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DIELDRIN (HEOD) METABOLISM IN

COCKROACHES AND HOUSE FLIES


J. O. NELSON AND F. MATSUMURA
Department o f Entomology
University o f Wisconsin
Madison, Wis. 53 706

When carbon-14-1abeled HEOD (dieldrin) is injected into the American


cockroach, German cockroach, and house fly, metabolism takes place forming
radioactive metabolites which have been isolated and identified by thin-layer
chromatographic methods. The major metabolite of dieldrin in the American
cockroach is cis-aldrindiol. This is the first report of cis-aldrindiol being formed as a
dieldrin metabolite and possible mechanisms for its formation are given. Other
dieldrin metabolites identified or characterized indicate that both hydrolytic and
oxidative systems are important in the metabolism of dieldrin by the three insect
species studied. The in vitro metabolism of HEOD-14C by the fat body from the
American cockroach also supports the above conclusion.

Dieldrin is one of the most persistent chlorinated hydrocarbon insecticide chemicals


known. Also, it is the source of some of the most frequently encountered pesticide
chemical residues in the U.S. waters (Lichtenberg et aL 1970). Thus, its metabolic fate in
various biological systems is of great concern to many scientists.

HEOD (dieldrin) metabolism has been studied in various mammalian and microbial
systems by several groups of workers (Korte and Arent 1965, Klein et aL 1968,
Matsumura e t al. 1968, Richardson et al. 1968, Matthews and Matsumura 1969, Fell et al.
1970, Matsumura et al. 1970). Structures for the identified metabolites reported by these
workers are given in Fig. 1. The nature of the metabolites isolated from in vivo studies
indicates that both hydrolytic (Korte and Arent 1965) and oxidative (Damico e t aL I968,
Richardson e t al. 1968) processes are important in mammals. Furthermore, in vitro
experiments with rats demonstrated the importance of oxidative, hydrolytic, and
glucuronic conjugation systems in dieldrin metabolism (Matthews and Matsumura 1969).
In microbial metabolism, the important metabolic processes are hydrolysis, skeletal
rearrangements, and, to a lesser extent, oxidation (Matsumura et al. 1968, Wedemeyer
1968, Matsumura e t al. 1970).

The metabolism of HEOD (dieldrin) in insects has been studied as a possible


mechanism of insecticide resistance (Winteringham and Harrison 1959, Brooks and
Harrison 1963). Mosquitoes are known to metabolize HHDN (aldrin) and HEOD (dieldrin)
224

Archives of Environmental Contamination and Toxicology,


Vol. 1, No. 3, 1973, © 1973 by
Springer.-Verlag New York Inc.
Dieldrin Metabolism in Roaches and Flies 225

to hydrophilic compounds (Korte et al. 1962, Gerolt, 1965). A dieldrin metabolite


obtained from mosquitoes is identified as aldrin glycol (Oonnithan and Miskus 1964) and
it is further identified as trans-aldrindiol (Tomlin 1968). Matsumura and Hayashi (I969)
demonstrate autoradiographic evidence of HEOD-14C metabolism by resistant and
susceptible German cockroaches but none of the metabolites are identified. Khan et al.
(1969) report the conversion of the photoisomers of aldrin and dieldrin to the identified
rat metabolite U-I (Fig. 1) by house flies and mosquito larvae.

In this study, HEOD-14C metabolism was examined in three insect species: American
cockroach, German cockroach, and housefly. HEOD (dieldrin) metabolites formed in vivo
were identified by thin-layer cochromatography with known dieldrin metabolites. The
metabolic processes involved were investigated with in vitro experiments.

