THE COMPARATIVE METABOLISM AND EXCRETION

OF HCE, A BIODEGRADABLE ANALOGUE OF

DIELDRIN, BY VERTEBRATE SPECIES
C. H. WALKER and G. A. EL ZORGANI 1
Department of PhyMology and Biochemistry
Reading University
Whiteknights, Reading, U.K.

The metabolism of HCE 2 has been studied both in vitro and in vivo in the rat,
rabbit, pigeon and Japanese quail. The rook, jackdaw, fulmar and domestic fowl
were only investigated in vitro. HCE was readily degraded to a h y d r o x y eP0xide
(HHC) by microsomal oxidation in all species, but there were marked interspecific
differences in hepatic microsomal hydrase activity towards this compound, the
rabbit showing the strongest activity, the pigeon showing no activity at all. There
were some interspecific differences in minor oxidative metabolites. After intraperi-
toneal injection of C 14 HCE, 50 percent of the dose was cleared from the body
within the stated times for the following species: - rabbit (two days) quail (four
days) and pigeon (eight days). Excreted C 14 appeared mainly in the feces with the
rat, b u t almost entirely in the urine with the rabbit. The principal metabolites in
vitro were also found in vivo with one possible exception, and the liver preparations
showed some promise for predicting the main primary metabolites in living animals.
The metabolism of HCE is compared with the metabolism of dieldrin. 3

Introduction

Dieldrin has proved to be an efficient insecticide when used for many purposes includ-
ing the spraying and dusting o f agricultural crops, the dressing of seed, and the dipping of
sheep. Unfortunately it has one serious disadvantage - strong biological persistence.
Dieldrin residues have come to be widely distributed in the living environment (Edwards
1970), and this has led to a marked reduction in use of the chemical in many developed
countries.

I n vitro studies with pig and rabbit liver microsomes (Brooks et al. t970) and centri-
fuged rat liver homogenates (Matthews and Matsumura 1969) have revealed that dieldrin
is only very slowly transformed by mixed function oxidation and hydration into water
soluble metabolites, and this would appear to be a major factor in determining this com-
pound's marked biological persistence.

1Now at Agricultural Research Corp. Wadi Medani, Sudan

2 ••2•3•4•9•9-hexa•h••r•-ex•-5•6-ep•xy-••4•4a•5•6•7•8•8a••ctahydr•-••4-rnethan•naphtha•ene
3HEOD (••2•3•4••••••-hexach••r•-6•7-ep•xy-••4•4a•5•6•7•8•8a-•ctahydr•-••4-end•-ex•-5•8-dimethan•-
naphthalene)

Archives of Environmental Contamination 97

and Toxicology, Vol. 2, No. 2, 1974, © 1974
by Springer-Verlag New York Inc.
98 C.H. Walker and G. A. E1 Zorgani

There are, however, certain structural analogues which are more rapidly metabolized
than dieldrin itself. One of these is HCE which displays similar insecticidal activity to
dieldrin for the housefly when synergized with sesamex, a compound which can repress
mixed function oxidation (Brooks and Harrison 1964). Like dieldrin, HCE can be metab-
olized both oxidatively and hydratively. Working with microsomes prepared from whole
houseflies, and from pig and rat liver Brooks (1966) has shown that HCE is metabolized
by mixed function oxidation to yield two hydroxy epoxides, one of which is produced
in considerably larger quantities than the other (Brooks 1966, 1969) (Fig. 1). Hydration
t o a trans-dihydrodiol also occurs in pig and rat liver microsomes (Brooks et al. 1970) but
not in housefly microsomes (Brooks 1966). The epoxide hydrase responsible for this
latter conversion is distinguishable from mixed function oxidases because it is not de-
pendent upon NADPH or 02 and is relatively insensitive to sesamex (Brooks e t al. 1970).
It appears to be similar to the epoxide hydrase found in the hepatic microsomes of guinea
pig, rat and rabbit which is active towards arene oxides (Oesch et al. 1970, 1971).

Rat and pig liver microsomes have shown considerably greater epoxide hydrase activity
than microsomes from the insect species so far tested (Brooks 1972). If the rat and the
pig are representative of higher animals, this may point to useful selective insecticidal
action by HCE and related compounds especially in the presence of sesamex and other
methylenedioxyphenyl synergists. The availability of hydration as an alternative to oxi-
dative attack should give some protection against insecticide/synergist combinations. In-
deed the extent to which HCE is synergised by sesamex in the housefly encourages this
point of view, and suggests strong dependence upon mixed function oxidation for effi-
cient elimination of the insecticide (Brooks 1969, 1972).

The greatest epoxide hydrase activity was shown by pig liver microsomes in the fore-
going work. Further investigations with this preparation and rabbit liver microsomes have

C1 OH
C I ~ O H
CIc1~ Hydration

C1 / ~NA C1 HCEtransdiol
HCE DPH/02
~ , Cl o C1 O
NADPH/02 ~11 NADPH/02
oU;h'ep2
! r;dClucts +U3
C1
C1 (OH) C1 (OH)2
HHC Dihydroxy HCE
Labelledcarbonatomsare markedwith a dot ( • )
Fig. 1. Metabolism of HCE by vertebrate liver microsomes.
 Metabolism and Excretion of HCE 99

demonstrated that hydration is stereoselective in the case of HCE and two other asym-
metrical epoxides, chlordene epoxide and one of the heptachlor epoxides (Brooks et al.
(1970). Of the two optical isomers of each epoxide, only the dextrorotatory (+)isomer
was readily hydrated. Hydration of HCE yielded an optically active diol which had
[o~]D23=+15+ 1.5 °.

A balance between oxidative and hydrative attack is a typical tbature of the metabo-
lism of the chlorinated epoxides related to dieldrin, and the study of this balance for
different compounds in different species should give useful guidance in the design of
selective and non-persistent insecticides. Since HCE shows some promise in this respect,
and is representative of the group in its metabolism, it was selected for the present investi-
gation. In vivo studies were carried out in addition to in vitro studies with two mam-
malian and two avian species (Walker and E1 Zorgani 1972). This approach enabled some
assessment to be made of the value of tn vitro studies for predicting events in the whole
animal.

