabolize secondary

insecticide products. Additionally, Kennaugh et al . (1993) reported similar results for PBO. They found that a 20-fold resistance level in Helicoverpa armigera (Hiibner) toward pyrethroids could be eliminated by PBO, however no evidence for increased permethrin detoxification by cytochrome P450 .

91 monooxygenases in the resistant strain was found. They concluded that synergism of pyretroid toxicity by PBO is not necessarily an indication of resistance due to increased detoxification by cytochromes P450.

It is known that many cytochrome P450 monooxygenase enzymes are involved in the metabolism of virtually all

insecticides (Hodgson 1985, Agosin 1985) , leading to insecticide detoxification or in some cases to activation of the molecule. Even when metabolism occurs through the action of several different cytochrome P450 isoforms (Scott

1991) , it remains unclear how many different P450s participate in resistance in a given strain (Berge et al

1998) . Propoxur is an insecticide that is exposed to diverse detoxifying reactions carried out by specific

cytochrome enzymes (Coon et al . 1996) and according with its chemical structure it is expected to have more oxidative reactions. In fact, the most important propoxur metabolites produced by the Marietta strain of German cockroach were oxidation products (W- hydroxymethyl propoxur, 2 -hydroxyphenyl N-methyl carbamate, and o- isopropoxyphenyl carbamate.

According to Tsukamoto and Casida (1967) the detoxification process constitutes only one of several mechanisms involved in insecticide resistance and .

92 metabolism. In certain cases, the pathway of degradation of insecticides in vivo may differ from that observed in in vitro enzyme systems. Topical bioassays in the Marietta strain of the German cockroach implied that propoxur hydrolysis was the principal route of detoxification, but in vitro metabolic studies refuted this notion. These data suggest that the putative esterase inhibitor, DEF, may inhibit microsomal monooxygenases as observed previously

(Valles et al . 1997, Scott 1991).

Further in vitro metabolic experiments were conducted to examine the effects of different tissue sources on propoxur metabolism. These results revealed that the most important tissue catalyzing the oxidative detoxification of propoxur was fat body. Fat body and gut are the primary detoxification tissues in most insects (Matsumura 1985)

The obvious discrepancy between the bioassay data

(showing that esterases are the primary detoxification route) and in vitro metabolic experiments (showing that only cytochrome P450 monooxygenases are involved in detoxification) prompted me to further investigate propoxur pharmacokinetics in the German cockroach. Experiments were conducted to examine the penetration rate of propoxur in the Marietta strain of German cockroach in the presence and absence of PBO and DEF. These studies showed that the 93 addition of the synergists, PBO or DBF, significantly decreased the rate of propoxur penetration in the Marietta strain of German cockroach. This effect was highly significant. The amount of propoxur reaching the inside of the synergist- treated cockroaches (120 minutes after treatment) was less than half that of propoxur- treated cockroaches alone. This observation is not without precedent. Bull and Pryor (1990) and Scott and Georghiou

(1986) also provided evidence that pretreatment with synergists slowed fenvalerate and permethrin penetration in house flies, respectively. Additionally, they reported increases in mortality attributable to synergist action.

Furthermore, Sun and Johnson (1972) were the first to describe quasi -synergism, an increase in insecticide toxicity due to accelerated penetration across the cuticular barrier as opposed to inhibition of detoxification enzymes. Although the PBO and DBF effect on propoxur penetration in the Marietta German cockroach is

different than that observed by Sun and Johnson (1972) , it nonetheless illustrates penetration rate changes due to the coapplication of chemicals. Although these data do not directly aid in explaining the disparate results between the bioassay data and in vitro metabolism experiments, they do show that DBF and PBO caused an equivalent decrease in .

94

propoxur metabolism in vivo (Table 5) . Hence, DBF inhibition of cytochrome P450 monooxygenases may partially explain the high synergism rate I observed in the bioassays

(Table 1)

In an attempt to further shed light on the mechanism (s) responsible for the synergism of propoxur toxicity by DBF, I proposed several working hypotheses which were subsequently tested experimentally. First, DBF could have been inhibiting cytochrome P450 monooxygenases resulting in enhanced toxicity. This hypothesis has already been addressed above and appears to play a participating role in the observed effect.

