EPA-600/1-76-003
January 1976
Environmental Health Effects Research Series
EFFECT OF DUCK HEPATITIS VIRUS ON
PESTICIDE TOXICITY
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating
to the tolerances of man for unhealthful substances or conditions.
This work is generally assessed from a medical viewpoint, including
physiological or psychological studies. In addition to toxicology
and other medical specialities, study areas include biomedical
instrumentation and health research techniques utilizing animals -
but always with intended application to human health measures.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.
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EPA-600/1-76-003
January 1976
EFFECT OF DUCK HEPATITIS VIRUS ON PESTICIDE TOXICITY
By
W. L. Ragland
Poultry Disease Research Center
University of Georgia
953 College Station Road
Athens, Georgia 30601
Grant No. R801800
Project Officer
Dr. Ronald L. Baron
Environmental Toxicology Division
Health Effects Research- Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement
or recommendation for use.
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ABSTRACT
This report presents the results of an investigation into the effect of
viral replication on hepatic endoplasmic reticulum on the activities of the
microsomal drug-metabolizing enzymes of liver. Two viruses which repli-
cate on hepatic endoplasmic reticulum without producing hepatic disease
were used. They were Newcastle disease vaccinal virus in chickens and
duck hepatitis virus in adult ducks. The microsomal enzymes assayed
were ethylmorphine N-demethylase, aryl hydrocarbon hydroxylase and
aniline hydroxylase. The soluble enzyme, nitroreductase, also was assayed
in chickens. Pesticide residue analysis was used to evaluate indirectly the
microsomal enzymatic conversion of DDT to DDD and the soluble enzymatic
conversion of DDT to DDE in ducks.
Viral replication did not increase the activities of the microsomal
enzymes and may have decreased the activities slightly. Viral replication
prior to chemical induction of enzymes in chickens by phenobarbital and
in ducks by DDT resulted in greater activities of the microsomal enzymes
but not the soluble enzymes than was obtained by chemical induction with-
out prior viral replication. Although the microsomal enzymes were induced
to greater levels by viral replication, pesticide residue analysis failed to
demonstrate increased clearance of residues from body tissues.
This report was submitted in fulfillment of Grant No. R801800 by the
Poultry Disease Research Center, University of Georgia under the sponsor-
ship of the Environmental Protection Agency. Work was completed as of
July, 1974.
m
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CONTENTS
Abstract iii
List of Figures v
Acknowledgments ^ vi i i
Sections
I Conclusions 1
II Recommendations 2
III Introduction 3
IV Analytical Methods 7
V Effect of Newcastle Disease Vaccinal Virus on the Induction
of Drug-Metabolizing Enzymes in Chicken Liver 23
VI Effect of Duck Hepatitis Virus on the Induction of Drug-
Metabolizing Enzymes in Duck Liver and on Tissue Residue
of DDT. 33
VII Effect of Newcastle Disease Vaccinal Virus on the Induction
of Drug-Metabolizing Enzymes and on Tissue Residue of
Dieldrin in Chickens. 51
VIII Discussion 54
IX References 59
X List of Inventions 62
iv
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FIGURES
No.
1. First order kinetics with time of N-demethylation of ethyl-
morphine by 1.0 ml of liver microsomes from chickens induced
with phenobarbital. 9
2. First order kinetics with enzyme concentration of N-demethyla-
tion of ethylmorphine in 15 min reaction. 10
3. First order kinetics with time of aryl hydrocarbon hydroxyla-
tion of benzo(a)pyrene by 0.3 ml liver postmitochondrial
supernatant fraction from chickens induced with 3-methyl-
cholanthrene. 11
4. First order kinetics with enzyme concentration of aryl hydro-
carbon hydroxylation of benzo(a)pyrene in 15 min reaction. 12
5. First order kinetics with time of aryl hydrocarbon hydroxyla-
tion of benzo(a)pyrene by 0.1 ml liver microsomes from ducks
induced with phenobarbital. 13
6. First order kinetics with time of aryl hydrocarbon hydroxyla-
tion of benzo(a)pyrene by 0.1 ml liver microsomes from ducks
induced with 3-methylcholanthrene. 14
7. First order kinetics with enzyme concentration of the aryl
hydroxylation of benzo(a)pyrene in 5 min reaction. 15
8. First order kinetics with time of aniline hydroxylation by 1.0
ml of liver microsomes from chickens induced with phenobarbital. 17
9. First order kinetics with enzyme concentration of aniline
hydroxylation in 20 min reaction. 18
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No. Page
10. First order kinetics with time of nitroreduction of p-nitrobenzoic
acid by 1.0 ml liver postmitochondrial supernatant fraction from
chickens induced with phenobarbital. 19
11. First order kinetics with enzyme concentration of p-nitrobenzoic
acid in 20 min reaction. 20
12. Effect of Newcastle disease vaccination on hepatic microsomal
ethylmorphine N-demethylase of eight-week-old pullets. 25
13. Effect of Newcastle disease vaccination on hepatic microsomal
aryl hydrocarbon hydroxylase of eight-week-old pullets. 26
14. Effect of Newcastle disease vaccination on hepatic microsomal
aniline hydroxylase of eight-week-old pullets. 27
15. Effect of Newcastle disease vaccination on hepatic nitroreductase
of eight-week-old pullets. 28
16. Effect of Newcastle disease vaccination on hepatic microsomal
ethylmorphine N-demethylase of eight-week-old cockrels. 29
17. Effect of Newcastle disease vaccination on hepatic microsomal
aryl hydrocarbon hydroxylase of eight-week-old cockrels. 30
18. Effect of Newcastle disease vaccination on hepatic microsomal
aniline hydroxylase of eight-week-old cockrels. 31
19. Effect of Newcastle disease vaccination on hepatic nitroreductase
of eight-week-old cockrels. 32
20. Feed consumption of DDT contaminated feed by ducks injected
with duck hepatitis virus or buffer. 34
21. Body weights at time of killing of ducks injected with duck
hepatitis virus or buffer and fed 900 ppm DDT. 35
22. Liver weights at time of killing of ducks injected with duck
hepatitis virus or buffer and fed 900 ppm DDT. 36
23. Changes in body weight during DDT treatment of ducks in-
jected with duck hepatitis virus or buffer. 37
24. Amount of microsomes per gm duck liver with time fed 900 ppm
DDT. 39
25. Activities of ethylmorphine N-demethylase in livers of ducks
injected with duck hepatitis virus or buffer and fed 900 ppm
DDT for 12 days. 40
vi
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No. Page
26. Activities of aryl hydrocarbon hydroxylase in livers of ducks
injected with duck hepatitis virus or buffer and fed 900 ppm
DDT for 12 days. 41
27. Activities of aniline hydroxylase in livers of ducks injected
with duck hepatitis virus or buffer and fed 900 ppm DDT for
12 days. 42
28. Total pesticide residue (DDT+DDD+DDE) in serum, bile and brain
of ducks injected with duck hepatitis virus or buffer and fed
DDT for 12 days. 43
29. Pesticide levels in serum of ducks injected with duck hepatitis
virus or buffer and fed DDT for 10 days. 44
30. Concentration of DDT and DDE in bile of ducks injected with
duck hepatitis virus or buffer and fed DDT for 12 days. 45
31. Concentration of DDE relative to total pesticide in bile of ducks
injected with duck hepatitis virus or buffer and fed DDT for
12 days. 47
32. Concentration of DDD and concentration of ODD relative to total
pesticide in bile of ducks injected with duck hepatitis virus or
buffer and fed DDT for 12 days. , 48
33. Ratio of DDD to DDE concentrations in bile of ducks injected
with duck hepatitis virus or buffer and fed DDT for 12 days. 49
34. Concentration of DDT, DDD, and DDE residues in brains of ducks
injected with duck hepatitis virus or buffer and fed DDT for
12 days. 50
35. Ethylmorphine N-demethylase and aryl hydrocarbon hydroxylase
activities of hepatic microsomes from dieldrin-contaminated
f
chickens treated with Newcastle disease vaccinal virus and
sodium phenobarbital. 53
vn
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ACKNOWLEDGMENTS
Mr. Tom Sparks of McCarty Enterprises, McGee, Mississippi is
thanked for supplying the dieldrin-contaminated chickens.