Materials a n d m e t h o d s
Animals. (1) Mature male rat, rabbit, pigeon and quail, in good condition, were used
throughout for both in vivo and in vitro studies. Wistar rats weighing 100-200 g and Old
English rabbits weighing 1.5-2 kg were obtained from Animal Supplies Ltd. Japanese
quail (Coturnix coturnix japonica) were bred in the department and were used between
six and nine weeks of age at a weight of 80-100 g. Pigeons (Columba livia) weighing
350-550 g were obtained from Harradine Farms, March, Cambs.

(2) Mature males of the following species were used for in vitro studies only: Jackdaws
(Comus monedula) and rooks (Corvus frugilegus) were supplied by Mr. F.S. Jones of the
Ministry of Agriculture, Fisheries and Food. Domestic fowl were reared in the depart-
ment. The fulmar petrel (Fulmarus glacialis) was a victim of oil pollution.

Preparation of homogenates and microsomes. All animals were killed by breaking the
neck, and the livers were quickly removed and washed. After removal of bile ducts, con-
nective tissue, and gall bladder (where present) the livers were sliced, weighed and
homogenized for two minutes in ice cold 1.15 percent potassium chloride solution (3 ml
per grain of liver) using an MSE stainless steel homogeniser. The homogenates so obtained
were centrifuged at 11,000 G for 30 rain at 0-4°C and the resulting supernatants were
drawn off with a 50-ml syringe. Microsomes were prepared by spinning 11,000 G super-
natants for one hour at t 05,000 G and 0-4°C. The supernatants were poured clear of the
microsomal pellets which were then resuspended in cold 1.15 percent KC1. The micro-
somal suspensions were spun for a further 30 rain at 105,000 G and 0-4°C. Finally the
microsomes were resuspended in 1.15 percent KCI to give the equivalent of 1 to 4 g of
liver per ml of suspension. Microsomal protein was determined by the Lowry method.

Incubation procedure. Reactions were carried out in 25-mi Edenmeyer flasks. The
flasks were incubated in a metabolic shaker operated at 30 cycles per rain, and at tem-
peratures of 37°C and 42°C for mammalian and avian species respectively.
100 C. tL Walker and G. A. E1 Zorgani

Using 11,000 G supernatants the flask contents were as follows :- 2 ml of supernatant

and 3 ml of phosphate buffer pH 7.4 containing 7.5/amoles of glucose-6-phosphate, 8
gmoles of nicotinamide and 1.2 /amoles of NADP. The phosphate buffer was made by
dissolving 5.5 mmoles of NaH2PO4 "2H20 and 4.4 mmoles of NaOH in distilled water and
making up to 100 ml after adjusting the pH to 7.4. Forty-/ag aliquots of substrates were
added dissolved in 20/al of absolute ethanol. Flasks were left open to the air when study-
ing oxidative metabolism; when anaerobic conditions were required, the flasks were
stoppered, purged with nitrogen, and glucose-6-phosphate, nicotinamide and NADP were
excluded from the medium.

The microsomal incubation medium was comprised of 0.5 rrd of microsomal prepara-
tion, 4 ml of reaction medium, and 0.2 ml of NADPH generating system containing 20
/amoles of glucose-6-phosphate, 1.8 units of glucose-6-phosphate dehydrogenase and 20
/amotes of NADPH dissolved in a reaction medium that contained 0.56 mmoles of
nicotinamide, 1.42 mmoles of KC1, 4.05 mmoles of NaH2POg2H20, and 4.4 mmoles of
NaOH, made up to 100 ml after adjusting to pH 7.4. When the NADPH generating mix-
ture was excluded, it was replaced by 0.2 ml of reaction medium. Substrates were added
as for 11,000 G supernatants. One-tenth-rot aliquots of 1.6 × 10-2M SKF 525A were
added where this inhibitor was used.

Reactions were stopped at the end of incubation by adding 1 ml of acetone in the case
of 11,000 G supernatants, and by partitioning immediately into the extraction medium
in the case of microsomes.

Extractions were carried out by transferring flask contents to 10-ml glass stoppered
tubes and partitioning three times into three ml of diethyl ether or 40:60 diethyl
ether:hexane. The organic extracts were drawn off with a 10-ml glass syringe and run
into 10-ml glass stoppered flasks. After making up to the mark, anhydrous sodium sul-
phate was added and the flasks stored at 4°C.

Collection of urine and feces. Rats were kept in metabowl cages designed for separate
collection of urine and feces. They were fed a B41 commercial diet.

Rabbits were housed in cages manufactured by Pullman Technical Services, Catalogue

No. 23/22. The cages were adapted for separate collection of urine and feces by making a
hole in a strong polythene sheet, and fitting an iron mesh into it, then fixing this beneath
the lower mantle upon which the animal rests, with a weight attached to the mesh to pull
it down. Urine passed through the mesh and collected in a dish while feces accumulated
on the polythene cover. Rabbits were fed No. 18 commercial diet and green vegetables
were provided regularly.

Pigeons were kept in the same type of cage as the rabbits, but without the attachment
for collecting urine. They were fed Pauls No. 2 poultry diet. Quail were kept in rat cages
(translucent RI model) in which food and water reservoirs were replaced by suitable flat
containers. They were fed a similar diet to pigeons. All animals were housed individually
and the ambient temperature was maintained at 21°C.
 Metabolism and Excretion of HCE I01

All treated animals received 10-30 mg/kg of C 14 HCE (specific activity 0.14/ac/mole)
in ethanolic solution (40 mg/ml) by intraperitoneal injection.

