Metabolic fate and toxic effects of one of the components of Tetradymia glabrata
by Sandra Keller Holian
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Chemistry
Montana State University
© Copyright by Sandra Keller Holian (1975)
Abstract:
Tetradymol is an hepatotoxin of moderate toxicity. It can survive in the animal system for at least
seven days and was located in all the organs examined.
Acute poisoning studies in mice have shown tetradymol caused dose dependent, centralobular necrosis.
The death time in control mice was 7.5 hours. The death time and the hepatic necrosis could be altered
after pretreatments with various compounds that altered the action of either the mixed function oxidase
enzymes or the conjugating enzymes.
Spectral binding studies have shown tetradymol to be a Type I binder to cytochrome P-450. This, along
with pretreatment studies indicated that it is metabolized via the mixed function oxidase system.
Pretreatment studies have shown the metabolite formed is more toxic than tetradymol. STATEMENT OF PERMISSION TO COPY
In presenting this thesis in partial fulfillment of the
requirements for an advanced degree at Montana State
University, I agree that the Library shall make it freely
available for inspection,
I further agree that permission
for extensive copying of this thesis for scholarly purposes
may be granted by my major professor, or, in his absence,
by the Director of Libraries,
It is understood that any
copying or publication on this thesis for financial gain
shall not be allowed without my written permission, .
Signature__________
Z
Date
, s/ 9
.... — -
-'
METABOLIC FATE AND TOXIC EFFECTS OF ONE OF
THE COMPONENTS OF TETRADYMIA GLABRATA
by
SANDRA KELLER HOLIAN
A thesis submitted in partial fulfillment
of the requirements for the degree
Of
.MASTER OF SCIENCE
in
Chemistry
Approved:
0
,.
ining CommitteeChairman, ExagEli
Head, Major Department '
Gradp tg/beah"
^
MONTANA STATE UNIVERSITY
Bozeman, Montana
June, 1975
ill
ACKNOWLEDGMENTS
The author would like to express her gratitude to the
following individuals for their patience, guidance and
assistance during the course of this research project.
Dr, P, W, Jennings, Graduate advisor
Dr, J 0 E, Robbins, Graduate advisor
Dr, W, Hill, Assistance in histology
.slide evaluation
Dr, J, Inhelder, Assistance in histology
slide evaluation
Gayle Callis, Preparing histology slides
She would like to give special recognition to her husband,
Andrij, who provided invaluable assistance in handling mice
after she developed a severe.allergy and provided moral
backing.
Acknowledgement is also due the National Institue of
Health for research funds
iv
TABLE OF CONTaNTS
page
LIST OF FIGURES 0
LIST OF TABLES
ABSTRACT
.
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INTRODUCTION .
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RESULTS AND DISCUSSION
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EXPERIMENTAL SECTION
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Reagents
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Instruments
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Pretreatment of mice
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Experiments . 0 0 O 0 • O O O
Quantitation of tetradymol
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APPENDIX
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Structures of pretreatment compounds e «
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Table of histology ■slides
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Death times 0 0 0 0 6 0 0 0 0 0 0 . 0
LITERATURE CITED
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« .-87
98
0 105
I
List of Figures
I6
page
5
Tetradymol mercuric chloride . .............. ...
Z0 Classic hexagonal lobule
e
0
3©
Flow diagram for liver ©
©
*
4«
Quadrant of a liver lobule
0 ©
©
5«
Structure of Ngaione
0
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Structure of Acetaminophen
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© ©
,
7.
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8
©
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.9
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7e Mixed function oxidase reactions
0
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Electron transfer and substrate oxidation
9©
Conjugation scheme for glutathione ©
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18
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e
© ©
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21
24
©
30
©
31
10©
Conjugation scheme for U DP-glucuronic acid
11©
Structure of glutathione
12©
Reaction of tetradymol with Ehrlicks reagent
13©
Tetradymol, four hours o
=
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41
14©
Tetradymol, six hours
©
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41
13©
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*
16
o © © © © © © . ©
Tetradymol,.eight hours
© ©
© ©
©
32
«
© ©
33
,
41
16©
Tetradymol, ten hours
o
©
,
©
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*
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41
17©
Normal mouse liver
©
*
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42
o
18© Sublethal tetradymol, eight hours
©
©
© ©
©
.42
19©
Hexobarbital, aniline, tetradymol spectral binding 44
20©
Tetradymol spectral binding
21,
22©
©
« .