Materials
Non-radioactive HEOD ( 1,2,3,4,1 0,10-hexachloro-6,7-epoxy-1,4,4a ,5,6,7,8a ,-octahy-
dro-l,4-endo,exo-5,8-dimethanonaphthalene, principal component of dieldrin) used was
obtained from Shell Chemical Company. It was labeled 99+% pure and it gave no
indication of contamination by thin-layer chromatography" (tic). HEOD-14C was obtained
from Amersham/Searle and was used after purification by thin-layer chromatography.
The specific activities of the HEOD-14C preparations used were 70.4 mCi/mM and 64.2
mCi/mM. Reduced nicotinamide adenine dinucleotide phosphate (NADPH2), adenosine
triphosphate (ATP), glucose-6-phosphate (G-6-P), and reduced glutathion (GSH) were
obtained from Nutritional Biochemical Corporation. Other materials used for the in vitro
work were crystalline bovine serum albumin (BSA) fraction V (Sigma Chemical
Company), nicotinamide (Eastman Organic Chemicals, Inc.), and Sesoxane (sesamex)
(Shulton Fine Chemicals, Shulton, Inc.). American cockroaches, Periplaneta americana
(L.), were from the stock culture of this laboratory. German cockroaches, Blattella
gerrnanica (L.), were from the dieldrin-resistant Fort Rucker pure strain (Matsumura et al.
1967). The house fly strain, Musca domestica (L.), was a dieldrin-resistant strain from
Corvallis, Oregon (Plapp and Hoyer 1967). Dieldrin resistance was maintained by
occasional selection with (dieldrin) for several years. The reference compounds used in
this study and shown in Fig. 1 were from the following sources: trans-aldrindiol was
synthesized by H 2SO 4 hydrolysis of dieldrin (Korte and Arent 1965); cis-aldrindiol was
synthesized by KMnO4 oxidation of aldrin (Korte and Arent 1965) and an authentic
sample was obtained from Shell Chemical Company; photodieldrin was produced by the
method of Robinson et al. (1966) and compared to an authentic sample supplied by J. D.
Rosen (Rutgers University); metabolites F and G (Fig. 1) were synthesized by and
obtained from Bieniek and Korte (1969); authentic samples of radioactive F-l, U-l, and
C-1 and non-radioactive F-1 and U-1 (Fig. 1) were isolated from rats by Matthews (1968).
226 J.O. Nelson and F. Matsumura

Methods
Treatment, extraction, and isolation of HEOD-14C metabolites from insects. Adult
cockroaches were injected intersegmentally between the third and fourth abdominal
sternites with 1 nmole HEOD-14C (70.4 mCi/mM) in 1 /A of 95% ethanol per roach.
Treated and untreated roaches were kept in glass containers without food or water. Times
of exposure varied with the experiment.

House flies, male and female, were immobilized with CO 2 and each fly was treated
topically with 0.1 nmole HEODA4C (64.2 mCi/mM) in 1 /A of ethanol on the ventral
abdomen. The flies were kept for 6 days in a battery jar and fed on a solution of sugar
water. (Approximately one half of the flies died during this period.)

Two basic extraction procedures were used throughout the studies. The following
procedure was used to evaluate the production of water-soluble metabolites. The treated
insects were homogenized in a Lourdes homogenizer with water and the homogenate was
filtered through borosilicate glass wool. The filtered homogenate was partitioned against
diethyl ether for cockroaches or against chloroform-ethanol mixture (9:1 by volume) for
house flies. The solid materials in the glass wool were washed with acetone. Aliquots from
each of the three fractions [water, ether (or chloroform-ethanol mixture), and acetone]
were counted for 14C-radioactivity, using liquid-scintillation counting (Table I).

In order to study the solvent-extractable metabolites, the following extraction


method was used. The treated insects were ground with a sodium stflfate-sand mixture
(1:1 by weight) and extracted 3 times with 50 milliliters of acetone. The extracts were
filtered using Buchner funnels, with suction. The extracts for each group were combined
and the solvent was removed through rotary evaporation. The residue was dissolved in 20

Table I. Recovery and Distribution of 14C-Radioactivity After in vivo


Incubation of HEOD- 14C with Female American Cockroaches

Radioactivity in fraction from


Fraction Control a animals Treated animals
Average Per cent Average Per cent
count/min. of total b count/min. of Total b
Water phase 876 0.33% 12,036 4.44%
Ether phase 3,726 1.41% 7,794 2.77%
Acetone extract 260,520 98.26% 251,470 92.79%
TOTAL 265,122 97.0% e 271,300 99.0% c
aThe control group was homogenized and TCA was added to the homogenate
before HEOD-14Cwas added.
bper cent of the total 14C-radioactivityrecovered.
cper cent of applied 14C<adioactivityrecovered.
Dieldrin Metabolism in Roaches and Flies 227

milliliters of water and extracted 3 times with equal volumes of diethyl ether. The
combined ether phases were concentrated through rotary evaporation and the residue was
partitioned three times with a hexane-acetonitrile mixture (1:1 by volume). Portions of
the hexane phase and acetonitrite phase were examined by thin-layer chromatography
and autoradiography to evaluate the presence of soluble metabolites in these phases. The
radioactivity of each spot was assessed by liquid scintillation counting of the silica gel
corresponding to the spot on the X-ray film. To isolate radioactive metabolites, the
concentrated acetonitrile fraction was spotted on thin-layer chromatographic plates. The
plates were developed with an ether-hexane mixture (1:1 by volume) to a distance of 15
cm from the origin. The metabolites were locat6d by the radioactive bands on the
autoradiographic X-ray films. The radioactive metabolites were recovered by scraping the
bands of silica gel and washing the silica gel with acetone in a fritted glass funnel. This tlc
purification procedure was repeated with different solvent systems until single tlc
radioactive spots were obtained.