Secondly, DBF could be inhibiting acetylcholinesterase resulting in enhanced toxicity of propoxur.

Acetylcholinesterase is an enzyme that degrades the neurotransmitter acetylcholine in the insect synapse and it is the primary target of organophosphates and carbamates.

Based on the inhibition experiments that I conducted,

German cockroach acetylcholinesterase was not further inhibited by the addition of DBF to the I 50 concentration of

propoxur (Table 9) . These data were not anticipated because

DBF is an organophosphorus insecticide which is commonly known to inhibit "C" esterases (i.e. cholinesterases, such

as acetylcholinesterase) . Inhibition of 95 acetylcholinesterase by the pesticide DEF had been previously demonstrated in fish. Strauss and Chambers

(1995) reported that the addition of 1 mM DEF inhibited acetylcholinesterase in all tissues evaluated. Apparently the Marietta strain of German cockroach is very sensitive

10'^ to propoxur because the I 50 was M. Comparatively,

acetylcholinesterase Bourget et al . (1996) found that the

10"^ propoxur I 50 for Culex pipiens was 5 x M. This mosquito species had two acetylcholinesterases, one of which was unaffected by this concentration of propoxur.

Another hypothesis to account for the synergism by DEF is the possible inhibition of P-glycoprotein in the cuticle. P-glycoprotein is a large transport glycoprotein present in some insect species that is involved in cuticular penetration of xenobiotics. It has been reported to be an important insecticide resistance mechanism in

certain insect species. For example, Banning et al . 1996a,

1996b showed that inhibition of P-glycoprotein by quinidine eliminated the resistance to thiodicarb in tobacco budworm,

Heliothis virescens F. However, I was unable to show any changes in propoxur penetration rate in the presence of quinidine in the Marietta strain of German cockroach.

Either, P-glycoprotein is not present in the German .

96

cockroach or it is unaffected by quinidine. Therefore, I was unable to further test my hypothesis.

The final hypothesis dealt with the possible contribution of sequestration. For example, I postulated that in a normal detoxification scenario in the German cockroach, propoxur may be bound by proteins which reduces its ability to arrive at the target site in a toxic dose.

It may be possible that pretreatment with DEF eliminated the ability of the cockroach to bind propoxur in subsequent treatments because the DEF had occupied all the sequestering protein sites. However, binding experiments

did not support this hypothesis (Figs. 5 and 6) . The vast majority of the propoxur incubated with cockroach protein sources eluted before the protein fraction. These data indicate that propoxur does not bind strongly to any particular protein. Again, this hypothesis is not without precedent. Several researchers have identified sequestering esterases in the German cockroach that were responsible for insecticide resistance (Prabhakaran and Kamble 1996, Scharf

et al . 1997b)

In conclusion, in vitro studies, showed that cytosolic and microsomal esterases did no metabolize propoxur, even when topical bioassays showed the opposite.

Propoxur was only detoxified by microsomal oxidases. 97

However, DEF inhibition of cytochrome P450 monooxygenases may partially explain the high synergism observed in bioassays. In vivo studies showed that synergist treatment

significantly reduced propoxur penetration, and they

virtually stopped internal breakdown of propoxur. The

addition of synergists to propoxur formulations may help to

decrease the amount of active ingredient used to control

propoxur resistant German cockroaches. . .

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Hussein Sanchez Arroyo was born in Pololcingo,

Guerrero, Mexico, on May 11, 1960. He spent his formative years in Mexico City, and upon graduating from high school he attended Universidad Autonoma Chapingo, Estado de Mexico, where he graduated in June, 1983, with his BS in agricultural parasitology. He completed his MS in entomology in December, 1987, at Colegio de Postgraduados where he is currently a professor. Hussein was awarded a grant from the

Mexican government for pursuing his Ph.D. in urban entomology at the University of Florida.

117 I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

P. G. Koehler Professor of Entomology and Nematology

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

Professor of Entomology and Nematology

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

5 . R. S. Patterson Professor of Entomology and Nematology

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

Valles Assistant Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertatijpn for the degree of Doctor of Philosophy.

R. J. Brenner Assistant Professor of Entomology and Nematology

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

0. N. Nesheim Professor of Food Science and Human Nutrition

This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the Degree of Doctor of Philosophy.

December, 2000

Dean, Graduate School