VTM
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SECTION I
CONCLUSIONS
The replication of Newcastle disease vaccinal virus in the livers of
chickens and the duck hepatitis virus in the livers of adult ducks facilitates
the induction of hepatic microsomal enzymes by subsequent chemical induc-
tion . The increased levels of activity do not persist and appear to be modu-
lated by other factors such as the viral replicatory cycle. Increased
clearance of pesticide from body tissues could not be demonstrated in virus-
treated birds even though the microsomal drug-metabolizing enzymes were
induced. Although replication of duck hepatitis virus in adult ducks
causes specific changes in the hepatic, microsomal, drug-metabolizing
enzymes, the results of the present investigation do not support the theory
that these changes are responsible for the protection against pesticide
toxicity provided by duck hepatitis virus.
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SECTION II
RECOMMENDATIONS
The specific interrelationships between viral replication on hepatic
endoplasmic reticulum and the drug-metabolizing enzymes of the hepatic
endoplasmic reticulum should continue to be investigated. Whether these
phenomena are of major importance from the point of view of environmental
protection or of more importance from a clinical health services point of
view must be evaluated by the appropriate governmental agencies. They
certainly are of considerable biological importance and should be investiga-
ted for the basic information to be gained if not for any other reason.
The effect of exposure to xenobiotic compounds on subsequent viral
replication should be investigated in addition to the effect of viral replica-
tion on induction of the drug-metabolizing enzymes.
In spite of the study reported here and the reports of others in the
literature, a good experimental model system for the study of the inter-
relationships between microsomal drug-metabolizing enzymes and viral
replication is still lacking. A good experimental model must first be found
and characterized before any substantial progress can be made in this
interesting area of research.
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SECTION III
INTRODUCTION
GENERAL
Xenobiotic compounds such as drugs, chemical pesticides and indus-
trial pollutants will induce elevated activities of a number of enzymes collec-
tively known as the drug-metabolizing enzymes. Since the first description
of drug-metabolizing enzyme activity by Mueller and Miller a great deal
of effort has been devoted to these enzymes and their inducers. So much
attention has been given to the chemical inducers that it has been assumed
by many investigators that chemical agents are the only significant modulators
of drug-metabolizing enzyme induction. Nevertheless, it is becoming
increasingly evident that profound interrelationships exist between certain
viruses and the hepatic enzymes which metabolize drugs and pesticides.
It is obvious that any infectious disease which causes frank hepatic necrosis
or severe cachexia will alter drug metabolism. There are scattered in
the literature, however, reports of specific effects of viral replication
on drug metabolism. The main characteristics which these viruses have
in common are (1) they are RNA viruses and (2) they replicate and/or
are assembled on hepatic smooth endoplasmic reticulum. Very pronounced
morphologic changes are produced by some of these viruses in the endoplas-
2
mic reticulum and, hence, it seems plausible, even likely, that significant
functional alterations would result from the morphologic changes. The
published reports of viruses affecting hepatic drug-mebabolism have been
summarized below.
Murine Hepatitis
3
Kato et al. reported that murine hepatitis viruses in Swiss mice
caused initially a stimulation of hexobarbital and strychnine metabolism
-------
which was followed by a steady decline in activity. Approximately 12
hours after infection the metabolism of these drugs had been increased
25% and 24 hours after infection the activity had dropped to control levels.
There was a steady decline in activity and 72 hours following infection
the metabolism of these drugs had been decreased 50% below control levels.
Beyond this point there was a steady increase and return to normal levels.
Duck Hepatitis
4
Friend discovered that injection of adult mallards with duck hepatitis
virus increased resistance to DDT and dieldrin. Not only was the number
of deaths from pesticide toxicity decreased but the length of time on the
contaminated feed required to produce deaths was prolonged and deaths
were observed only during the last several days of the experiment. Had
the ducks been kept on the feed regimen longer, the mortality curve may
have been identical to controls, only delayed in its appearance. Considering
the possibility that an interaction between viral replication and the pesticide-
metabolizing enzymes of the hepatic smooth endoplasmic reticulum might
be the basis for the protection, Ragland et al. determined the effect of
duck hepatitis virus replication in adult ducks on ethylmorphine N-demethyla-
tion by postmitochondrial supernatant fractions of liver. Mallard drakes
were inoculated with duck hepatitis virus 48 hrs prior to exposure to DDT
in the diet for 7 days. At 7 days the level of enzyme activity was lower
in birds inoculated with the virus and fed a normal diet than in normal
birds fed control diet. Enzyme activity in non-virus-treated birds fed
DDT was induced about 4 x over normal birds fed a normal diet. Enzyme
activity in the virus-treated birds fed DDT was about 22% (based on per
gm liver) to 37% (based on per total liver) higher than in the non-virus-
treated birds fed DDT.
Infectious Hepatitis B of Humans
C
Doshi e_t al_. obtained liver biopsy material from patients in the
necrotic and regenerative phases of infectious hepatitis and measured
several parameters, one of which was pentobarbital hydroxylase. The
authors found that whereas pentobarbital hydroxylase activity was markedly
reduced during the necrotic phase of hepatitis it was greatly increased
during the regenerative phase. The decrease in the necrotic phase was
-------
understandable and had been anticipated but the increased activity which
followed was considered noteworthy as most investigators have found that
drug-metabolizing enzymes cannot be induced with chemicals during hepatic
regeneration. In speculating on the cause of the unexpected stimulation
of enzyme activity the authors mentioned the possibility of a specific effect
of viral replication. They further speculated that the elevated enzyme
activity may explain the difficulty experienced in the sedation of convalescent
hepatitis patients with barbiturates.
JUSTIFICATION AND OBJECTIVE OF THE PRESENT STUDY
The observations are significant in that they indicate that chemicals
may not be the only important environmental contaminants which alter
drug-metabolism. As our primary concern in infectious diseases has shifted
from acute and chronic infectious disease to attenuated infections (masked,
latent, inapparent infectious, microbial persistence, etc.) ,we have become
involved with the subtle, long-term effects (such as oncogenesis) of infectious
agents which do not produce overt infectious diseases. Such conditions
are known to occur. Persistent viral hepatitis in man is not an inconsequen-
tial matter. Another interesting example is the Ulster strain of Newcastle
disease virus. Ireland has remained free of Newcastle disease for years,
but when serologic surveillance of Irish birds was instituted, it was learned
that although Ireland was free of the disease, it was not free of the virus.
The apparent paradox was solved by the isolation and characterization
of the Ulster strain which is a non-pathogenic but immunogenic strain
of Newcastle disease virus whose existance had never been even suspected.
In this report we will demonstrate that vaccinal Newcastle disease virus
is another virus which alters hepatic drug-metabolism. There may be
additional viruses yet undetected which may have similar effects in various
species, including man. But it is not necessary to conjure up undiscovered
viruses. Most of the live virus vaccines, including human vaccines, utilize
RNA viruses which replicate on hepatic smooth endoplasmic reticulum,
and they may be affecting the hepatic drug-metabolizing enzymes. The
assumption that there are viruses which do affect hepatic drug-metabolism
in man, and even evidence that hepatitis type B virus does, the mechanisms
-------
involved need to be ascertained in order that the significance of their inter-
action with other non-infectious, environmental contaminants, i.e. chemicals,
can be evaluated.
Thus, the present study was undertaken to examine further the
interrelationship between duck hepatitis virus and pesticide toxicity.