In vitro metabolism of HEOD-14C by tissues of female American cockroaches. Female


American roaches were dissected under insect saline (Yamasaki and Narahashi 1959) and
the fat body and hindgut was removed. When the hindgut was used, it was cut open and
rinsed of its contents in a buffer solution (0.15 M potassium phosphate, 0.05 M sucrose,
pH 7.4). The tissues were immediately placed in ice cold buffer solution. Homogenization
was accomplished in a Potter-Elvehjem homogenizer fitted with a teflon pestle; the pestle
was turned by a Lourdes homogenizer at a rheostat setting of 25% (900 rpm) for
approximately 1 minute.

The homogenate was centrifuged at 0-2°C for 20 minutes at 10,000 x g. The resulting
crude supernatant was used as the microsomat fraction. (It also contained the soluble
portion of the cells.) After centrifugation, the microsomal fraction was diluted to a
volume with buffer so that 0.5 ml of microsomal fraction contained the equivalent of one
cockroach fat body or hindgut.

A typical incubation mixture consisted of 1 nmole of HEODA4C (70.4 mCi/mM)


added with 1 /21 of ethanol, 0.5 ml microsomat fraction, various cofactors added in
phosphate buffer, and sufficient buffer solution to bring the incubation volume to 2 ml.
The system was incubated for 2 hours at 37°C with mild shaking in a Labline metabolic
shaker. The reactions were stopped by extracting 3 times with 2 ml of diethyl ether. The
extraction was accomplished in 1.6 x 15 cm test tubes by vigorous agitation on a Vortex
Jr. vibrator followed by centrifuging in 15-ml centrifuge tubes to complete separation of
the emulsion formed. Portions of the aqueous and ether phases (0.5 ml) were taken for
scintillation counting. The ether phases were evaporated to dryness under a gentle stream
of air. The residues were dissolved in a small amount of acetone and spotted on tlc plates
for analysis of organic soluble metabolites. Radioactive spots on the developed tic
plates were located by means of autoradiography. The amount of radioactivity present
228 J.O. Nelson and F. Matsumura

was quantitatively determined by scraping the silica gel corresponding to the spot and
counting with liquid scintillation counting.

Results and discussion


In vivo metabolism of HEOD-14C by American cockroaches
Treatment and extraction. Two female American cockroaches were injected with 1 ~tl
each of HEOD-laC (1 x 10 .3 M in ethanol), kept for 24 hours without food or water,
homogenized in water and the homogenate was extracted and analyzed as described in
the Methods section. Results of liquid scintillation counting of each fraction are
presented in Table I. Recoveries of the applied 14C-radioactivity are essentially complete:
99.0% for the treated and 97.0% for the control. It is apparent that a portion of the
14C-radioactivity from the treated roaches is water soluble. The difference in
water-soluble radioactivity, between the treated and control roaches, suggests that
binding to water soluble components is involved in the treated roaches and that this
finding is not an artifact.

A qualitative examination of the 14C-radioactivity present in each fraction using


thin-layer chromatography [solvent system: ether-hexane mixture (1:1 by volume)[ and
autoradiography reveals the presence of several dieldrin metabolites in the acetone extract
of the treated roaches. There is not any indication of HEOD-14C breakdown in any of the
control-group fractions. Both the ether and water phases from the treated roaches
contain some radioactive material which remains at the origin on the tlc plate. Also, the
ether phase also has a radioactive spot at rf = 0.26. These results show that American
cockroaches do metabolize dieldrin in vivo and suggest that, possibly, some conjugation
system is involved with the dieldrin metabolism.

Isolation of radioactive metabolites. American cockroaches were treated with


HEOD-lgC, held for 10 days, ground with sodium sulfate and sand, extracted with
acetone, and the extract was concentrated and partitioned, using the procedures
described in the Methods section. Qualitative examination of each fraction by thin-layer
chromatography and autoradiography indicates the presence of at least eight radioactive
metabolites in the acetonitrile fraction from the treated females and seven from the
treated males (Fig. 2). Only unchanged HEOD (dieldrin) was detected in acetonitrile
fraction from the control group, and in the water and hexane phases of all three groups.