©
© ©
.
45
Hydroxylation of salicylamide
© ©
.
© ©
.
49
Salicylamide spectral binding
© ©
,
« ©
©
50
vi
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55
Phenobarbital, four hours
.
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0
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24.
Phenobarbital, six hours
.
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25.
3-methylcholanthrene, six hours
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26 e
SKF-525A, six hours
e
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27 o
Piperonyl butoxide, eight hours
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Salicylamide, six hours
29.
Cysteine, six hours
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31.
.
.
.
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.
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Diethylmaleate, eight hours
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Olive oil, eight hours
.
.
32.. Ethanol, eight hours
.
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33,
Plot of death time vs SKF-525A dose
34.
Double reciprocal plot of death time
O• O
tetradymol dose
.
.
.
VS
35.
Death time vs reciprocal of Cyt . P-450
36.
Metabolic cage
.
.
.
.
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63
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71
vii
List of Tables
page
Io
Plant extract sheep feeding
O
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20
Mouse feeding
e
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Mechanistic classification <
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Direct hepatotoxicity criteria .•
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5.
Indirect hepatotoxicity criteria
9
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Hypersensitivity criteria
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Substrates and pathways for cytochrome P- 4 5 0
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80
Compounds causing Type I or II spectral changes
9«
Acid lability of t'etradymol
.
.
.
.
O
O
10«,
Tetradymol stability in stomach
Ho'
Tetrddymol recovery from organs
12.
Elimination of tetradymol
13.
Death time
.
14.
15.
16.
.
e ?: o
.
28
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Varying SKF-525A dose .
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Varying'tetradymol dose
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■17.
Elution pattern for Alumina Column
.
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Tetradymol stability at different pH .
.
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77
19.
Tetradymol recovered from organs
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79
20.
Elimination of tetradymol
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viii
Abstract
Tetradymol is an hepatotoxin of moderate toxicity. It
can survive in the animal system for at least seven days and
was located in all the organs examined.
Acute poisoning studies in mice have shown tetradymol
caused dose dependent, centralobular necrosis. The death
time in control mice was 7«5 hours. The death time and the
hepatic necrosis could be altered after pretreatments with
various compounds that altered the action of either the
mixed function oxidase enzymes or the conjugating enzymes*
Spectral binding studies have shown tetradymol to be a
Type I binder to cytochrome P-450. This, along with
pretreatment studies indicated that it is metabolized via
the mixed function oxidase system. Pretreatment studies have
shown the metabolite formed is more toxic than tetradymol.
INTRODUCTION
Tetradymol is a toxic constituent isolated from
Tetradymia glabrata by Dr0 Sam Reeder.
It is a member of the
Compositae family, Senecio tribe, resembling sage brush and
found in a broad region covering an area north to
Washington, east into Wyoming, west to California and south
to the Utah-Arizona border.
It was first shown to be
responsible for death in sheep on the Nevada ranges by
%
Fleming.
Further, it was known at that time to cause a ■
reversible phenomenon called "Big Head", the symptoms of which
were facial and ear tissue swelling.
For more information on
"Big Head" the reader is referred to Brown.
From 1918 to 1922 Fleming and his colleagues conducted
feeding experiments and a brief chemical study in which the
more pertinent facts necessary for killing sheep were
ascertained:
(I) under scarce food conditions sheep would
eat the new growth of T0 glabrata. normally they would not,
and an adult sheep could eat up to 2% of its body weight per
day without apparent harm; (2) since the lethal dose could be
fed over a relatively long period it was thought the toxic ,
principle was slowly eliminated; (3 ) death was attributed to.
hepatodysfunction and cardiac, failure; (4 ) the toxic
constituent was contained in petroleum ether and acetone
extracts from the green plant."*"
Considering this work and.a later investigation by
2
Clawson and H u f f m a n , ^ the problem of isolating the toxic
constituent of T0 Klabrata was undertaken by Drs0 S e K e
Reeder^ and J 0 C0 Hurley.^1 Two toxic compounds were isolated
tetradymol and tetradymadiol 6-isobutyrate, the following i s .