Substrates. HCE was synthesized as described by Brooks and Harrison (1964). The re-
crystallised product had a melting point of 222-224°C and gave a single peak when ex-
amined by gas chromatography. The infrared absorption maxima are reported in Table I,
and correspond to those given by an authentic sample of HCE. C14-labelled HCE was pre-
pared from C14 hexachlorocyclopentadiene supplied by The Radiochemical Centre,
Amersham. Because of the small quantities involved, the procedure for HCE synthesis was
modified in the following way: A benzene solution containing 8 mg ofC14 hexachloro-
cyclopentadiene of activity 0.25 mc was carefully evaporated down in its container
ampoule. The brown residue was dissolved in 140 mg of freshly distilled hexachloro-
cyclopentadiene and 60 mg of cyclohexa-1, 3-diene, and the resulting mixture transferred
to a 15-ml glass tube with a quickfit joint. After adding a few crystals of quinol the tube
was fitted with a cold finger condenser and transferred to an oil bath at 120°C for 10
rain. The resulting olefine was recrystallized and epoxidized as before, and the final
product purified by thin layer chromatography on Kieselgel G using 1:100 acetone:

Table I. Chromatographic characteristics o f liCE and its metabolites

Thin layer chromatography

Kieselgel G. 3:1
Benzene:ethyl acetate Gas chromatography RRTs b
Compound Relative Rf valuesa Apiezon L SE52

HCE 4.45 0.73 0.68

U1 3.90 1.08 1.30 (1.40)
U2 3.30 1.76 (1.50) 2.90 (1.90)
HHC 3.10 1.55 (1.28) 1.93 (1.47)
U3 2.5 2.42 (1.25) 3.45 (1.47)
Dihydroxy
epoxide 1.7 2.70 (2.49) 3.70 (3.48)
DIOL 1.0 1.85 (1.60) 2.35 (1.87)

aThin layer chromatography-relative Rf values are calculated with respect to the trans-
dihydrodiol.
b Gas liquid chromatographic relative retention times are calculated with respect to HEOD
(dieldrin).
The relative retention times are for columns that had been operating for 2 months or
more and represent average values. Young columns showed shorter relative retention
times for most compounds.
Values for trimethyl silyl derivatives are given in brackets.
The characteristic infra-red absorption maxima for samples of HCE and its metabolites
are: HCE; 870, 1300, 1450, 1600: HCE trans-dihydrodiol; 750, 1250, 1600, 2950,
3400: HCE main hydroxy epoxide (HHC); 760, 1250, 1600, 2900, 3500 cm -1.
102 C.H. Walker and G. A. E1 Zorgani

hexane as mobile solvent. The yield was 25 mg of HCE of specific activity 1.16/ac/mg. It
gave a single peak on two different gas chromatographic columns. Its purity was checked
by re-running on TLC and confirming that the specific activity remained unchanged. A
sample of the ( - ) enantiomer of HCE was supplied by G.T. Books.

The trans-5,6.diol of HCE (1,2,3,4,9,9-hexachloro-l,4,4a,5,6,7,8,8a-octahydro-l,4-

methanonaphthalene trans, 5,6 diol) was prepared as described by Brooks et al (1970).
Identity and purity were checked by gas chromatography and infra red spectroscopy
(Table 1). A sample of HHC, the main hydroxy epoxide formed from HCE, was obtained
by biosynthesis as described later in the text.

Analytical procedures. Thin layer chromatography (TLC) was carried out on 20 × 20

cm plates coated with Kieselgel G or H of 250/a thickness, activated in an oven at 120°C
for two hours. Five- to ten-p1 aliquots of solutions of substrates and metabolites in ethyl
acetate or 40:60 diethyl ether:hexane were applied to the plates. So far as possible the
quantity of each applied compound was kept in the range of 2-20 og per spot. The plates
were run in 2:1 or 3:1 benzene:ethyl acetate, and spots were located by spraying with
0.5% w/v silver nitrate in ethanol and irradiating with germicidal (254 m/a) ultraviolet
light.

Column chromatography was used for cleaning up extracts of urine, feces and bird
droppings. Florisil (Hopkin and Williams chromatography grade) was activated for 3
hours at 130°C. After cooling in a desiccator it was equilibrated with 0.6 ml of water
per 20 g of Florisil. Four-g lots of Florisil were packed into glass columns (10 × 0.5 cm)
and pre.washed with petroleum ether (b.p. 40-60°C). After adding 0.5-1 ml of concen-
trated extract of urine, feces or droppings, the column was eluted with 12 ml of petro-
leum ether (b.p. 40-60°C) and this fraction discarded. This was followed by successive
elutions with 25-ml aliquots of solvent systems of increasing polarity. The fractions so
isolated were examined by gas liquid chromatography', thin layer chromatography, and
where appropriate by scintillation counting.

Gas liquid chromatography (GLC) was carried out using Perkin Elmer F11 gas chro-
matographs fitted with electron capture detectors (both tritium and nickel 63 sources
were used). All-glass columns (0.25 inch id and varying lengths) were fitted, and these
were packed with either of two different stationary phases as follows:

1) 2.5% Apiezon L and 0.5% epikote 1001 resin on 80-100 mesh chromosorb W oper-
ated at 180°C.
2) 2.5% SE52 and 0.5% epikote 1001 resin on 80-100 mesh DMCS treated chromo.
sorb W operated at 165°C.

Some extracts were injected directly but, in all cases, metabolites containing hydroxy
groups were converted into their trimethyl silyl ethers and examined by GLC. The silylat-
ing reagent was prepared by mixing 4 ml of dry Analar grade pyridine with 0.5 mt of
hexamethyl disil~ane and 0.25 ml of trimethyl chlorosilane (the last two compounds sup-
plied by Koch-Light Laboratories Ltd.).
 Metabolism and Excretion of HCE 103

Samples of dry solutions in organic solvents containing 1-10/ag of hydroxy compound

were taken just to dryness under dry nitrogen in glass quickfit tubes, and treated with 0.2
ml of silylating reagent. They were quickly stoppered after blowing dry nitrogen over the
surface for 4 to 5 seconds. The tubes were held at 50°C for 1 hr, before blowing down to
dryness with nitrogen, making up to a suitable volume with hexane, and injecting into the
gas chromatograph. (Blowing was terminated immediately when the samples were dry.)