In order to identify each radioactive metabolic product it was necessary to completely


purify each radioactive spot and to employ some of the tic solvent systems listed in Table
II. In many preliminary experiments, reference compounds were also spotted along with
the isolated metabolites. Thus, isolated radioactive metabolites and similar authenic
reference compounds were spotted side by side for critical matching of the rf values in
each of the five solvent systems used.
Dieldrin Metabolism in Roaches and Flies 229

Table II. Rf Values for HEOD (dieldrin} Metabolites of HEODA4Cfrom American


Cockroaches, and Authenic Reference Compounds in Various Solvent Systems

Metabolite Rf value in indicated solvent system


or Ether- Hexane- Methylene Chloroform- Benzene-ethyl
compound hexane (1: 1) acetone (4:1) chloride ethanol (9:1) acetate (3:1)
trans-Aldrindiol 0.07 0.08 0.00 0.31 0.07
Metabolite B 1 0.05 0.08 0.00 0.29 0.07
Metabolite Bz 0.04 0.08 0.03 0.38 0.09
Metabolite B3 0.04 0.08 0.03 0.44 0.09
cis-Aldrindiol 0.19 0.17 0.05 0.53 0.18
Metabolite D 0.19 0.17 0.05 0.53 0.18
Metabolite E 2 0.22 0.24 0.41 0.83 0.39
Metabolite E 3 0.23 0.24 0.66 0.88 0.55
U-1 0.23 0.24 0.67 0.88 0.55
Metabolite H 0.31 0.19 0.I1 0.66 0.45
C-1 0.31 0.29 0.17 0.80 0.45
Metabolite J1 0.31 0.29 0.17 0.80 0.45
Metabolite J2 0.31 0.29 0.30 0.77 0.45
F-1 0.3t 0.29 0.30 0.77 0.45
G 0.49 0.45 0.59 0.87 0.65
F 0.29 0.24 0.57 O.82 0.56
Photodieldrin 0.35 0.26 0.63 0.88 0.58
HEOD (dieldrin) 0.62 0.53 0.79 O.88 0.73

The major metabolite, metabolite D (Fig. 2), was found to match cis-aldrindiol (Fig. 1)
in all critical matching tests with the five solvent systems. Non-radioactive contaminants,
which had induced "tailing" of the radioactive metabolite D, were removed using the
chloroform-ethanol (9: I by volume) solvent system. The resulting purified metabolite D
gave a symmetrical spot with reproducible rf values for all the solvent systems used. This
is the first report of cis-aldrindiol being formed from dieldrin by any biological system.
Judging from the quantities of this compound found (in relation to other metabolites) in
both the male and female American cockroaches (17.05% and 39.73% of the total
radioactivity in the acetonitrile fractions, respectively), this is by far the most important
metabolic product of dieldrin in the American cockroach.

In order to study other major metabolites, it is necessary to choose the right solvent
systems which can clearly resolve some of the closely related candidate compounds.
Matthews and Matsumura (1969) report that compounds F-1 and C-1 are similar in their
chromatographic behavior except in a tlc system with methylene chloride as the solvent
system. In the American cockroach, there are two radioactive metabolites which,
respectively, match each of these compounds: metabotite Jl, a minor metabolite,
matches C-I ; metabotite J2, a major metabolite, matches F-t (Table II). Metabolite B1
matches trans-aldrindiol (Fig. 1), a metabolite of HEOD (dieldrin), which is known to
230 J.O. Nelson and F. Matsumura

form in several biological systems (Korte and Arent t965, Tonflin 1968, Wedemeyer
1968, Matthews and Matsumura 1969, Feil et al. 1970). Also identified is metabolite E3
which matches U-1 but is present in only small quantities. The presence of compounds
C-t and U-1 indicates the existance of an oxidative system in American cockroaches,
similar to that described by Matthews and Matsumura (1969) in rats. However, the small
quantities of these metabolites found suggest that this system is of relatively minor
importance for dieldrin metabolism in the American cockroach.

el el CI

~,,~c, vo~ c,. . C'pc, Y ~°


CI H OH

trans-Aldrindiol cis-Aldrindiol F-1

CI el CI
C l ~ Cl~,~
?c, v _o c,~a_~c,..
~ oC, c, c, o

Metabolite G Metabolite F Photodieldrin

c,C~ CI
c , ~ ~ c , ~ .~ CI

C I y -"O c~~o cy~ .7--o


H "1"OH Ct "
O

U-1 C-1 HEOD

Fig. 1. Chemical structures of various known HEOD (dieldrin) metabolites and


derivatives. The structure of compound C-1 is tentative.
Dieldrin Metabolism in Roaches and Flies 231

Chemical characterization of metabolite H. Another major metabolite, metabolite H


(rf = 0.31), does not match any of the known compounds in all five thin-layer
chromatographic systems. Its chromatographic behavior is similar to the known
monohydroxylated compounds. Since insufficient quantities of the metabolite were
available for spectral characterization, attempts were made to characterize it by
subjecting it to certain chemical reactions. The results of the reactions were determined
by the changes in rf values found for the radioactive reaction products. Radioactive
dieldrin and compound F-I, a known monohydroxy dieldrin metabolite, were also
reacted under the same conditions to serve as controls for the experiments.