It was necessary to modify the research as the project was funded for only
one year instead of the three years as proposed. The effects of viral replica-
tion of duck hepatitis virus in adult mallard drakes on several hepatic
drug-metabolizing enzyme activities and on tissue residues of DDT were
evaluated. The protocol was changed to include an investigation into the
effects of Newcastle vaccine virus replication on enzyme activities of chickens
since it is not a virus with preferential replication in liver and, if the
results were positive, they would provide evidence that specific effects
of viral replication on drug-metabolism is not confined to a small group
of hepatitis viruses.
-------
SECTION IV
ANALYTICAL METHODS
PREPARATION OF POSTMITOCHONDRIAL SUPERNATANT AND MICROSOMAL
FRACTIONS OF AVIAN LIVER.
All birds were killed by decapitation using a guillotine. Livers
were excised free of adhering tissue, rinsed, blotted and weighed. All
tissue preparations were maintained at 0-4 C. Liver weights were recorded
and 10 g portions taken for homogenization in 30 ml 0.15M KC1 using ten
strokes of a motor-driven teflon pestle in a Potter-Elvehjem homogenizer.
The resulting 25% (w/v) homogenates were centrifuged in a Sorval SS-
34 or Beckman JA20 rotor for 20 min at 10,000 x g max. The postmitochon-
drial supernatant fractions were decanted and aliquots saved for assay
of nitroreductase activity.
The greater portion of the postmitochondrial supernatant fraction
was spun for 20 min at 346,000 x g max in the Type 65 rotor of a Beckman
L2-65B ultracentrifuge. The clear supernatant fractions were discarded
and the microsomal pellets suspended in 0.15 M KC1 to the volumes of
the postmitochondrial supernatant fractions from which they were derived.
One milliliter of microsomal suspension usually contained 4-6 mg protein
7
(assayed by the method of Lowry, et td. using bovine serum albumin,
fraction V, as standard) and represented 300-350 mg fresh liver.
Enzyme Assays
Procedures were derived for assaying the activities of several drug-
metabolizing enzymes of avian liver. They were based on procedures
used for mammalian systems but modified to assure substrate excess and
first order kinetics with time and enzyme concentration. All assays of
-------
drug-metabolizing enzymes were carried out in 25 ml Erlenmeyer flasks
in a metabolic shaker bath maintained at 37 C. Except as noted, reaction
vessels were shaken at 120 cycles per min under air.
Ethylmorphine N-Demethylase
The N-demethylation of ethylmorphine by 1.0 ml of post mitochondria!
supernatant fractions or 1.0 ml of microsomes was assayed according to
o
the method of Sladek and Mannering. The formaldehyde produced was
9
measured by a modified method of Nash as described by Anders and Manner-
ing, except that a 5.5% rather than a 5.0% ZnSO.- 7H-O solution was
employed. First order kinetics were observed with time (Fig. 1) and
enzyme concentration (Fig. 2).
Aryl Hydrocarbon Hydroxylase
The assay procedure for the determination of aryl hydrocarbon (benzo-
(a)pyrene) hydroxylase activity was essentially that of Nebert and Gelboin.
Adjustments were made for a total reaction volume of 3 ml, and postmitochon-
drial supernatant fractions or microsomes were used instead of the cell
homogenate. In addition, bovine serum albumin, fraction V, (final concentra-
12
tion, 1 mg/ml) was included to solubilize the substrate (Alvares et al.).
When enzyme activities were assayed with fractions from chicken liver,
0.3 ml of enzyme was used and the reaction time was 15 min. First order
kinetics with time and enzyme concentration are demonstrated in Figs.
3 and 4. The amount of enzyme was reduced to 0.1 ml microsomes and
the reaction time was shortened to 5 min when assays were performed
with duck liver fractions in order to assure first order kinetcs (Figs.
5,6, and 7). Reactions were stopped with 3 ml cold acetone. Ten milliliters
of hexane were added and the mixtures incubated an additional 10 min
with shaking. A 2 ml sample of the organic phase was extracted with 6
ml IN NaOH and the products assayed in a Turner model 111 spectrofluorometer
equipped with filters selected for activation (Wratten 47B, maximum trans-
mittance at 430 nm) and fluorescence (Wratten 58, maximum transmittance
at 530 nm). Since several related products are measured in this assay,
the enzyme activities were recorded in arbitrary fluorescence units rather
13
than using the 8-hydroxy derivative as a standard. This method of
-------
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10 15
REACTION TIME, min.
20
25
Figure 1. First order kinetics with time of N-demethylation of ethylmorphine
by 1 .Q ml of, liver microsomes from chickens induced with phenobarbital.
-------
2.0
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o
X
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ENZYME CONCENTRATION, ml MICROSOMES
1.0
Figure 2. First order kinetics with enzyme concentration of N-demethylation
of ethylmorphine in 15 min reaction. Liver microsomes were prepared from
chickens induced with phenobarbital.
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REACTION TIME, min.
15
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Figure 3. First order kinetics with time of aryl hydrocarbon hydroxylation
of benzo(a)pyrene by 0.3 ml of liver postmitochondrial supernatant fraction
from chickens induced with 3-methyl-cholanthrene. A NADPH generating
system was used.
11
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O
LJ
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ID
O
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IT
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ENZYME CONCENTRATION, ml MICROSOMES
Figure 4. First order kinetics with enzyme concentration of the aryl hydro-
carbon hydroxylation of benzo(a)pyrene in 15 min reaction using NADPH
generating system. Liver microsomes were prepared from chickens induced
with phenobarbital.
12
-------
1000
246
REACTION TIME,
8
10
mm.
Figure 5. First order kinetics with time of aryl hydrocarbon hydroxylation
of benzo(a)pyrene by 0.1 ml of liver microsomes from ducks induced with
phenobarbital. NADPH was added to the reaction mixture.
13
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REACTION TIME, min.
Figure 6. First order kinetics with time of aryl hydrocarbon hydroxylation
of benzo(a)pyrene by 0.1 ml of liver microsomes from ducks induced with
3-methyl-cholanthrene. First order kinetics were obtained with 2 and 10
fold dilutions of microsomes. First order kinetics with enzyme concentration
is also demonstrated by amount of product formed for each of the three
enzyme concentrations assayed. A NADPH generating system was used.
14
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ENZYME CONCENTRATION, ml MICROSOMES
Figure 7. First order kinetics with enzyme concentration of the aryl hydro-
carbon hydroxylation of benzo(a)pyrene in 5 min reaction using NADPH.
Liver microsomes were prepared from ducks induced with phenobarbital.
15
-------
recording the enzyme activity has been discussed and justified by Hansen
14
and Fouts.
Aniline Hydroxylase
The reaction mixture used in the assay of aniline hydroxylase activity
contained, in a total of 5 ml, 50 mM phosphate buffer, pH 7.4, 10 mM aniline
(added in 0.1 ml acetone) ,0.4 mM NADP, 4 mM glucose-6-phosphate,
2 U glucose-6-phosphate dehydrogenase, 5 mM MgCl, and 1 ml microsomes.
The reactions were incubated for 20 min, stopped with 2.5 ml 20% TCA
and centrifuged to remove precipitated protein. The p-aminophenol produced
in the reaction was assayed by the method of Imai and Omura as follows:
1 ml of clarified reaction mixture was mixed with 1 ml 1% phenol in 0.5
M NaOH and 1 ml 1 M NagCO,. After 20 min the product was assayed spectro-
photometrically at 620 nm. First order kinetics with time and enzyme
concentration are demonstrated in Figs. 8 and 9.