The oxidative metabolism of HCE proved to be relatively complex. Since methods for
the chemical synthesis of oxidative metabolites were lacking, quantitative assessment was
attempted by radio-counting samples derived from in vitro experiments. Twentyqtl alp
quots of 2 mg/ml of C14 HCE (specific activity 0.4 #c/mg) in ethanol were added to in-
cubation media prepared in the usual way. Extracts of incubation media were either run
directly on thin4ayer plates or following clean-up by Florisil column chromatography.
Where column chromatography was used, the following fractions were collected: 20:80
diethyl ether:hexane, 40:60 diethyl ether:hexane and 23:1:1 diethyl ether:methanol:
acetone. On thin layer plates the metabolites appeared as dark blue-purple spots after
color development, and these areas were scraped from the plate and extracted with ace-
tone. Where necessary the samples of metabolites were re-run on thin-layer plates in
another solvent system to improve clean-up. Acetone extracts from thin layer plates were
reduced to dryness under nitrogen and the residues dissolved in hexane. The hexane solu-
tions were examined by gas-liquid chromatography on two different columns before and
after silylation, prior to determining radioactivity by scintillation counting.

Where a reasonable quantity of C14-labelled metabolite was obtained in this way, it

was set aside as a standard after carrying out a further check on purity. This was done by
establishing that the gas chromatographic peak height measured for a standard quantity
of radioactivity was not changed after further clean-up on thin layer plates (i.e. constant
specific activity was confirmed after successive clean-up steps). The chromatographic
characteristics of HCE and its metabolites are summarized in Table I.

Extraction of urine, feces, and bird droppings. Initially, samples of feces and bird
droppings were dried for 12 hours at 45°C before powdering, weighing and subsampling.
Subsamples were extracted with a 2:1 acetone:hexane mixture in a soxhlet apparatus for
48 hours. The extracts were evaporated to dryness and redissolved in 20 ml of acetone.
Coextracted proteinaceous material was precipitated by adding 10 ml of ten percent
aqueous ferrous chloride and the mixture extracted three times with 50-ml portions of
hexane. The hexane extracts were reduced to 20 ml before Florisil column clean-up.
Where appropriate, subsamples were taken for scintillation counting before clean-up.

Poor recoveries of radioactivity were obtained in early studies with rat, quail and
pigeon, and later samples of feces and droppings were mixed directly with ethanol with-
out drying following a procedure described by Baldwin (1971) for extracting dieldrin
metabolites from feces. Samples were adjusted to approximately pH6 with dilute hydro-
chloric acid, then heated on a water bath at 70°C for 10 min. Solid material was centri-
fuged down and the supernatant drawn was off with a syringe and filtered through a
glass wool plug. The procedure was repeated twice to give 200-260 ml of ethanol extract
104 C . H . Walker and G. A. E1 Zorgani

for each sample and the volume was measured. Ethanol extracts were subsampled for
scintillation counting. Twenty- to 50-ml aliquots were reduced to dryness under a slow
stream of oxygen-free nitrogen. With quail each residue was taken up in 5 ml of 1N sul-
phuric acid, and the resulting solution partitioned three times into 5-ml portions of
diethyl ether. The ether fraction was dried over anhydrous sodium sulphate. The residual
aqueous phase was blown over with nitrogen, boiled under reflux for two hours, and re-
extracted three times with 5-ml portions of diethyl ether. The ether fraction and the
residual aqueous fractions were submitted for scintillation counting. The dry ether frac-
tions were evaporated to dryness and the residues dissolved in hexane prior to Florisil
column clean-up.

Urine was extracted three times with its own volume of diethyl ether after adjusting
to pH4.0 with glacial acetic acid. The combined ether extracts were dried over anydrous
sodium sulphate prior to evaporation to dryness and the residues were each dissolved in
20 ml of hexane. A subsample of the aqueous layer remaining after ether extraction was
mixed with an equal volume of 2N sulphuric acid and refiuxed for 2 hr, before repeating
the extraction with ether as described above. The final hexane solutions resulting from
ether extracts were cleaned up by column chromatography and, where appropriate, their
radioactivity was determined by scintillation counting.

The following were examined by liquid scintillation counting: Urine samples, organic
extracts of urine, feces, bird droppings and liver preparations, and solvent fractions result-
ing from column chromatographic separation. One-half-ml aliquots were added to 10 ml
of dioxan based liquid scintillator K354 (Koch Light) in a 20-ml counting vial. The
samples were counted on a Tracer Lab instrument. Efficiency of counting was determined
by use of an external standard, making reference to a quenching curve. A standard solu-
tion of C 14 DDT was used for constructing the quenching curve.

Infrared spectroscopy. HCE was incorporated into a potassium bromide disc, while
HHC and HCE trans-dihydrodiol were dissolved in carbon tetrachloride and introduced
into micro cells. The spectra were measured on a Unicam SP200 instrument.

Results
In vitro studies. All the values cited in Tables II-V represent averages for three or more
replicate incubations. The percentage of oxidation and hydration of HCE in 11,000 G
supernatants /and liver microsomes under standard conditions are reported in Tables II
and III. The percentage of aldrin epoxidized to dieldrin by the same preparations is also
given where measured. The exclusion of NADPH from microsomal preparations, and the
exclusion of oxygen and NADPH from supernatants strongly inhibited oxidation.

With both microsomes and supernatants from the rabbit, and supernatants from the
rat and quail, diol formation increased when oxidative metabolism was inhibited. This is
apparently the result of greater availability of substrate, since all of these preparations
showed strong oxidizing activity (31-73 percent conversion of substrate) in the presence
of NADPH and oxygen.
 Table II. Oxidation and hydration of HCE and epoxidation of aldrin by liver microsomes

Mg microsomal HCE metabolism Aldrin metabolism

protein per % HCE not % HCE converted Estimated % % Aldrin
Species Cofaetors incubation converted to diol oxidation a expoxidized

+NADPH 11 16 73
Rabbit 3.6
-NADPH 41 56 3
+NADPH 71 1 28 97
Rat 8.0
-NADPH 96 1 3

Quail +NADPH 4.8 24 0.4 15 48

-NADPH 98 0.4 1
+NADPH t0 0 90 est 25
Pigeon -NADPH 5.8 80 0 20 ~"

+NADPH 2.25 38 12 50
Rook -NADPH 75 15 10 51
Jackdaw - 12 3 85 80
Fulmar - 27 2 71 53 o
¢3
aFor atl samples fortified with NADPH, % oxidation of HCE was estimated by subtracting (% HCE recovered unchanged + % HCE
converted to diol) from 95%, which was the total recovery % of HCE + metabolites estimated by radio counting. For samples
without NADPH, % oxidation was estimated where possible from quantity of HHC formed, this being the only identifiable
metabolite.
Microsomes:
Incubation medium comprised 4.0 ml reaction medium + 0.5 ml microsomes + 0.2 ml NADPH generating mixtures (0.2 ml reaction
medium for non-oxidative runs) + 20 #1 of 2 mg/ml substrate. Incubation time 30 minutes, incubation temperatures 37°C for
mammals, 42°C for birds.
106 C. H. Walker and G. A. E1 Zorgani