In order to study the probability of acetylation (Fell et at. 1970), one-half of the
isolated radioactive metabolite H (approximately 60,000-70,000 cpm) was treated with a
mixture of 0.2 ml of acetic anhydride and 3 /11 of triethyl amine. The mixture was

HEOD 26.53% 99.90%


(Dieldrin) 60.54%

J l~ 4.39% 6.84%

H 7.22%
1,00% 2.08%
E
~) 2.16% 4.14%

D 17.05% 39.73%
5.13%
B
t• 3.14% 1~ 6.44%
A @ 2.°5% 4.40% [ ~

Male American Female American Female American


roach (treated) roach (treated) roach (control)

Fig. 2. Representation of an autoradiogram of the thin-layer chromatographic separation


of in vivo HEOD-14C metabolites from American cockroaches. Solvent systems:
ether-hexane mixture (1 : 1 by volume). The control group was treated with HEOD-lac
after killing by freezing.
232 J . O . Nelson and F. Matsumura

allowed to stand at room temperature for 2 days then heated on a steam bath for 30
minutes; water and ether were added and the mixture was mixed well. The ether layer
was removed and evaporated to dryness under a gentle stream of dry N2. The residue was
picked up in ether and spotted on a tlc plate along with untreated metabolite H.
Radioactive HEOD (dieldrin) and radioactive F-1 were treated under identical conditions
to serve as controls for the reaction. The tlc plate was developed in a solvent system of
me thylene chloride.

The acetylation products from compound F-1 and metabolite H give single spots with
higher rf values than the reactants: rf = 0.12, 0.18, 0.33, and 0.53 for metabolite H,
metabolite H acetate, compound F - l , and compound F-1 acetate, respectively. HEOD
(dieldrin) is unchanged by this reaction. The acetate derivatives are converted to the
original materials by hydrolysis with 0.5 N alcoholic KOH (10 min at approximately
80°C), thus confirming the presence of a hydroxyl group on metabolite H.

An attempt to oxidize metabolite H to an aldehyde or ketone, using the oxidizing


mixture described by Feil et al. (1970), did not change the rf of metabolite H. The results
of the chemical characterization experiments indicate that metabolite H is a
m o n o h y d r o x y derivative o f HEOD (dieldrin) with the hydroxyl moiety in a tertiary
position.

Table IIl. Distribution of Radioactivity in Female American Cockroach Tissues After


Treatment with HEOD-14C

Tissue Recovered radioactivity, %, after


or
material Contact a Injection

Foregut 6.80 5.91 3.21 2.65


Midgut 1.45 1.69 1.27 1.21
Hindgut 5.25 6.66 12.76 9.40
Caecae 1.31 1.07 1.49 1.83
Malpighian tubules 0.16 0.93 0.47 0.36
Fat Body 11.00 12.82 20.25 11.00
Nerve Cord 0.20 0.46 1.30 1.50
Reproductive organs 1.19 2.10 7.09 6.14
Cuticle and muscle 27.81 30.96 37.39 55.07
Excreta 2.04 b 5.52 b
Body fluids 11.57 7.96 8.94 10.71
Rinse of petri dish 31.21 29.42 0.32 0.14

aCockroaches were exposed to a residual film of HEOD-14C in a glass container.


bNo excreta was found in the container.
Dieldrin Metabolism in Roaches and Flies 233

Tissue localization of HEOD (dieldrin) metabolism in the American cockroach. Four


female American cockroaches were treated with HEOD-lgC; two by injection of one
nanomole each of HEOD-14C in 1 /al of ethanol and two by contact with one nanomole of
HEODJ4C each in petri dishes. After 24 hours of exposure, the roaches were dissected
and the tissues extracted with diethyl ether. An aliquot of each extract was assayed by
liquid scintillation counting and the remainder was spotted on tlc plates. The percentage
of radioactivity found in each tissue is given in Table III. It is clear that the percentages of
radioactivity present in abdominal tissues of cockroaches which received abdominal
injections are high. Of much more interest is the distribution of metabolites among the
tissues (Fig. 3). It is apparent that most of the HEOD metabolites are found in the
cockroach hindgut; no HEOD is found in the excreta. This is contrary to the finding of
Cohen and Smith (1961) in their work with locusts. They report that locusts injected
with HEOD-36C1 excrete only unchanged HEOD.