Nitroreductase
The reaction mixture used for assay of nitroreductase consisted
of the following: 100 mM phosphate buffer, pH 7.4,2 mM p-nitrobenzoic
acid, 0.2 mM NADPH, 0.5 mM FMN, 5mM each of cysteine, niacinamide
and MgCl9, and 1 ml enzyme postmitochondrial supernatant fraction in
^ .
a total volume of 5 ml. The reaction flasks were fitted with sidearms which
held the enzyme until the reactions were initiated. The flasks were equili-
brated to 37 C, gassed with N,, tightly capped, and tipped to mix the
contents. They were shaken at 60 cycles per minute for 30 min. Reactions
were stopped with 5 ml cold 10% TCA and centrifuged to remove the precipi-
tated protein. The p-aminobenzoic acid produced in the reaction was
determined by the method of Bratton and Marshall as modified by Juchau
except that absorbance was read at 545 nm as specified by the original
authors. First order kinetics with time and enzyme concentration are
demonstrated in Figs. 10 and 11.
PESTICIDE RESIDUE ANALYSIS
Extraction Procedures
Fat tissue - Samples of abdominal fat, taken from chickens adulterated
with dieldrin, were prepared for gas-liquid chromatography as follows:
16
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gp-6
I 0.5
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S°-3
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10
REACTION TIME, min.
15
20
Figure 8. First order kinetics with time of aniline hydroxylation by 1.0 ml
of liver microsomes from chickens induced with phenobarbital.
17
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o
10
(O
o
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0.9
0.8
0.7
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ENZYME CONCENTRATION, ml MICROSOMES
Figure 9. First order kinetics with enzyme concentration of aniline hydroxy-
lation in 20 min reaction. Liver microsomes were prepared from chickens
induced with phenobarbital.
18
-------
o
200r-
o
o
cr
Q.
10 20
REACTION TIME, min.
30
Figure 10. First order kinetics with time of nitroreduction of p-nitrobenzoic
acid by 1.0 ml of liver postmitochondrial supernatant fraction from chickens
induced with phenobarbital.
19
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lOOi—
- 80
|9
* o
3^60
K
o
ID
Q
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m
Q 40
20
0 0.2 0.4 0.6 0.8 1.0
ENZYME CONCENTRATION, ml POST-
MI TO CHONDRIAL SUPERNATANT FRACTION
Figure 11. First order kinetics with enzyme concentration of p-nitrobenzoic
acid in 20 min reaction. Liver postmitochondrial supernatant fractions
prepared from chickens induced with phenobarbital.
20
-------
1 g samples were macerated to a dry powder with 2 g anhydrous N
The mixture was extracted with successive 15 ml portions of hexane by
vigorous mixing in a 45 ml centrifuge tube for 60 sec on a vortex mixer.
The combined extracts were concentrated to 20 ml by evaporation and cleaned
18
up by the acetonitrile partitioning method described by Jones and Reddick
19 20
as modified by Mills and further refined by Samuel. Volumes were
scaled down approximately and pure hexane replaced pet ether. The final
sample in washed hexane was evaporated to dryness and the residue taken
up in 10 ml hexane for chromatographic analysis.
Serum - DDT and its metabolites DDD and DDE were extracted from blood
serum of exsanguinated mallards by two methods. The first was a simplifica-
21
tion of Dale's procedure. It involved the extraction of 1 ml serum with
5 ml hexane in a centrifuge tube vortexed rapidly for 3 min. The hexane
phase was sampled directly after 5 min centrifugation at 800 x g max.
The second extraction method, which gave somewhat higher recoveries,
was adapted for pesticides from the lipid extraction procedure of Bligh
22
and Dyer. Samples of 1 ml serum was extracted with 5 ml 1:1 chloroform-
methanol by rapid vortexing for 60 sec. Ten milliliters distilled water
were added and the mixture vortexed an additional 10 sec. After centrifuga-
tion for 15 min, 100 yl of the chloroform layer were removed, evaporated
to dryness, and reconstituted with hexane.
Brain - Samples of 0.5 g duck brain were macerated to dryness with 1
g Na0SO. in a watch glass. Further extraction was done as with the serum
it *t
samples.
23
Bile - The lipid extraction method of Folch using 2:1 chloroform-methanol
was adapted to small samples of bile. Samples of 200 y 1 duck bile containing
DDT were combined with 4 ml CHCU and 2 ml MeOH in 45 ml centrifuge
tubes and vortexed rapidly for 2 min. Ten milliliters water were added
and the vortexing continued for 15 sec. Centrifugation for 5 min at 800
x g max. separated the two phases, and aliquots of the chloroform layer
were treated as described above.
Chromatographic Technique
The gas cnromatograph was a Tracor model 222. It was equipped
C O
with a Ni electron capture detector, a linearizer, and a Linear Instruments
21
-------
model 252 integrating recorder. Columns were of glass, U-shaped, 1.8
m x 3.5 mm i.d. The carrier gas was 95% argon/5% methane at 40 p.s.i.
and passed through a molecular sieve drier cartridge. All samples were
injected in 5 y 1 pesticide grade hexane.
The column used for the assay of dieldrin residues was packed with
1.5% OV-17, 1.95% QF-1 on 100-120 mesh chromosorb W. Carrier flow
was 75 ml/min with 25 ml/min purge. Column oven, inlet and detector
temperatures were 225 C, 240 C, and 300 C respectively. A 5% OV-1 column
was used intermittently for verification of peak indentities.
The column used for DDT and its metabolites contained 1.5% OV-1,
2.5% QF-1 on 100-120 mesh chromosorb W, H.P., acid-washed and DMCS-
treated. Oven, inlet and detector temperature were maintained at 195
C, 220 C and 250 C, respectively. Carrier flow was routinely 70/30.
The percent recovery of each pesticide or metabolite was determined
individually by quantifying recoveries of known amounts of standards
after extraction from the corresponding body fraction of ah untreated control.
All pesticide standards were obtained from the U.S. Environmental Protection
Agency, Pesticides and Toxic Effects Laboratory, Research Triangle Park,
N. C. The degree of purity of each standard was established in our laboratory
before use.
22
-------
SECTION V
EFFECT OF NEWCASTLE DISEASE VACCINAL VIRUS ON THE INDUCTION OF
DRUG-METABOLIZING ENZYMES IN CHICKEN LIVER.
One-day-old broiler type chicks which had not been treated with
any vaccine or drug were housed in modified Horsfall-Bauer units and
maintained in filtered air under positive pressure. They were fed growing
ration feed containing no chemical additives and had free access to water.
When the birds were 8 weeks old they were vaccinated intraperitoneal-
c
ly with 5 X 10 chick embryo LD-fl of the LaSota B-l strain of Newcastle
disease virus per kilogram body weight or equivalent volumes of sterile
diluent. Three days later the birds were given intraperitoneally 40 mg
sodium phenobarbital per kg body weight or equivalent volumes of sterile
saline daily for 3 days. The birds were killed on the 7th day by decapitation
and hepatic microsomes were prepared for assay of the 3 microsomal enzymes,
N-demethylase, aryl hydrocarbon hydroxylase and aniline hydroxylase.
Aliquots of the postmitochondrial supernatant fraction were used to assay
the soluble enzyme, nitroreductase.
The enzyme activities observed in pullets are given in Figs. 12-
15 and the activities in cockrels are given in Figs. 16-19. The levels
of the drug-metabolizing enzymes in vaccinated birds were not markedly
different from the levels in the unvaccinated saline-treated controls.
Activities of the 3 microsomal enzymes were elevated considerably ("induced")
by treatment with phenobarbital. On the other hand, nitroreductase activity
in the postmitochondrial supernatant fraction was not elevated by phenobarbi-
tal in non-vaccinated birds. The activities of the microsomal enzymes
were considerably greater in the vaccinated birds given phenobarbital
than in the non-vaccinated birds given phenobarbital. Ethylmorphine
23
-------
N-demethylase was 84% higher in pullets and 41% higher in cockrels.
Aryl hydrocarbon hydroxylase was 51% greater in pullets (p< 0.05) and
153% greater in cockrels (p< 0.001) . Aniline was 39% greater in pullets
and 18% greater in cockrels. On the other hand, the activity of nitroreductase
was 44% lower in pullets (p< 0.05) and 20% lower in cockrels.