Table III. Oxidation and hydration o f HCE and epoxidation of aldrin by 11,000 G
supernatantsa derived from liver homogenates

% HCE Estimated
Incubation % HCE not converted % HCE % Aldrin
Species conditions converted to diol oxidized epoxidized

Cofactors/O 2 5 20 75 87
Rabbit No cofactors/N 2 73 25 < 2 12

Cofactors/O 2 48 13 39 78
Rat No cofactors/N 2 84 15 <~ 2 3

Cofactors/O 2 67 2 31 65
Quaff No cofactors/N 2 96 3 <~ 2 3

Cofactors/O2 5 0 95 58
Pigeon No cofactors/N 2 98 0 < 2 2

alncubation medium comprised 2 ml supernatant + 3 ml phosphate buffer pH 7.4 (with

cofactors for oxidative conditions; without cofactors under anaerobic conditions for non-
oxidative conditions). Incubation times: 90 minutes for HCE, 30 minutes for HHDN;
incubation temperatures as for microsomes. Estimation of percentage oxidation - as for
microsomes.

Table IV. Metabolism of liCE under oxidative conditions

Rabbit Rat Pigeon Quail

Microsomes
% HCE metabolized 73 27 27 17

Different Metabolites as a Percentage of Total Metabolites

Trans diol 29 5 0 8

Hydroxy epoxide (HHC) 33 95 90 92

Other oxidative products (UI) 20

(U2)

Secondary metabolites )
which can be formed ) (U3) 4
from hydroxy epoxide )
in presence of NADPH ) (Dihydroxy HCE) 10 a 4 a

aFormed from HCE oxidatively by microsomes where more than 70% of the substrate had
been converted.
Substrate 40 /~g C 14 labelled HCE (Sp. activity 0.5/ac/mg). NADPH generating mixture
included. Incubation time 30 rain.
 Metabolism and Excretion of HCE 107

Table V. Metabolism o f liCE under oxidative conditions (11,000 G supernatants)

Rabbit Rat Pigeon Quail

% HCE metabolized 95 48 95 67

Different Metabotites as a Percentage o f Total Metabolites

Symbol or
number
Main hydroxy epoxide (HHC) 37 55 42 67

Metabolites of unknown structure (U1) 18

formed under oxidative conditions (w2) 11

Secondary metabolites formed from (U3) 3a a a

hydroxy epoxide in presence of 02 (Dihydroxy HCE) 12 a a 34 a a

aFormed from HHC under oxidative conditions

Although all species showed quite strong mixed function oxidase activity, the capacity
for epoxide hydration was extremely variable between species, with the rabbit, rat and
rook producing the most diol followed by the jackdaw, quail and fulmar petrel. The
pigeon showed no measurable hydrase activity. Rabbit microsomes did not hydrate the
( - ) enantiomer of HCE to any measurable extent.

The relative amounts of hydration and oxidation carried out by microsomes and
11,000 G supernatants were similar for the rabbit, quail and pigeon. In the rat, however,
relatively more hydration occurred in the I 1,000 G supernatants than in the microsomes.
In all cases the 11,000 G supernatants achieved more oxidation and hydration per unit
weight of liver than did the corresponding microsomal fractions.

The effect of pH on the balance between oxidation and hydration was studied using
11,000 G supematants prepared from rabbit liver (Fig. 2). The rate of oxidation was
relatively slow between pH 4.5 and 6.5 and the rate of hydration was of a similar order.
At pH 7.5, however, the rate of oxidation greatly increased and the rate of hydration
correspondingly decreased, probably as a result of competition for substrate. Further in-
creases in pH brought about a decline in the amount of oxidation with little change in the
amount of hydration until pH 10.5 was reached when it suddenly increased. Thus maxi-
mal oxidation relative to hydration was found in the vicinity of pH 7.4.

Turning now to the oxidative metabolism of HCE in vitro the major hydroxy epoxide
HHC was the most abundant metabolite in both microsomes and I 1,000 G supernatants
of all species and accounted for 43-89% of the total oxidative metabolites (Tables IV
and V). Two further oxidative products were limited to particular species - one to the
rabbit (U1) and one to the pigeon (U2). Finally there were two other compounds which
occurred in microsomes and 11,000 G supernatants where the metabolism of HCE was
108 C.H. Walker and G. A. E1 Zorgani

greater than 73 percent; these were dihydroxy HCE and U3. Dihydroxy HCE is a product
of oxidative attack upon HHC (Walker and Zorgani (1973), and U3 is also a secondary
metabolite.

In vivo studies. The recovery of C14 in urine and bile, and in extracts of feces and
droppings is shown graphically in Figure 3, each point representing an average of three or
more animals. The maximum rate of loss of radioactivity in urine and extracts of feces
was noted on the first day after dosing for the rabbit and on the fourth day after dosing
for the rat. There was a striking difference between the two species with regard to the
preferred route of excretion. The rabbit excreted 92 percent of administered radio-
activity in the urine alone within 5 days, while less than one percent was found in the
feces. In the rat 34.8 percent of administered radioactivity was found in the feces, while
only 3.8 percent was found in the urine. The extraction of C14 from rat feces was ineffi-
cient here but in subsequent experiments with ethanol extraction 59-71% of the C 14 dose
was recovered within 8 days (Kenny 1973).

In the case of the quail and the pigeon, extraction of droppings using 2:1 acetone:
hexane proved to be very inefficient. After 21 days collection only 11 and 9 percent of
the administered radioactivity were recovered in the case of the quail and the pigeon,
respectively. In later experiments, employing ethanol extraction, 85 and 74 percent of
the radioactivity were recovered after 15 days in the quaff and pigeon, respectively
(Fig. 3).