Metabolite H, as previously characterized, appears in the fat body, cuticle and muscle,
and reproductive organs, and, to a lesser extent, in all of the gastric tissues. A small
amount of metabolite which matches cis-aldrindiol also appears in the first three
previously mentioned tissues.

German cockroach in vivo metabolism o f HEOD (dieldrin)


Dieldrin metabolism was also studied in German cockroaches as described above for
the American cockroaches. The tic pattern of HEOD-14C metabolites from German
cockroaches (Fig. 4) is similar to that of the American cockroaches (Fig. 2). Indeed, the
isolated lgC-metabolites from German cockroaches match the same reference compounds
matched by the American cockroach metabolites. A radioactive spot also matched
metabolite H of the American cockroach as previously characterized.

The difference between the two species of cockroaches lies in the quantity of
metabolites produced. Visual inspection of the tlc autoradiograms, as represented in Fig.
2 and Fig. 4, shows more overall metabolism in the American cockroaches. While
cis-aldrindiol is the major metabolite for American cockroach, it is only a minor
metabolite for German cockroaches. On the other hand, compound F-1 is the most
important metabolite in German cockroaches. Apparently, the same metabolic systems
are present in both species but the importance of each system to HEOD (dieldrin)
metabolism varies with the species.

In vivo metabolism of HEOD (dieldrin) by house flies


Metabolic activities of house flies to degrade HEOD (dieldrin), as shown in Fig. 4 in
terms of the percentages of metabolites produced, are much less than those found in
cockroaches. The isolated radioactive metabolites match the following reference
compounds in thin-layer chromatography: F-l, C-1, U-l, and trans-aldrindiol. The
t,a

HEOD
(Dieldrin)
® @ ® ® ~ @

©
Z

e~

B g
A O
Foregut Midgut Hindgut Gastric Malpighian Fat Nerve Reproductive Muscle Excrete Body
caecae tubules body cord organs and cuticle fluids

Fig. 3. Representation of an autoradiogram showing the distribution of radioactive metabolites among the tissues
of a HEODA4C-treated American cockroach. Solvent system: ether-hexane mixture ( l : l by volume).
Dieldrin Metabolism in Roaches and Flies 235

HEOD Dieldrin 95.32%


(Dieldrin) / 90.56%
E O 0.91%

F O 2.49% D ~ 1.09%

E ~ 1.58% C 0.99%

D ~ 1.66%

C Q 0.70%

B ~ 1.86%
B ~ 0.36%

A O 1.15% 1,33%
A C)

German Housefly
cockroach

Fig. 4. Representation of an autoradiogram showing the thin-layer chromatographic


separation of HEOD-14C metabolites from German cockroaches and house flies. Solvent
system: ether-hexane mixture (1:1 by volume).

pertinent rf values are summarized in Table IV. Not any radioactive spots match
cis-aldrindiol or metabolite H of the American cockroach. These results indicate that
HEOD (dieldrin) metabolism in vivo by the house fly parallels the rat's oxidative and
hydrolytic metabolism of HEOD as described by Matthews and Matsumura (1969). The
house fly strain used is known to have a low level of microsomal oxidase activity
(Schonbrod et al. 1968) and this fact probably accounts for the low level of HEOD
(dieldrin) metabolism.

In vitro metabolism o f HEOD-14C by the American cockroach


The in vivo experiments with American roaches show that metabolism of HEODA4C
does occur. To investigate the processes involved in tt~s metabolism, in vitro experiments
were performed. A fat-body preparation, as described in the Methods section, was used
236 J.O. Nelson and F. Matsumura

for most of this work: it consisted of the supernatant from the homogenized fat body in
buffer obtained by centrifugation at 10,000 x g for 20 minutes. [A similar crude
supernatant has been used by other workers (Nakatsugawa and Dahm 1962) as a source
for the microsomal and soluble fractions.] A preliminary experiment showed that
HEOD-14C was metabolized by the crude fat-body supernatant. Addition of NADPHz,
ATP, and glucose-6-phosphate, three cofactors which Matthews and Matsumura (1969)
indicated had stimulated in vitro metabolism of dieldrin by rat liver microsomes, also
stimulated metabolism of dieldrin by the crude fat-body supernatant. A representation of

HEOD
(Dieldrin)