24
-------
CI40
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—
—
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T
T
CONTROL NDV
PB
NDV+PB
Figure 12. Effect of Newcastle disease vaccination on hepatic microsomal
ethylmorphine N-demethylase of eight-week-old pullets. NDV = Newcastle
disease vaccinated group. PB = phenobarbital induced group. NDV + PB =
group vaccinated with Newcastle disease virus and subsequently induced
with phenobarbital. Data are mean activities + std error of the mean for
5 chickens.
25
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26
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CONTROL NDV
PB NDVtPB
Figure 13. Effect of Newcastle disease vaccination on hepatic microsomal
aryl hydrocarbon hydroxylase of eight-week-old pullets. NDV = Newcastle
disease vaccinated group. PB = phenobarbital induced group. NDV + PB =
group vaccinated with Newcastle disease virus and subsequently induced
with phenobarbital. Data are mean activities + std error of the mean for
5 chickens.
26
-------
75
c
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65
260
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Q.
in
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CONTROL NDV
PB
NDV+PB
Figure 14. Effect of Newcastle disease vaccination on hepatic microsomal
aniline hydroxylase of eight-week-old pullets. NDV = Newcastle disease
vaccinated group. PB = phenobarbital induced group. NDV + PB = group
vaccinated with Newcastle disease virus and subsequently induced with
phenobarbital. Data are mean activities + std error of the mean for 5
chickens.
27
-------
9
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1
1
CONTROL NDV PB NDV+PB
Figure 15. Effect of Newcastle disease vaccination on hepatic nitroreductase
of eight-week-old pullets. NDV = Newcastle disease vaccinated group. PB =
phenobarbital induced group. NDV + PB = group vaccinated with Newcastle
disease virus and subsequently induced with phenobarbital. Data are
mean activities + std error of the mean for 5 chickens.
28
-------
ISO
170
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CONTROL NDV
PB
NDWPB
Figure 16. Effect of Newcastle disease vaccination on hepatic microsomal
ethylmorphine N-demethylase of eight-week-old cockrels. NDV =
Newcastle disease vaccinated group. PB = phenobarbital induced group.
NDV + PB = group vaccinated with Newcastle disease virus and subsequently
induced with phenobarbital. Data are mean activities + std error of the
mean for 5 chickens. ~
29
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CONTROL NDV
PB
NDVtPB
Figure 17. Effect of Newcastle disease vaccination on hepatic microsomal
aryl hydrocarbon hydroxylase of eight-week-old cockrels. NVD = Newcastle
disease vaccinated group. PB = phenobarbital induced group. NDV + PB =
group vaccinated with Newcastle disease virus and subsequently induced
with phenobarbital. Data are mean activities + std error of the mean for
5 chickens.
30
-------
90
85
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T
CONTROL NDV
PB
NDV+PB
Figure 18. Effect of Newcastle disease vaccination on hepatic microsomal
aniline hydroxylase of eight-week-old cockrels.. NDV = Newcastle disease
vaccinated group. PB = phenobarbital induced group. NDV + PB =
group vaccinated with Newcastle disease virus and subsequently induced
with phenobarbital. Data are mean activities + std error of the mean for
5 chickens.
31
-------
c7
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C"l ^F»
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CONTROL NDV
PB
NDVtPB
Figure 19. Effect of Newcastle disease vaccination on hepatic nitroreductase
of eight-week-old cockrels. NDV = Newcastle disease vaccinated group.
PB = phenobarbital induced group. NDV + PB = group vaccinated with
Newcastle disease virus and subsequently induced with phenobarbital.
Data are mean activities + std error of the mean for 5 chickens.
32
-------
SECTION VI
EFFECT OF DUCK HEPATITIS VIRUS ON THE INDUCTION OF DRUG-METABO-
LIZING ENZYMES IN DUCK LIVER AND ON TISSUE RESIDUE OF DDT.
Adult flighting mallard drakes were divided into two groups of 45
birds each. Two days prior to exposing the ducks to DDT, one group
was injected intraperitoneally with 2x10' chick embryo LD_fl of duck
hepatitis virus and the other group was injected with an equal volume
(0.2 ml) of 0.85% NaCl in 0.1 M sodium-potassium phosphate buffer, pH
7.4. The birds were fed a chick growing ration containing no feed additives
ad libitum. On day "0" the birds were exposed to DDT by replacing the
feed with the same feed to which p,p'-DDT (Aldrich Chemical Co., Milwaukee,
WI) had been added with thorough mixing to a level of 900 ppm. The
average intake of DDT was about 50 mg/kg/day. Just prior to the change
in feed, all birds were weighed (bird weights ranged from 1.0 - 1.5 kg)
and 5 from each group were killed. Blood serum, bile and brain were
collected and stored at -26 C for pesticide residue analysis as described
in Section IV. Liver was collected for enzyme analysis as described in
Section IV. Five birds from each group were weighed, killed and the
same samples taken for analysis on days 1,3,5,7,8,9 and 12.
Feed consumption was recorded (Fig. 20), however, because of
the feeding habits of ducks this type of data is less reliable for ducks than
for other species. Thus, the more pronounced variations must be considered
lightly and the most reasonable conclusion is that there was no major difference
between the virus-treated and control birds in the amount of feed eaten.
The body weights and liver weights at the time the ducks were killed
are given in Figs. 21 and 22. Changes in body weight relative to the birds'
weights at day 0 have been plotted in Fig. 23. The virus injected birds
33
-------
160
o
*
o
3
o
•V
o»
S 80
2
(O
z
o
o
o 40
u
UJ
u.
1
I
3 5 79
DAYS FED 900 ppm DDT
12
Figure 20. Feed consumption of DDT contaminated feed by ducks injected
with duck hepatitis virus (broken line) or buffer (solid line) .
34
-------
5 789
DAYS FED 900 ppm DDT
12
Figure 21. Body weights at time of killing of ducks injected with duck
hepatitis virus (broken line) or buffer (solid line) . Each point is the mean
weight for 5 ducks.
35
-------
3 5 789
DAYS FED 900 ppm DDT
12
Figure 22. Liver weights at time of killing of ducks injected with duck
hepatitis virus (broken line) or buffer (solid line) and fed 900 ppm DDT,
Each point is the mean weight for 5 ducks.
36
-------
579
DAYS FED 900 ppm DDT
Figure 23. Changes in body weight during DDT treatment of ducks in-
jected with duck hepatitis virus (broken line) or buffer (solid line). Each
point is the mean value for 5 ducks.
37
-------
lost on the average 45 gin or 3.5% of their body weight, whereas the control
birds lost on the average 3.4 gm or 0.3% of their body weight. The virus
injected birds killed on the last day had lost 5% of their body weight and
the control birds killed on the last day had lost 2% of their body weight.
Increases in the amount of microsomes per gm liver (Fig. 24) and
in the activities of the microscomal drug-metabolizing enzymes (Figs.
25-27) both provide evidence of induction by DDT. Microsomal content
did not increase as rapidly in the livers of ducks injected with duck hepatitis
virus until about the eighth day when the rate of increase exceeded that
of the control birds.
The differences in microsomal content are not reflected in the enzyme
data in Figs. 25-27 since the data is recorded on the basis of microsomal
protein The activities of all 3 enzymes were greater in the virus-treated
birds for the first 3 days with one exception and that was N-demethylase
which was lower on day 1 (p < 0.05). On day 5, however, the activities
of all 3 enzymes were lower in the virus-treated birds, but on days 7 and
8 were again higher than those of control birds. The point of greatest
statistical significance was day 8 when all three enzyme activities were
greater in the virus-treated birds at the 0.05 to 0.01 level. On day 9 the
activities had fallen to the same levels as the control birds but they had
increased again by day 12.
The results of pesticide residue analysis are given in Figs. 28-34.