Of the radioactivity excreted in rabbit urine only 35 t o 40 percent was extractable

directly by ether. Most of the remainder partitioned into ether after refluxing with acid
suggesting that most of the excreted material was in the form of conjugates. Acid hy-
drolysis released considerable quantities of HHC and a polar compound not previously
encountered (see last paragraph, this section).

100- x - - x - - x Oxidation
= - = Hydration
X.~X
80-

,- X

o~ 60-
.o ~ X
40-

20-
lP

0
4.0 5.0
I
6.0
I
7.0
I
8.0
I
9.0
|
10.0
I

pH
11.0
'I

Fig. 2. The effect of pH on the hydration and oxidation of HCE by rabbit liver l l,O00 G
supernatant.
% Total 25- % Total dose 50% of dose
dose recovered Excreted within
recovered Pigeon % Total
20- 20- Rat 39
per dose
day Rabbit 93 2 days
15- recovered
15- . Pigeon 74 8 days
per day
85 4 days
10- 10-

5- 5-

0 I I I I I 1' I I I 0 I '' I I I I 1 I
0 2 4 6 8 10 12 14 t6 18 0 2 4 6 8 10 12 14
Days after dosing Days after dosing
cr
% Total
dose 4 0 - *%.
recovered
per day 35- ;e Rabbit Extracts of feces
and droppings N
30- • c~
% Total Bile
dose 25- 25- •
recovered ,, , . . • • • Urine
per day 20- Rat 20- "
o Cb
15- 15'

10- 10- "o

eo
5- e•
• o o
,*°eeeeeeoeeeom
0 i t i J l I I I I
0 2 4 6 8 10 0 2 4 8 6
Days after dosing Days after dosing
Cannulation of bile duct - more than 40% of dose eliminated within 4 days in bile and urine
Fig. 3. Recovery of injected C 14 in urine, bite, and extracts of feces and droppings. o
~o
110 C.H. Walker and G. A. E1 Zorgani

After picking up the residues from ethanol extracts of quail droppings in dilute acid,
52 percent of the total excreted radioactivity partitioned directly into ether (hencefor.
ward called 'ether-extractable material'), while a further 9 percent was extractable after
refluxing, and 9 percent remained in the aqueous layer. Thus, some 30 percent of the
extracted radioactivity was not accounted for in these fractions. Also much highet pro-
portion was recovered as ether-extractable material from droppings collected in the first 4
days (88 percent) compared with those collected during the rest of the experiment (2t to
30 percent). In the latter case, a considerable amount of radioactivity was easily volati-
lized, most notably from extracts which were reduced by rotary evaporation. Here most
of the missing radioactivity was found in the distillate. Loss of material during reduction
of volume was probably the main reason for the rather low recoveries when extracts were
evaporated under nitrogen. Ether-extractable material was cleaned up by column chro-
matography and the separate fractions collected and counted (Table VI). Fraction 1 was
mainly HHC while fractions 2 and 3 consisted principally of more polar compounds.

The metabolites isolated from urine, feces and droppings are summarized in Table VII,
and the estimated quantities are expressed as a percentage of the original dose given.
Material obtained by partition into ether after acid hydrolysis is included here. To deter-
mine the quantities of metabotites, cleaned-up fractions were run on thin layer plates
and appropriate areas removed and extracted. The radioactivity was then determined for
each metabolite, and expressed as a percentage of the total radioactivity recovered from
the thin-layer plate for the fraction in question. From this it was possible to estimate the
total quantity of metabolite recovered. In certain cases complete separation of metabo-
lites was not achieved, and here percentages are only given within estimated limits. Pure
samples of HHC and diol were used for direct determination of these compounds in
Florisil fractions, and the results so obtained were in reasonable agreement with those
determined by radio counting (Table VII).

HHC proved to be the most abundant isolated metabolite for all species investigated,
whereas the diol was only found in small quantities in rabbit urine and in rat and quail
droppings. Dihydroxy HCE occurred in reasonable quantities in both rabbit urine and
quail droppings, while U3 was found in rabbit urine only. In addition to these compounds
which were also produced under in vitro conditions several other metabolites more polar
than HHC were found. By far the most abundant of these was the polar compound noted
earlier which was released after acid hydrolysis of rabbit urine, and accounted for 36 per-
cent of the original dose. It is possible that this compound is an artifact produced during
acid hydrolysis since it was not found in the first ether extract of urine.

Discussion
In the in vivo experiments the average percentage of the injected C14 recovered in sol-
vent extracts of feces and droppings and in urine was 38.6, 92.4, 74 and 85 percent for
the rat, rabbit, pigeon and quail respectively (Fig. 3). With improved extraction, 59-71
percent of the dose was recovered from rat feces within 8 days (Kenny 1973). With the
exception of the early results with the rat, these values do not differ greatly from the
recovery percentages encountered in in vitro experiments, although there were significant
 Table VI. Quail droppings." Separation of liCE metabolites from ether fractions by Florisil column chromatography

Radioactivity present in ether extractable

Period material as a percentage of total ethanol - Radioactivity recovered in Florisil fractions expressed
of collection extractable radioactivity as a percentage of total radioactivity applied to column
e
Fraction 2
Fraction 1 23:1:1 Fraction 3
20% v/v Ether:Hexane Ether: Methanol: Acetone Absolute Ethanol

0-4 days 88 50 35 8
¢3
5-7 days 21 17 30 44

9-12 days 30 10 32 8

13-15 days 28 15 - 48

Metabolites Some HHC, DIOL, Dihydroxy HCE

found in Mainly HHC Dihydroxy HCE and other
fraction and other unknown unknown
compounds compounds
112 C.H. Walker and G. A. E1 Zorgani

losses during subsequent clean-up. Half of the injected dose was cleared within the stated
periods for the following species: rabbit (2 days), quail (4 days) and pigeon (8 days).
With cannulated rats, more than 40 percent of the injected dose was cleared in the bile
and urine within 4 days of treatment (Kenny 1973). The half-lives of HCE should come
well within these times. By contrast dieldrin has a half-life in adipose tissue of 10.3 days
in male rats (Robinson et al. 1969) and 47 days in pigeons (Robinson et al. 1967). This
difference is probably explained by the fact that HCE is degraded much more rapidly by
liver preparations than is dieldrin (Brooks et al. 1970, Matthews and Matsumura 1969).
Relatively rapid metabolism in vitro is associated with relatively rapid clearance in vivo.