CI Cll NI Nil

Fig. 5. Representation of tlc autoradiograms showing & vitro metabolism of HEOD-14C


by fat-body supernatant from female American roaches. Ether-soluble metabolites were
separated by tlc [solvent system: ether-hexane mixture (1:1 by volume)], detected by
auto-radiography, and quantitated by liquid-scintillation counting. Values given are
percentages of the recovered radioactivity C I and C II are control incubation mixtures
containing 1.0 ml of 30% fat body supernatant, 10 nM HEOD-14C, and 4.0 ml phosphate
buffer, pH 7.1. N I and N I I , in addition to the control mixture, contain 9.6 /aM
NADPH2,3.6/aM ATP, and 6.5/aM G-6-P.
Table IV. Rf Values ,for HEOD-14C Metabolites from House Flies, Authentic Reference Compounds, and HEOD (Dieldrin)

Metabolite a or eom ~ound


FIF-A HF-B trans- HF-C U:l c-1 rlF-D Metab. H [-IF-D 2 F-1 F-E[ nEO e-~
Solvent system Aldrin- (Ameri- (dieldrin) 3.
diol can)

Ether-Hexane
(1:1) 0.00 0.08 0.07 0.24 0.2z 0.33 0.32 0.32 0.33 0.33 0.56~ 0.62
Hexane-Acetone
(4:1) 0.00 0.08 0.08 0.25 9.26 0.30 0.31 0.20 0.30 0.30 0.38 0.53
O
Methylene
Chloride 0.00 0.00 0.00 0.64 9.65 0.18 0.18 0.11 0.30 0.30 O.78 0.78
Chloroform-
Ethanol (9:1 ) 0.13 0.30 0.31 0.90 0.9( 0.46 0.46 0.68 0.81 0.81 0.87 0,88
Benzene-Ethyl
Acetate (3:1) 0.00 0.09 0.10 0.59 0.5c~ 0,5C 0.50 0.47 0.50 0.50 0.73 0.74
aHF stands for house flies.

b3
o3
--..I
238 J.O. Nelson and F. Matsumura

the tlc autoradiogram showing these results is given in Fig. 5. The radioactivity present in
each spot was also measured by liquid scintillation counting. The radioactive in vitro
metabolites were isolated and spotted on tlc plates with the in vivo metabolites and
reference compounds. The results of this experiment show that the major hz vitro
metabolite is metabolite H and that compound C-1 is the second most abundant product
in vitro. Other minor metabolites found in vitro include trans-aldrindiol, cis-aldrindiol,
and compound U-1.

The addition of the cofactors mentioned above greatly stimulates the production of
metabolite H (approximately a 240% relative increase). Also stimulated by the cofactors
is the formation of compounds C-t and U-1 which show 49.4% and 97.5% relative
increases, respectively. The production of trans-aldrindiol is essentially unaffected by the
cofactors while that of cis-aldrindiol decreases by 57.5%.

In another experiment, the effect of NADPH 2 alone on the in vitro metabolism


of HEOD (dieldrin) was studied. Five replicates each of the control and of
NADPH2-fortifled incubation mixtures yielded the following results: the presence of
NADPH2 as the sole cofactor stimulates metabolite H production by only 26.4%;
production of compounds C-1 and U-1 is stimulated by NADPH 2 alone to the same
relative percent increases as in the experiment with the three cofactors (47.6% and 94.0%,
respectively). As before, the relative amount of trans-aldrindiol produced is unaffected by
the cofactor. The amount of cis-aldrindiol, on the other hand, does not show any
decrease with NADPHz. It is apparent from these results that production of compound
C-1 is controlled by a NADPHz-dependent system and that the production of metabolite
H, although it is moderately stimulated by NADPH2 alone, is greatly stimulated by the
N A D P H 2 ATP glucose-6-phosphate combination. Trans-aldrindiol production is un-
affected by any of the cofactors used.

The effect of sesamex on the rate of/n vitro metabolism was studied. Various amounts
of sesamex were added to NADPH2-fortified incubation mixtures. The ether-extractable
metabolites were separated by thin-layer chromatography, detected with autoradi-
ography, and the radioactivity present in each spot was measured with liquid scintillation
counting. Changes in the relative amounts of the metabolites produced was the criterion
for judging the sesamex sensitivity of the metabolic systems involved. Results are
presented in Fig. 6. No strong pattern of sesamex inhibition is seen; however, metabolite
H shows a slight but steady decline with increasing inhibitor concentration.