The total pesticide residue (DDT + ODD + DDE) in serum, bile and brain
is given in Fig. 28. Total pesticide concentrations in serum and brain
accumulated throughout the course of the experiment. Concentration of
total pesticide in the bile increased for 4-5 days after which it remained
relatively constant. Although total pesticide continued to accumulate in
serum, most of it was non-metabolized DDT with only slight increases
in DDE and DDD (Fig. 29). The relative concentrations of ODD in serum
decreased from 12 and 18% to 8% of total pesticide and the relative concentration
of DDE decreased from 22 and 28% to 14% of the total pesticide.
In bile, the level of DDT increased for 3-4 days after which it remained
relatively constant (Fig. 30). There was no difference between experimental
and control groups. There also was no difference between DDE levels
38
-------
22i-
3579
DAYS FED 900 ppm DDT
12
Figure 24. Amount of microsomes per gm duck liver with time fed 900 ppm
DDT. Broken line = ducks injected with duck hepatitis virus. Solid line =
ducks injected with buffer. Each point is the mean value for 5 ducks.
39
-------
3 57 9
DAYS FED 900 ppm DDT
Figure 25. Activities of ethylmorphine N-demethylase in livers of ducks
injected with duck hepatitis virus (broken line) or buffer (solid line) and
fed 900 ppm DDT for 12 days. Each point is the mean activity + std error
of mean for 5 ducks.
40
-------
5 78
DAYS FED 900 ppm DDT
Figure 26. Activities of aryl hydrocarbon hydroxylase in livers of ducks
injected with duck hepatitis virus (broken line) or buffer (solid line) and
fed 900 pmm DDT for 12 days. Each point is the mean activity + std error
of mean for 5 ducks. ~
41
-------
3 5 789
DAYS FED 900 ppm DDT
12
Figure 27. Activities of aniline hydroxylase in livers of ducks injected with
duck hepatitis virus (broken line) or buffer (solid line) and fed 900 ppm
DDT for 12 days. Each point is the mean activity + std error of mean for
5 ducks.
42
-------
SERUM
BRAIN
BILE
013579 12
DAYS FED 900 ppm DDT
Figure 28. Total pesticide residue (DDT + DDD + DDE) in serum, bile and
brain of ducks injected with duck hepatitis virus (broken line) or buffer
(solid line) and fed DDT for 12 days. Each point is mean value for 5 ducks.
43
-------
12
10
- 9
Q
8
01
il
CO
LU
a:
5
24
Q.
3
2
4 7
DAYS FED 900 ppm DDT
10
DDE
ODD
Figure 29. Pesticide levels in serum of ducks injected with duck hepatitis
virus (broken line) of buffer (solid line) and fed DDT for 10 days. Each
point is mean value for 5 ducks.
44
-------
lOt-
DDT
579
DAYS FED 900 ppm DDT
• DDE
12
Figure 30. Concentration of DDT and DDE in bile of ducks injected with
duck hepatitis virus (broken line) or buffer (solid line) and fed DDT for
12 days. Each point is mean value for 5 ducks.
45
-------
in the two groups (Fig. 30). The rate of increase leveled off after several
days but appeared to continue to rise slowly which is more evident in Fig.
31. The level of DOD increased continually and was consistently greater
in the experimental group after 7 days and consistently greater after 3
days when expressed as percent of total pesticide (Fig. 32) . The ratio
of DDD to DDE in bile was greater in the experimental group from the first
day (Fig. 33).
The residues of DDT, DDD, and DDE in brain are given in Fig.
34. The concentrations of all 3 residues continued to accumulate. The
relative concentration of DDE increased slightly from 10% of total at day
1 to 15-21% on day 12. The relative concentration of DDD remained constant
at about 5% of total.
46
-------
6
3579
DAYS FED 900 ppm DDT
12
Figure 31. Concentration of DDE relative to total pesticide in bile of ducks
injected with duck hepatitis virus (broken line) or buffer (solid line) and
fed DDT for 12 days. Each point is mean value for 5 ducks.
47
-------
16
14
12
10
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o 8
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S 6
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01
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5 78
DAYS FED 900 ppm DDT
12
Figure 32. Concentration of DDD and concentration of ODD relative to total
pesticide in bile of ducks injected with duck hepatitis virus (broken line) or
buffer (solid line) and fed DDT for 12 days. Each point is mean value for
5 ducks.
48
-------
l.8r-
0.2
0 I
3579
DAYS FED 900 ppm DDT
Figure 33. Ratio of DDD to DDE concentrations in bile of ducks injected with
duck hepatitis virus (broken line) or buffer (solid line) and fed DDT for 12
days. Each point is mean value + std error of mean for 5 ducks.
49
-------
-112
57 9
DAYS FED 900 ppm DDT
12
Figure 34. Concentration of DDT, ODD and DDE residues in brains of ducks
injected with duck hepatitis virus (broken line) or buffer (solid line) and
fed DDT for 12 days. Each point is mean value for 5 ducks.
50
-------
SECTION VII
EFFECT OF NEWCASTLE DISEASE VACCINAL VIRUS ON THE INDUCTION OF
DRUG-METABOLIZING ENZYMES AND ON TISSUE RESIDUE OF DIELDRIN IN
CHICKENS.
Commercial broiler type pullets which had been accidentally contamina-
ted with dieldrin were obtained in April, 1974 from McCarty Enterprises,
McGee, Mississippi. The birds were offered to us with the hope that vaccina-
tion with Newcastle disease vaccinal virus would provide a means of accelera-
ting the excretion of tissue residues. The birds had been fed the contaminated
diet from 1 to 19 days of age when the feed was changed to an unadulterated
ration. Five birds had been killed at 19, 24 and 30 days of age and body
fat assayed for dieldrin content by Woodsen-Tenent Co. of Memphis, Tennessee.
The average dieldrin residues were 1.20, 0.80, and 0.95 ppm respectively.
Five birds were killed at 52 days of age and abdominal fat samples
assayed for dieldrin as described in Section IV. Twenty birds were separated
into 4 groups of 5 birds each. The birds in two groups were injected intra-
g
peritoneally with 5 x 10 chick embryo LD5Q of the LaSota B-l strain of
Newcastle disease virus. Three days later and for 2 additional days the
birds in one of the virus groups were injected intraperitoneally with 40
mg sodium phenobarbital in sterile saline / kg body weight and the birds
in the other group were injected with equivalent volumes of sterile saline.
The birds in the two non-virus groups were injected on the first day with
sterile diluent and the birds in one group were treated with phenobarbital
on the third through the fifth day while the other group was injected with
sterile saline. Birds in all four groups were killed on the 7th day and
51
-------
samples taken for residue analysis and assay of hepatic microsomal ethylmor-
phine N-demethylase and aryl hydrocarbon hydroxylase.
Vaccination resulted in increased activities of both microsomal enzymes
but they were not increased as much by vaccination as they were by pheno-
barbital (Fig. 35) . In contrast with the results recorded in Sections V
and VI, viral replication followed by phenobarbital treatment did not produce
any greater increase in enzyme activities than with phenobarbital treatment.
Dieldrin residues (Table 1) were essentially the same in all of the
birds regardless of treatment.
Table 1. CONCENTRATIONS OF DIELDRIN IN ABDOMINAL FAT OF CHICKENS
ACCIDENTALLY CONTAMINATED WITH DIELDRIN
Treatment group
Untreated
Saline only
NDV
Phenobarbital
[DV + Phenobarbital
Day killed
0
21
21
21
21
Concentration, ppma
0.24 + 0.03
0.11 + 0.01
0.14 +0.01
0.15 + 0.03
0.14 + 0.01
Mean + standard error of the mean for 5 ducks.
52
-------
v. 6°r-
0
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Figure 35. Ethylmorphine N-demethylase and aryl hydrocarbon hydroxylase
activities of hepatic microsomes from dieldrin-contaminated chickens treated
with Newcastle disease vaccinal virus and phenobarbital. Data are mean
activities + std error of mean for 5 chickens.