In the pigeon there was an increase in the rate of clearance about the fifth day after
injection. In the birds showing this effect, there was also a sharp fall in food intake at the
same time, suggesting that it may be connected with mobilization of fat depots. Alter-
natively it may be due to enzyme induction.

HHC in the free and/or conjugated state was the principal metabolite in all cases in
vitro, and was excreted by all species in vivo. It was the main excreted metabolite in the
rabbit, and the most abundant of the metabolites isolated from rat feces and quail
droppings (Table VII). The diol was found in the feces, droppings or urine of the rat,
rabbit and quail; relatively less diol with respect to other metabolites was found in the
urine than in liver preparations with the rabbit. Dihydroxy HCE was produced in vivo by
rabbit and quail; the failure to isolate it from rat feces may be connected with in-
efficiency of extraction (Table VII). U3 was only found in rabbit urine. Thus, in vitro
studies showed some promise for prediction of primary metabolites of HCE in vivo.

Although the main primary metabolites produced in vitro in the rabbit and quail were
also found in vivo, there were differences in the metabolic pattern between the two situa-
tions. Relatively less diol was found in vivo than in vitro with the rabbit. There was evi-
dence of labelled material (including HHC) existing in conjugated form(s) in rabbit urine
and quail droppings. Indeed more than 50 percent of the total injected dose in the rabbit be-
came extractable from urine only after acid hydrolysis. By contrast most of the products
of HCE in microsomes and liver supematants were immediately extractable, indicating
little or no conjugate formation here. (It is desirable to add suitable cofactors to study
conjugations in microsomal systems and supernatants, as in the studies on dieldrin
metabolism by Matthews and Matsumura 1969). Not surprisingly a wider range of com-
pounds was excreted than was produced in vitro and it is possible that some of the
metabolites in feces and bird droppings are produced by microbial action.

The formation of dihydroxy HCE in liver microsomes and 11,000 G supematants by

secondary oxidative attack on HHC has been described elsewhere (Walker and E1 Zorgani
1973). This reaction was not found to occur until much of the HCE substrate had dis-
appeared, suggesting that HCE competes effectively with its more polar metabolite for
sites of mixed function oxidation. It was found only in relatively small amounts in quail
droppings and rabbit urine, and it would be interesting to know whether the conjugation
of HHC in vivo limits its oxidation. It is likely that HHC undergoes some enterohepatic
circulation in the rat, and the question arises as to whether HHC returned to the liver
 Table VII. Excreted HCE metabolites - estimated percentage o f original dose to animals

Dihydroxy
Species HHC U1 U3 HCE DIOL Others Total Notes

Rabbit 42 5.0 1.0 6.3 2.2 36 a 92 Nearly all C 14 excreted in urine.

Figures quoted here include ether
extractable material before and after
acid hydrolysis

Rat 29 0 0 1.9 6.7 37.5 This includes all the material extracted
by 2:1 acetone:hexane from feces
¢3

Quail 20(18) 0 0 6 <2(1) 21 48 This represents the material P,

~.,°
partitioning into ether after reduction O

of ethanol extracts to zero

Pigeon 5.7 3 8.7 This only represents material extracted ¢3

by 2: t acetone:hexane from droppings

aMost of this accounted for by a single compound produced only by acid hydrolysis
Figures in brackets directly determined by gas chromatography

%0
114 C.H. Walker and G. A. E1 Zorgani

tends to be conjugated, oxidized or both. In view of this, it is desirable to follow the

time course of reaction until most of the substrate has gone so that any secondary oxida-
tion products can be identified, if in vitro studies are to be used to predict events in vivo.

The difference between the rat and the rabbit with regard to preferred route of excre-
tion has also been reported for dieldrin (Baldwin 1971, Korte and Arent 1965, Baldwin
et al. 1972) trans chlordane, and heptachlor epoxide (Klein 1965) and may be explained
by the work of Hirom et al (1972) and Smith (1971). The study of a range of organic
anions revealed that different species have different threshold values with respect to the
molecular weight of compounds that can be excreted in the bile. Below the threshold mole-
cular weight most excretion occurs via the urine while above it significant excretion
occurs via the bile ( > 10%) (Hirom et al. 1972). It has been suggested that a secondary
form of bile is produced by selective reabsorption of relatively small molecules and ions
from primary bile (Clark et al. 1971). The rabbit was found to have a much higher thresh-
old value (475 -+ 50) than the rat (325 -+ 50), and HHC has a molecular weight of 385
which falls between these two thresholds. Thus HHC and its conjugates may be expected
to be excreted in the bile of the rat. In the rabbit, free HHC should be excreted in the
urine. On the other hand there is evidence for HHC conjugates in rabbit urine, and the
question arises as to whether there are conjugates above the threshold molecular weight
in urine, and if so whether they are formed in the liver. The possibility of explaining this
difference in preferred excretory route for other organochlorine metabolites on the same
grounds has been discussed (Walker 1973).

This interspecific difference raises some important issues. Where metabolites and con-
jugates are excreted in the bile there is the possibility of microbial action and reabsorp-
tion. Microbial action can produce more toxic compounds than were originally excreted
(e.g. the thyrotoxic metabolite of chloramphenicol)(Smith 1971). It can also cause the
breakdown of conjugates with concomitant release of metabolites that are easily absorbed
from the gut and recirculated to the liver (Smith 1971). Furthermore, material excreted
in the bile takes longer to be expelled from the body than material excreted in the urine.
Accordingly the removal of metabolites is likely to be more rapid and efficient via the
urine than v& the feces, and this may partially explain the more rapid clearance of HCE
by the rabbit than by the rat. However, it should also be borne in mind that the rabbit
showed greater oxidative and hydrative activity in the liver preparations.