In another experiment the effects of other cofactors and materials on the in vitro
metabolism of HEOD-14C were studied. The results show that bovine serum albumin
(BSA) increases the overall level of HEOD-14C metabolism, an effect similar to that found
by Tsukamoto and Casida (1967) with housefly microsomes. The other cofactors and
materials (nicotinamide, MgC12, reduced glutathione, ascorbic acid, and NADP) have
Dieldrin Metabolism in Roaches and Flies 239

little or no effect on the production of HEOD:4C metabolites. Metabolites of HEOD


(dieldrin) in cockroaches are summarized in Figure 7.

In order to study the tocation of dieldrin metabolizing enzymes, hindgut tissue


homogenates were prepared using a technique similar to the one used for the fat body
homogenate. A hlndgut was dissected from a female American cockroach under insect
saline, thoroughly rinsed of its contents in buffer, then homogenized and centrifuged to
give a hindgut crude supernatant. The pattern and level of HEOD (dieldrin) metabolism
from the hindgut homogenates are very similar to that from the fat-body homogenates.
Thus, there appears to be little qualitative difference in the ability of hindgut- and
fat-body microsomal preparations to metabolize HEOD (dieldrin).

In conclusion, it appears that hydrolytic metabolism of HEOD (diel~trin) to cis- and


trans-aldrindiol, particularly in the hindgut, is the most important degradative route for
dieldrin in cockroaches. Oxidative metabolism also occurs but to a lesser extent in
American and German cockroaches.

Metabolite
c
J=o
H=Zx
c~ D=o
m" 40 B=X

.£2

3o
C

20
O

10
~5
E

0 , ! P ......... I
0 0.01 0.05 0.10 0.20

#M of sesoxane/tube

Fig. 6. Graph of the effect of sesamex on the relative amounts of HEODJ4C metabolites
produced by fat-body supernatants from female American cockroaches.
240 J.O. Nelson and F. Matsumura

The basic pattern HEOD (dieldrin) metabolism in the house fly, on the other hand,
appears to be rather similar to that found previously in mammals. Since this manuscript
was prepared, an article on the dieldrin metabolism in house tlies has appeared (Sellers
and Guthrie 1972). They too, have identified trans-aldrindiol as one of the HEOD

Cl

Metabolite H

f el
F-1

CIcc'~ . ¢ ~ ~

c'~J/c , _L C

cl CI

Dieldrin~lp ~ CI cis-Aldrindiol

c a_W- ° .
H"~"OH ~ CI
C-1 *'It trans-Aldrindiol

Clio, o

u-1
Fig. 7. Pathways for HEOD (dieldrin) metabolism in the American and German
cockroaches. The change denoted by the broken arrow has not been established.
Dieldrin Metabolism in Roaches and Flies 241

metabolites in the house fly. The pattern of dieldrin metabolism found by these workers
appears to be very similar to the ones reported here, judging by the rf values published for
the various metabolites.

Formation of cis-aldrindiol in the female American cockroach is interesting in the light


of the absence of such evidence in any other biological systems, as reported so far. It has
been suggested that trans-aldrindiol is formed in biological systems as a result of the
initial protonation of the epoxide ring with the subsequent hydroxylation (nucleophilic
attack) from the opposite direction as in the case with the chemical reaction of acid
hydrolysis of HEOD (dieldrin).

There are several possibilities to explain the biological formation of cis-aldrindiol.


First, since it has been shown in this study as well as in others (Matthews and Matsumura
1969, Brooks and Harrison 1969) that this ring opening system in animals involves
hydrolytic enzymes, it is not unreasonable to assume that a one-step hydration reaction
takes place from the direction of the ring (exo-position), instead of the two-step reaction
mentioned above. The second possibility is that, on the assumption that a two-step
reaction occurs, the direction of the later nucleophilic attack is sterically restricted to
favor the cis- formation. Such a steric factor has been discussed by Chau and Cochrane
(1970), who chemically produced cis-aldrindiol from HEOD (dieldrin) by first treating
HEOD with acetic acid-sulfuric acid mixture and then hydrolyzing the acetate.

Whatever the actual enzymatic mechanism of cis- formation, there is still an entirely
different possibility: the female American cockroach possesses a weak epimerase (H. B.
Matthews, personal communication) which could be active in other animals in converting
cis-aldrindiol into trans-aldrindiol. In that case, the formation of cis-aldrindiol could be
the common first step of the biological reaction.

Acknowledgments
This study was supported, in part, by research grant No. EP-R-801060 from the
Environmental Protection Agency and by the Division of Research, College of
Agricultural and Life Sciences, University of Wisconsin.

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Manuscript received October 14, 1972; accepted January 20, 1973