a brief summary of Reeder1Sy work with tetradymol,
From whole plant feeding experiments on sheep it was
shown that:
(I) feeding 1% of body weight for three days
resulted in death; (2) brdmsulphalein clearance time was
greatly lengthened; (3 ) blood serum ammonia levels were
elevated three to six times in poisoned Sheep0^
points indicate hepatodysfunction,
The last two
To test cardiac
dysfunction electrocardiograms were monitored on all sheep
resulting in no marked changes being observed.
Autopsies were performed on all sheep that were poisoned
The results are summarized below: 6
1,
Liver tissue demonstrated panlobular necrosis
localized in the centralobular area,
2,
Kidney tissue showed some general congestion and
■ swelling and hyperemia especially in the
medulary portion,
3»
Varying degrees of congestion were reflected in
■ the lungs with some emphysema and bronchiolar
hemorrhage,
4«
Cardiac tissue was not greatly different from
' 3
normal revealing some congestion and a few
subepicardial hemorrhages.
It was concluded that the toxic principle was a
hepatotoxin and did not greatly effect the heart.
The results of plant-extract feedings are shown in
■ 7
Table I.
This Table shows the percentage of plant weight
to body weight was similar to whole-plant feedings and for
the hexane or acetone extracts, the BSP clearance time and
blood serum ammonia level changes were comparable.
Results
of feeding hexane or actone extracts were similar in dosage
level and hepatic.damage incurred indicating the toxic
constituent was successfully extracted by these solvents.
Since sheep, were a large and expensive laboratory animal,
other smaller animals were tested resulting in similar gross
changes in the livers.
It was decided that mice would be
used for further toxicity experiments.
Preliminary separation and feeding experiments of the
crude extract with mice indicated two different toxins.
One of the toxins, tetradymol, was isolated and its structure
was confirmed by X-ray crystallographic determination of the
O
mercuric chloride derivative shown in Figure I.
Extractions with hexane were made with both ground and
unground plant material.
Since grinding the material did not
result in the isolation of more tetradymol, it was assumed the
TABLE -I7
Plant Extract Sheep Feeding Experiments
Sheep
Y/t. Age Extract
number lbs.■yrs. fed
__ '
______;
__.
_______ .
_____
Extract % pi ant.'.'NE^ level
BSP-clearance0 Result,
from
of body
pounds weight ._____ ;
____ |
_____ .
_________ :
__________
98
I
Acetone
4.4
E-665
104
I
Hexane
5.0
4.8 . 3/14 5/13 7/14
E-634
103
.I
Pentane
•of ethanol
2.5
2.5
3/6
release
E-6 28
104
I
Ethanol•
remains
2.5
2.5
3/6
release
Plant
extracted
with-
■4.5
3/12 5/18
3/36 5/79
E-636
5/44 7/39
death
v. ill00
sacrificed
pounds'fed
H-641
102
I.
Acetone
2.2
2.2
3/5
re-use
H-671
90
I
Hexane
2.5
2.8
3/8
release
.release
I ' Ethanol
100
3/6
2.5
2.5
H-655
♦Recorded as a fraction with the day of the feeding experiment when the.
level
test was run in the numerator and the.g/ml of NH^ found in the serum recorded as
the denominator
0Recorded as above with the day in the numerator and the T1 in minutes recorded as.
the denominator.
^
00V. ill meaning very sick, actually down and on the verge of death.
5
toxin was a surface compound and further hexane extractions
were made on
unground plant material.
An LD^q of 170 to 333mg/kg was determined for the pure
tetradymol compound by feeding experiments with mice.
The
results are shown in Table 2.^
Because preliminary feeding experiments indicated
tetradymol was a hepatotoxin, the liver would be an important
organ in considering what happens to tetradymol and the
effects of tetradymol poisoning.
Therefore, a brief review of
the liver structure and function will be presented.
The liver contains hundreds of lobules which are the
basic functional unit.
These lobules are basically hexagonal
in shape being longer than wide.
The liver lobule is
constructed around a central vein and is composed principally
of many hepatic cellular plates.