The metabolism of HCE resembles that of dieldrin in certain respects. Pig and rabbit
liver microsomes can hydrate dieldrin to a trans-dihydrodiol in the absence of NADPH
and oxygen, but only very slowly (Brooks et al. 1970). This diol is the main excreted
metabolite of dieldrin in the rabbit (Korte and Arent 1965), and is also excreted by the
rat (Baldwin et al. 1972). In studies of dieldrin metabolism with 10,000 G supernatants
derived from rat liver homogenates incubated in the presence of NADPH and oxygen,
Matthews and Matsumura (1969) reported that the main metabolites were oxidation
products, only small quantities of diol being produced. One of these oxidation products
was 9-hydroxy-dieldrin which appears to be the main excreted metabolite in the feces,
while another was a pentachloroketone which is excreted in the urine. Nine-hydroxy-
dieldrin can be conjugated to form a glucuronide in vivo (Baldwin 1971) and by rat
 Metabolism and Excretion of HCE 115

liver microsomes (Matthews and Matsumura 1969). Thus HCE and dieldrin can be metab-
olized by both hydration and oxidation, hydration tending to be stronger in the rabbit
than in the rat. The main oxidation product found in both cases is a monohydroxy
epoxide. On the other hand HCE is metabolized more rapidly than dieldrin in vitro and
the principal primary oxidation product HHC is converted to other, more polar com-
pounds, under conditions favouring oxidative attack (Robinson et al. 1967).

The hydrative capacity of supernatants and liver microsomes towards HCE shows
similar interspecific variations to those found for the related compound HEOM (Zorgani
et al. (1970). In both cases the pigeon shows the poorest activity, the rabbit shows the
greatest activity, and two members of the Corvidae, the rook and the jackdaw, show
more activity than the other birds tested. HEOM was hydrated more rapidly than HCE in
all cases. In view of these interspecific differences in HCE hydration, it would be interest-
ing to investigate the effect of an inhibitor of microsomal mixed function oxidation (e.g.
sesamex) upon the acute LDsos for HCE in this range of species. The magnitude of any
synergistic effect is likely to depend upon the extent to which hydration can provide an
alternative detoxication pathway to microsomal mixed function oxidation.

Acknowledgments

The authors are grateful to the Science Research Council for a grant for the purchase
of a gas chromatograph, to Mr. F.S. Jones (M.A.F.F.) for supplying rooks and jackdaws,
and to Dr. G.T. Brooks for gifts of C14 labelled hexachlorocyclopentadiene and the ( - )
enantiomer of HCE.

References

Baldwin, M. K.: Ph.D. Thesis, Univ. of Surrey (1971).

Baldwin, M. K., J. Robinson, and D. V. Parke: A comparison of the metabolism of HEOD
(dieldrin) in the CF1 mouse with that in the CFE rat. Food Cosmet. Toxicol. 10,
333 (1973).
Brooks, G. T.: Progress in metabolic studies of the cyclodiene insecticides and its rele-
vance to structure-activity correlations. World. Rev. Pest Control 5, 62 (1966).
Brooks, G. T.: Investigations with some biodegradable dieldrin analogues. Proc. 5th Br.
Insect. Fungicide Conf. 472 (1969).
Brooks, G. T.: Pathways of enzymatic degradation of pesticides. Environ. Quality Safety
1,106 (1972).
Brooks, G. T. and A. Harrison: The effect of pyrethrin synergists especially sesamex on
the insecticidal potency of hexachlorocyclopentadiene derivatives (cyclodiene insec-
ticides) in the adult housefly. Biochem. Pharmacol. 13, 827 (t964).
116 C.H. Walker and G. A. E1 Zorgani

Brooks, G. T., A. Harrison and S. E. Lewis: Cyclodiene epoxide ring hydration by micro-
somes from mammalian liver and houseflies. Biochem. Pharmacol. 19,255 (1970).
Clark, A. G., P. C. Hirom, P. Millburn and R. T. Williams: Absorption of some organic
compounds from the bitiary system of the rat. J. Pharm. Pharmacol. 23, 150
(1971).
Edwards, C. A.: Persistent Pesticides in the Environment. London: Butterworth (1970).
E1 Zorgani, G. A., C. H. Walker and K. A. Hassall: Species differences in the in vitro
metabolism of HEOM - a chlorinated cyclodiene epoxide. Life Sci. 9, 415 (1970).
Hirom, P. C., P. Millburn, R. L. Smith and R. T. Williams: Species variations in the
threshold molecular weight factor for the biliary excretion of organic anions. Bio-
chem. J. 129, 1071 (1972).
Kenny, J.: unpublished results (1973).
Klein, W.: Metabolism of chlorinated insecticides. Radioisotopes in the detection of pesti-
cide residues. F.A.O./I.A.E.A. Div. of Atomic Energy in Agr., Vienna~(1965).
Korte, F. and H. Arent: Metabolism of insecticides IX. Isolation and identification of
dieldrin metabolites from urine of rabbits after oral administration of dieldrin 14C.
Life Sci. 4, 2017 (1965).
Matthews, H. B. and F. Matsumura: Metabolic fate of dieldrin in the rat. J. Agr. Food
Chem. 17, 845 (1969).
Oesch, F., C. R. Creveling, D. M. Jerina and J. W. Daly: Purification and properties of a
hepatic epoxide hydrase. Fed. Proc. 29,473 (1970).
Oesch, F., N. Kaubisch, D. M. Jerina and J. W. Daly: Hepatic epoxide hydrase. Structure-
activity relationships for substrates and inhibitors. Biochem 10, 4858 (1971).
Robinson, J., A. Richardson and V.K.H. Brown: Pharmacodynamics of dieldrin in
pigeons. Nature 213, 734 (1967).
Robinson, S., M. Roberts, M. K. Baldwin and A.I.T. Walker: The pharmaco-kinetics of
HEOD (dieldrin)in the rat. Food Cosmet. Toxicol. 7, 317 (1969).
Smith, R. L.: Excretion of drugs in the bile. Handbook of Experimental Pharmacology
XXVIII/1,354. Springer Veflag (1971).
Walker, C. H.: Comparative aspects of the metabolism of pesticides. Environ. Qual. Safety
3. In press (1973).
Walker, C. H. and G. A. E1 Zorgani: Comparative metabolism of dieldrin analogues.
Environ. Qual. Safety 1,248 (1972).
Walker, C. H. and G. A. E1 Zorgani: Metabolism of a dieldrin analog - secondary oxida-
tion by liver microsomes. Life Sci. 13, 585 (1973).