53
-------
SECTION VIII
DISCUSSION
Duck hepatitis viral replication in the livers of adult ducks causes
specific, subtle changes in the hepatic microsomal drug-metabolizing
enzymes. Previously reported results were confirmed in the present
study. Not only does duck hepatitis virus produce these changes in duck
liver but Newcastle disease vaccinal virus produces similar changes in
chicken liver. It now appears that, when the reported effects of murine
3 6
hepatitis virus and human hepatitis virus type B are taken into considera-
tion , specific effects on microsomal drug-metabolizing enzymes may be
characteristic of many viruses which produce hepatitis. Of even greater
interest (and importance) is the observation that viruses such as Newcastle
disease vaccinal virus which replicate on hepatic endoplasmic reticulum
without causing hepatic disease may also specifically alter the hepatic
drug-metabolizing enzymes.
Replication of Newcastle disease virus in the absence of a chemical
inducer did not result in any consistent increases in enzyme activities.
Although the results were variable and occasionally the microsomal enzyme
activities were slightly higher than controls, the trend was to activities
slightly lower than controls similar to the effects of duck hepatitis virus
in ducks. Although the replication of Newcastle disease virus in dieldrin-
contaminated chickens (not subsequently induced with phenobarbital)
resulted in higher enzyme activities than in the control birds (Fig. 35),
none of the birds can be considered naive in regard to chemical induction.
All had been exposed to dieldrin and still carried significant pesticide
residues in their tissues. Although the activities of the two microsomal
enzymes were well below the maximum inducible levels and were surely
54
-------
in the process of returning from higher to normal levels, they still were
well above the levels of normal birds. Thus, it must be considered that
the birds' microsomal enzymes were still responding to the chemical inducer,
dieldrin, or specific changes in the endoplasmic reticulum produced in
response to dieldrin were still present and, thus, the higher enzyme activities
from viral replication were due to an additive effect not possible in naive
birds.
Although the levels of the drug-metabolizing enzymes in chickens
were not increased by viral replication in the absence of a chemical inducer
(phenobarbital), the activities of the microsomal enzymes were considerably
greater in the vaccinated birds given phenobarbital than in nonvaccinated
birds given phenobarbital. This "superinduction" or "facilitation of induction"
was statistically significant in the case of aryl hydrocarbon hydroxylase
in both males and females. The lack of statistical significance at the 5%
level in the case of the other two enzymes is due in part to the relatively
small sample size. The percent increase is impressive, however, particular-
ly for N-demethylase, and implies biological significance. Furthermore,
the inductive or "facilitative" effect of vaccination probably was more striking
in the case of aryl hydrocarbon hydroxylase because phenobarbital is
a weak inducer of this enzyme. Since phenobarbital at the dosage used
in this study is a strong inducer of N-demethylase and aniline hydroxylase,
the levels of these enzymes were already induced to such high levels by
phenobarbital in the nonvaccinated birds that it would be difficult to demon-
strate statistically significant differences between the phenobarbital group
and the "superinduced" levels in the vaccinated group. A weak chemical
inducer of these enzymes would have to be used in order to establish statisti-
cally significant differences by viral "superinduction" (viral "facilitation"
of induction may be a better term since viral replication in the absence
of a chemical inducer does not produce increased enzyme activity) .
The activities of the soluble enzyme, nitroreductase, were not
increased significantly by phenobarbital, virus or virus plus phenobarbital.
The activities in the virus plus phenobarbital group were lower than the
phenobarbital group rather than higher. The reason for the decreased
activity is not apparent.
55
-------
In the duck experiments, the three microsomal enzymes were signi-
ficantly higher in the virus treated group than in the non-virus group
on the 8th day. Aniline hydroxylase and N-demethylase were also higher
on the 7th day and the data appear to be consistent with the previously
reported higher activities on the 7th day. In the preliminary work several
years ago, increases were also noticed on the 3rd day (Ragland et al_.,
unpublished observations) but data were not collected on the 3rd day in
a subsequent experiment which served as the basis for the published report.
It is interesting to observe that the microsomal enzymes were also increased
on the 3rd day in the present study, thus, confirming the previously observed
increases on days 3 and 7. The enzyme activities at other intervals of
time were altogether different and not at all what had been anticipated on
the basis of previous results. Nevertheless, since increased activities
have been observed in the virus group on day 3 on two occasions and on
day 7 or 8 on three occasions, we can be fairly confident that facilitation
of enzyme induction by virus occurs at these times. The activities of
the three enzymes were higher in the virus group on days 3 and 7-8, and
on day 12 they appeared to be on the rise again. On days 1, 5 and 9 the
activities in the virus group had fallen to or below the levels of the control
group (that is, the chemically induced group). It was as if the rise and
fall in enzyme activity in the virus group is on some schedule regulated
by the viral replicatory cycle. This is an intriguing speculation but at
the present time must remain that - speculation.
The observed effects of viral replication on enzyme activities or
pesticide residues cannot be attributed to generalized marasmic consequences
of viral replication. There was no major difference in feed consumption
by the two groups and no striking differences in body weight. Although
the losses in body weight would have been costly to a commercial grower
the losses were not severe enough to cause profound biological consequences
in the birds. The variation in liver weight was greater but the analytical
data cannot be ascribed to these differences. The results cannot be explain-
ed on the basis of differences in body weight or liver weight, as the ratios
of the virus group to the control group at days 3 and 8 are reversed and
thus would be contradictory. Finally, the general health of the birds
could not have been greatly impaired as the livers of birds in both groups
56
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were able to increase their microsomal content (Fig. 24). Although the
birds in the virus group were initially less efficient in responding to the
DDT by increasing the synthesis of endoplasmic reticulum, the cause was
a specific viral effect rather than generalized debilitation as evidenced
by greater synthesis of endoplasmic reticulum in the virus group later
on.
The total pesticide in brain and serum continued to increase throughout
the experiment. Total pesticide in bile leveled off by the 5th day (Fig.
28) indicating that the livers were handling as much pesticide as they
could. That the ducks were taking in more pesticide than they could handle
is substantiated further by the fact that most of the pesticide in brain and
in serum was DDT. Conversion of DDT to DDD and DDE was stimulated
and both metabolites accumulated in brain and serum at approximately
the same rate.
Excretion of DDD and DDE in bile was different. Although both
were excreted in bile in increasing amounts, the excretion of DDD was
progressively greater than DDE throughout the experiment (Fig. 33).
The greatest significant difference was only at the 10% level, but if there
were no difference between virus and control groups, the chance that
7
DDD excretion would be greater at every interval examined is 2 , or one
chance in 128. The results are consistent with the hypothesis that viral
replication on endoplasmic reticulum alters the microsomal enzymes specifi-
24
cally. The enzymes which convert DDT to DDD are microsomal whereas
DDT is converted to DDE by a soluble enzyme.
25
According to Hill et al. ca. 20 ppm DDD + DDT in brain is a lethal
concentration and ca. 10 ppm is associated with clinical signs of intoxication.
The brains of ducks in this study contained up to 11.5 ppm which was
associated with mild hyperexcitability and there were no deaths.
4
Friend reported that the total pesticide residue in brains of ducks
treated with virus prior to feeding DDT were markedly lower than in untreat-
ed birds fed DDT. Only several brains were assayed, however, and we
have been unable to confirm his observation in this larger study. It is
therefore questionable at the present time that the resistance to DDT toxicity
4 5
afforded to ducks by prior exposure to duck hepatitis virus ' is due
57
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to enhanced detoxification by induced microsomal enzymes even though
the enzyme activities were elevated at several intervals following exposure
to the virus. In addition, vaccination of dieldrin-contaminated chickens
for Newcastle disease failed to effect increased clearance of the pesticide
even though the microsomal enzymes had been induced. The pesticide
load in the chickens had decreased to such a low level before vaccination
that the conditions under which the experiment was conducted were not
ideal and probably were inadequate for a proper test of the efficacy of
viral replication to increase clearance.