The plates are usually two
cells thick and radiate centrifugically from the central vein.
Lying between the hepatic plates are the small bile canaliculi
and around the plates are the liver sinusoids.
On the
periphery of the lobule are portal areas which contain the bile
6
TABLE 29
Mouse Feeding Experiments
Materials
and/or carrier
mg/kg of
toxin
Ie Pure (II)
5.0% ethanol in
n-hexane
2o
"
3.
4.
5.
460
3.8
9
360
280
190
160
3.6
3.5
3.1
4.0
. 8
8
8
8.
6e 50% ethanol in
.n-hexane
7o
0.0
3.3
8
50
0.0
5.0
9
50
Se .Crude extract
in propylene
glycol
2000-3000
3.0-4.5
9o Crude extract
in n-hexane
10.
"
2100-3300
4.4-5.8
1100-1200
I.9-2.0
4-
25
lie N-hexane ■
12.
"
0.0
0.0 .
4 o0-4o9
7.0-8.6
8
7.
00
00
13. Sublimed (II)
.in n-hexatie
14.
"
750-580
4 o 8-6.2
7"
100
330
3.3
9
100
.330
3.3
9
100
170
282
200
140
100
3.3
3.9
3.9
3.9
3.9
11
5
5
5
5
45
■ 40
00
00
00
15. Pure (II) in
n-hexane
16.
"
17.
"
18.
"
19.
"
20.
"
ml/kg total
volume
Number of % dying in
animals
one week
'
'5,
25
• 50
63
75
50
.
12
Pure (II) refers to sublimed , base washed tetradymol.
.
• 00
100
'
7
central
v e i n of
lobule
s e p t a join
at angles
Figure ?.
Tow-power photomicrograph illustrating the
classic hexagonal lobule.10
8
7r>/> cr^ Cf /Z-'./r:Ir P /o.
/
i i i l c Lrv ccm.ciLictxlu.^ f l o w s o n to w c m d
a n d KepaVvc a'Plery enters sinusoids w h e r e
it -flows t o w a r d s central vein
of loViXi)e
Figure 3«
Blood and exocrine flow in liver lobule. 12
Q
Jiver
p Qrench-Xjma
central
vein
in he r e
Figure 4 .
limiting
plate
sinusoid
sheets of
liver cells
appear
as cords
spaces
Quadrant of a liver lobule.
sinxrsoids
branch
of portal
vein
inhere
10
duct, hepatic artery, and portal vein,1^ s-11
Three views of
the liver lobule are illustrated in Figures 4 ^ and if,
The liver lobule has a dual blood supply:
(I) blood
flows in from the portal vein through the liver sinusoids,
empties, into the hepatic Veins and hence into the vena cava
(this food-laden blood comes from the intestine); (2 ) oxygen
rich blood flows in from the hepatic artery through the
sinusoids and empties, into the hepatic v e i n s , ^
The exocrine secretions of the hepatic cells are drained
away by the bile canaliculi which carry the secretions from ■
the central area of the lobule to the bile duct and. is even­
tually emptied into the intestine,
• The functions of the liver are numerous and intricate;
therefore, only a brief summary will be presented.
The hepatocytes can store a variety of compounds; such
as, glycogen, amino acids, proteins, iron and vitamins.
These
can then be released into the system when they are n e e d e d , ^
As a result, the liver then performs not only a storage function
but also, a regulatory function.
For example, the liver takes
glucose from the blood and stores this as glycogen.
When
glucose concentration begins to fall it is returned to the.
blood, which is called the glucose buffer function of the
liver,
20,21 . Another way the liver maintains.normal blood
glucose levels is through gluconeogenesis which converts amino
11
acids and lactic acid to glucose,
PO Pl
*
The liver performs important functions in protein
metabolism;
■
(I) deamination of amino acids; (2) formation of
urea for ammonia removal; (3 ) formation of plasma proteins;
(4 ) interconversions; such as, transaminations, among the
different amino acids and other 'compounds,
The liver has a. protective function in detoxifying,
various compounds.
ammonia to urea.