58
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SECTION IX
REFERENCES
Mueller, G. C., and J. A. Miller. The Metabolism of Methylated
Aminazo Dyes. II. Oxidative Demethylation by Rat Liver. J. Biol.
Chem. 202:579-587 (1953).
Huang, S. -N., and V. Groh. Immunoagglutination Electron Micro-
scopic Study on Virus-Like Particles and Australia Antigen in Liver
Tissue. Lab. Invest. 29(4): 353-366 (1973).
Kato, R., Y. Nakamura, and E. Chiesara. Enhanced Phenobarbital
Induction of Liver Microsomal Drug-Metabolizing Enzymes in Mice
Infected with Murine Hepatitis Virus. Biochem. Pharmacol. 12: 365-
370 (1963).
Friend, M. and D. O. Trainer. Duck Hepatitis Virus Interactions
with DDT and Dieldrin in Adult Mallards. Bull. Environ. Contam.
Toxicol. 7:202-206 (1972).
Ragland, W. L., M. Friend, D. O. Trainer, and N. E. Sladek. Inter.:
action Between Duck Hepatitis Virus and DDT in Ducks. Res. Comm.
Chem. Pathol. Pharmacol. 2(2): 236-244 (March 1971).
Doshi, J., A. Luisada-Opper, and C. M. Leevy. Microsomal Pento-
barbital Hydroxylase Activity in Acute Viral Hepatitis. Proc. Soc.
Exp. Biol. Med. 140:492-495 (1972).
Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.
Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.
193:265-275 (1951).
59
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8. Sladek, N. E. and G. J .Mannering. Induction of Drug Metabolism.
I. Differences in the Mechanisms by which Polycyclic Hydrocarbons
and Phenobarbital Produce Their Inductive Effects on Microsomal N-
Demethylating Systems. Mol. Pharmacol. 5:174-185 (1969).
9. Nash, T. The Colorimetric Estimation of Formaldehyde by Means of
the Hantzsch Reaction. Biochem. J. 55:416-421 (1953).
10. Anders, M. W., and G. J. Mannering. Inhibition of Drug Metabolism.
I. Kinetics of the Inhibition of the N-demethylation of Ethylmorphine
by 2-diethylaminoethyl 2, 2-diphenylvalerate HC1 (SKF 525-A) and
Related Compounds. Mol. Pharmacol. 2: 319-327 (1966) .
11. Nebert, D. W., and H. V. Gelboin. Substrate-inducible Microsomal
Aryl Hydrocarbon Hydroxylase in Mammalian Cell Culture. II. Cellu-
lar Responses During Enzyme Induction. J. Biol. Chem. 243(23):
6250-6261 (1968).
12. Alvares, A. P., G. Schilling, A. Garbut, and R. Kuntzman. Studies
on the Hydroxylation of 3, 4-Benzpyrene by Hepatic Microsomes.
Effect of Albumin on the Rate of Hydroxylation of 3, 4-Benzpyrene.
Biochem. Pharmacol. 19:1449-1455 (1970).
13. Wattenberg, L. W., J. L. Leong, and P. J. Strand. Benzpyrene
Hydroxylase Activity in the Gastrointestinal Tract. Cancer Research
22: 1120-1125 (1962).
14. Hansen, A. R., and J. R. Fouts. Some Problems in Michaelis-Menten
Kinetic Analysis of Benzpyrene Hydroxylase in Hepatic Microsomes
from Polycyclic Hydrocarbon-Pretreated Animals. Chem.-Biol.
Interact. 5: 167-182 (1972).
15. Schenkman, J. B., H. Remmer, and R. W. Estabrook. Spectral
Studies of Drug Interaction with Hepatic Microsomal Cytochrome. Mol.
Pharmacol. 3:113-123 (1967).
16. Bratton, A. C., and E. K. Marshall, Jr. A New Coupling Component
for Sulfanilamide Determination. J. Biol. Chem. 128:537-550 (1939).
17. Juchau, M. R. Studies on the Reduction of Aromatic Nitrogroups in
Human and Rodent Placental Homogenates. J. Pharmacol. Exp. Ther.
165:1-8 (1969).
60
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18. Jones, L. R., and J. A. Reddick. Separation of Organic Insecticides
from Plant and Animal Tissues. Anal. Chem. 24: 569-571 (1952) .
19. Mills, P. A. Detection and Semiquantitative Estimation of Chlorinated
Organic Pesticide Residues in Foods by Paper Chromatography.
J. Assoc. Offic. Anal. Chem. 42: 734-740 (1959).
20. Samuel, B. L. An Improved Screening Method for Chlorinated and
Thiophosphate Organic Insecticides in Foods and Feeds. J. Assoc.
Offic. Anal. Chem. 49:346-353 (1966).
21. Dale, W. E. Hexane Extractable Chlorinated Insecticides in Human
Blood. Life Sciences 5: 47-54 (1966) .
22. Bligh, E. G, and W. J. Dyer. A Rapid Method of Total Lipid Extrac-
tion and Purification. Can. J. Biochem. Physiol. 37: 911-917 (1959).
23. Folch, J., M. Lees, and G. H. S. Stanley. A Simple Method for the
Isolation and Purification of Total Lipids from Animal Tissues. J.
Biol. Chem. 226:497-509 (1957).
24. Morello, A. Induction of DDT-metabolizing Enzymes in Microsomes
of Rat Liver after Administration of DDT. Can. J. Biochem. 43:1289-
1293 (1965).
25. Hill, E. F., W. E. Dale, and J. W. Miles. DDT Intoxication in Birds:
Subchronic Effects and Brain Residues. Toxicol. Appl. Pharmacol.
20:502-514 (1971).
61
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SECTION X
LIST OF INVENTIONS
1. No inventions.
2. The following abstract of a paper presented at the fourteenth annual
meeting of The Society of Toxicology, Williamsburg, Virginia,
March 9-13, 1975, will be published in the Society's journal.
Ragland, W. L. and S. J. Buynitzky. Effects of Viral Replication
on Drug Metabolism. Toxicology and Applied Pharmacology, in press.
62
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT N
EPA-6T
-76-003
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
EFFECT OF DUCK HEPATITIS VIRUS ON PESTICIDE TOXICITY
5. REPORT DATE
January 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
W. L. Ragland
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Poultry Disease Research Center
University of Georgia
953 College Station Road
Athens, Georgia 30601
10. PROGRAM ELEMENT NO.
1EA078
11. CONTRACT/GRANT NO.
R801800
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report presents the results of an investigation into the effect of viral
replication on hepatic endoplasmic reticulum on the activities of the microsomal drug
metabolizing enzymes of liver. Two viruses which replicate on hepatic endoplasmic
reticulum without producing hepatic disease were used. They were Newcastle disease
vaccinal virus in chickens and duck hepatitis virus in adult ducks. The microsomal
enzymes assayed were ethylmorphine N-demethylase, aryl hydrocarbon hydroxylase and
anyline hydroxylase. The soluble enzyme, nitroreductase, also was assayed in chickens
Pesticide residue analysis was used to evaluate indirectly the mocrosomal enzymatic
conversion of DDT to ODD and the soluble enzymatic conversion of DDT to DDE in ducks.
Viral replication did not increase the activities of the microsomal enzymes and
may have decreased the activities slightly. Viral replication prior to chemical
induction of enzymes in chickens by phenobarbital and in ducks by DDT resulted in
greater activities of the microsomal enzymes but not the soluble enzymes than was
obtained by chemical induction without prior viral replication. Although the
microsomal enzymes were induced to greater levels by viral replication, pesticide
residue analysis failed to demonstrate increased clearance of residues from body
tissues.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS c. COSATI Held/Group
Drugs
Metabolism
Enzymes
Hepatitis Viruses
Pesticides
Viral replication
06A
06T
060
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
70
20. SECURITY CLASS (Tills page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
63
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