One example is the transformation of
Further, the liver will transform and/or
conjugate undesirable products or compounds that are absorbed
from the intestine which otherwise might prove deleterious to
the body.19’20
A wide range of compounds comprise the collection of
hepatotoxic agents ranging from simple molecules, such as,
carbon tetrachloride to such complex compounds as steriods,
Their deversity is reflected not only in their structure but
also in their modes and degrees of action.
The following
discussion is to aid in identifying and classifying these
agents into a more unified system.
In Table 3, hepatotoxic agents are classified by the
presumed mechanism of hepatotoxicity.
The criteria for distinguishing between the types of
hepatotoxins listed above are summarized in Tables 4 , 3, and
6,
The compounds that are intrinsic hepatotoxins were
12
Table 320
Mechanistic Classification of Hepatotoxins
I.
Ho
Intrinsic hepatotoxins
A0
Direct - injure liver cells
directly and other organs
Be
Indirect - injure liver cells
by diverting,'blocking, or
competitively inhibiting
essential metabolites
Host Idiosyncracy
A*
Hypersensitivity
Be
Metabolic abnormality in host
Table Zf22’2^
Direct Hepatotoxicity Criteria
Ie
Brief interval between exposure and liver damage
2o
Toxicity dose related
3o
Distinct liver lesions and. often other organs
Zt-o
Experimentally reproducible
5o .High incidence
6o
Protoplasmic poisons
7o
Histological change is the same in man
and predictable from animal experiments
13.
Table 5
Indirect Hepatotoxicity Criteria
I,
Same as for Direct except for 3 and 6
20
Hepatic necrosis or other damage produced by:
3o
ae
Competition with essential metabolites
be
Selective, binding of essential metabolites
or nutrients
Ce
Inhibition of specific ensyme functions
Selective interference with hepatic secretory or
excretory mechanisms without parenchymal damage
Table 625?26
Hypersensitivity Criteria
Ie
Sensitization period (1-4 weeks) or previous
exposure
2e
Recurrence of liver damage on readministration
3«
Cross-, hypo-, or desensitization may be produced
4o
Dose independent
5e
Low incidence of occurrence
6»
High incidence of rash, fever, eosinophilia
7«
Coincidence of blood dyscrasins
8o
Histology consistent with hypersensitivity
9.o
Injury not produced in animal species studies
14
originally grouped in a large class of industrial chemicals or
solvents, these being such things as carbon tetrachloride,
chloroform, halogenated hydrocarbons, etc^
27
In addition,
some drugs have been shown to cause direct hepatotoxicity,
A brief summary of necrosis will be presented before
discussing compounds that cause liver damage.
Hepatic necroses may be roughly classified by distri­
butions
(I) focal necrosis, ie,, small necrotic foci di,stri- .
buted without any constant relationship to particular areas of
the liver lobules; (2) zonal necrosis, in which the involved
areas are in fairly constant relationship to a particular part
of the liver lobules and are referred to as central, midzonal,
and peripheral; (3 ) diffuse necrosis in which hepatic
parenchyma cells are destroyed over massive areas.
pg
In central...or centralobular necrosis, the most common of
zonal necroses, the lobule surrounding the central vein is
necrosed.
This form of.necrosis may be caused by a variety of
chemical poisons, such as, carbon tetrachloride, chloroform,
29
and trinitrotoluene, '
Midzonal necrosis involves the middle
regions of the liver lobule and is apparent with yellow fever.
Phosphorus poisoning causes peripheral necrosis which is
localized around the portal area of the l o b u l e , .
The first sign of necrosis is swelling in. the cells where
more severe changes will be apparent later.
This will be
15
followed by cytoplasmic vacoular degeneration or fatty
infiltration*
Nuclear changes will become apparent such as
karyolysis, pykiiosis, and karyorrhexis and cell definition
will be lost as the cell dies*"^
Resistance of hepatic cells varies with their metabolic
and nutritional state*
Adequate stores of glycogen or
adequate amounts of methionine, choline or other vitamin
complex components appear to give some protection from
po
injurious influences*
As stated earlier numerous chemicals cause liver injury
but little is known of the mechanism by which these chemicals
produce such injury*
51
'
'
■
The next section is a discussion of
some compounds that produce liver necrosis (classification,
distribution), how necrosis may be altered and a suggestion of
why necrosis occurs*
In 1961, Denz and Hanger^ isolated and characterized
the liver toxin from the leaves of the Ngaio tree (Myoporum
Letum)*'
Various domestic animals would readily eat the" tree's
leaves, the result of which was fatal.
This poisoning resulted
in liver damage, icterus, and photosensitivity*
The toxic
principal was identified as a sesquiterpene ketone, called
ngaiorie; the structure is given in Figure 5 *
was isolated from the Ngaio oil*
510mg/kg in mice*
This compound,
The LD^q of the oil was
The toxin caused zonal liver necrosis.
16
r-T/CH;
^
CH^-C-CH^CH^
Figure 5»
Structure of Ngaione^
usually midzonal, and death in mice.
The compound's LD^q
was demonstrated to be 300mg/kg, a hepatotoxin of moderate
toxicity.
Seawright and 0'Donahoo,
in 1971, did a more complete
study of the histology of ngaione.
They administered
intragastrically a LD^0 dose of ngaione then sacrificed
four mice at each time period, I, 3, 6, 12, 24, and 48 hours
after poisoning.
Under the light microscope midzonal damage
was not seen until three hours, with the electron microscope
midzonal changes were present in one hour.
The damage
visible by light microscope started as fine cytoplasmic
vacoules and progressed through to complete necrosis by twelve
hours.
After this some regenerative changes were apparent as
indicated by phagocytic activity of macrophages and the livers
were apparently normal after nine days.
In 1972, Seawright and Hrdlicka^ reported that
pretreating with phenobarbital, an inducer of mixed function
oxidase enzyme synthesis, or SKF-525A, a binding inhibitor of
17
cytochrome P-450, effectively changed the LD^q of ngaione and
changed the zonal necrosis in mice,. Aftor pretreating with
phenobarbital the LD^q was increased to 370mg/kg and the
necrosis was moved from midzonal to peripheral.
Pretreating
with SKF-525A increased the LD^q to 530mg/kg and changed the
necrosis to the centralobular region,
Seawright concluded
from these results there was a concentration gradient of the
mixed function oxidase enzymes in the lobule and a critical
concentration was necessary for the toxic effects.
In view
of this, SKF-525A moved the critical concentration closer to
the central area, by binding to the peripheral and midzonal
cytochrome P-450,' resulting in the necrosis being reflected in
the centralobular area,
Phenobarbital, by inducing enzyme
synthesis, moved this critical concentration to the peripheral
region and resulted in necrosis in this region.
For hepato-.
toxic damage to occur a critical concentration ratio of toxin
to mixed function oxidase enzymes (cytochrome P-450) was
necessary.
After reports.that overdoses of acetaminophen caused
hepatic necrosis in man, Mitchell, et a
l
examined the .
histology and the results of various pretreatments on this
necrosis, ' The structure of acetaminophen (4-hydroxyacet-'
■anilide) is given in Figure 6,
Acetaminophen caused centralobular necrosis in mice and
18
OH
C-CH
Figure 6.
Structure of acetaminophen
rats similar to that in man and was shown to be doge
dependent.
By pretreating with phenobarbital the incidence
and severity of necrosis was potentiated.
Piperonyl butoxide,
a binding inhibitor of cytochrome P-450, and cobaltous chloride,
an inhibitor of mixed function oxidase enzyme synthesis, when
used in pretreating the animals prevented necrosis.
The
effect of pretreatment on the rate of acetaminophen
disappearance from liver and plasma showed no alteration with
phenobarbital and cobaltous chloride but was slowed with
piperonyl butoxide.
Mitchel concluded a metabolite covalently
bound in the liver macromolecules caused the liver necrosis
and that pretreating resulting in alteration of the necrosis
also altered binding of the metabolite.'^’^
Mitchell, et al.,
57
examined the possibility of
glutathione protection of acetaminophen necrosis by pretreating
with cysteine, an inducer of glutathione synthesis, or diethylmaleate, a binding inhibitor of glutathione.
Pretreatment
with cysteine reduced necrotic damage and diethylmaleate
pretreatment potentiated the necrosis indicating a
19
protective role by glutathione,,
Mitchell concluded that the
metabolite did not cause necrosis until glutathione avail­
ability was depleted by conjugation of the metabolite,
38
" .
Brodiev suggested in 196? that chemically inert
chemicals may cause necrosis by a covalent linkage between.
a metabolite and various macromoleculese
Brodie, et alOJ
supported his suggestion, in. 1970, by showing that ^ O b r o m o benzene was covalently bound at sites of necrosis.
39
They
concluded that the liver can convert stable organic compounds
to toxic agents which cause necrosis by covalent bonding.
They
showed the importance of microsomal enzymes in this conversion
by pretreating with phenobarbital, potentiating necrosis, and
SKF-525A, decreasing necrosis caused by bromobenzene.
Later
38
in 1971, Mitchell, et al,,
published a report supporting
Brodie's conclusions, '
In 1973, Zampaglione, et al«,^
continued to show the
role of- detoxifying enzymes in bromobenzene necrosis,
Pretreating with phenobarbital increased bromobenzene metabolism
and potentiated necrosis, conversely, SKF-525A pretreatment
slowed metabolism and prevented necrosis.
The interesting
point of-this publication was the effect of 3-methylcholanthrene
pretreatment, an inducer of mixed function oxidase enzyme
synthesis.
Although ^irmethylcholanthrene increased the
metabolism of bromobenzene, it provided protection against
20
the necrosis*
According to Zampaglione, et al«, this
protection resulted from an alteration in the pathways ofbromobenzene metabolism*
During the previous discussion it is apparent that the
mixed function oxidase enzyme system, specifically cytochrome
P-450> is intimately involved in the toxicity of chemicals* ■
The following is a review of cytochrome P-450 as to mechanism,
multiplicity, effectors, and the spectroscopic properties*
When foreign compounds, particularly lipophilic compounds,
are introduced to the body and go into the liver they may be
oxidized by the mixed function oxidase system*
Cytochrome
P-450 is considered to be a terminal hydroxylase in this
system*
Cytochrome P-450 can catalyze a number of mixed
function oxidase reactions, three of which are illustrated in
Figure 7.
The diversity of chemicals modified by cytochrome P-45O
catalyzed oxidations is staggering and include conversions
such as:
(I) multiple positions of hydroxylation on steroid
molecules; (2) oxidative conversion of heme to bile pigments;
and (3) omega oxidation of fatty acids*^
In table ? are
representative substrates for cytochrome P-450 catalyzed
reactions and their reaction pathways*
The function of cytochrome P-450 is to activate molecular
oxygen for introduction into a compound resulting in a more
21
Codeine
Morphine
CH2O
NADP+
Monomethyl-Z1-aminoantipyrine
Z1-Aminoantipyrine
N H -C O -C H 5
NADP
N A DPII+H
Acetanilide
Figure 7.
H2O
p-Hydroxyacetanilide
Three types of mixed function oxidation reactions,41
22
Substrates and Pathways for Cytochrome P-450^
Pathway
Substrate
Aromatic Hydroxylation
3$.4-benzpyrene
Zoxazolamine
Acetanilide
Estradiol
Aliphatic Hydroxylation
Hexobarbital
Testosterone
Fatty Acids
• N-Dealkylation
Aminopyrine
Meperidine
Imipramine
O-Dealkylation
Codeine
Acetophenitidin
S-Dealkylation
Chlorpromazine
polar product.
.
Intensive efforts have been made to elucidate
the mechanism by which oxidative transformations catalyzed by
cytochrome P-450 occur.
Eetabrook has proposed the following
six steps as occurring in the reduction and oxidation of
1.
The reversible interaction of a substrate molecule
with a low spin form of ferric cytochrome P-45O
accompanied by the formation of a high spin form of
the ferric-substrate complex of cytochrome P-450;.
2.
The one electron reduction of the high spin form
of the ferric-substrate complex of cytochrome P-45O
to a ferrous-substrate complex.
3»
The reversible interaction of oxygen with the
ferrous-substrate complex of cytochrome P-450 to ..
form an oxygenated or oxy-ferrous-substrate complex.
4o
A second one electron reduction step required to
23
generate an intermediate which is as yet undefined;
5o
A proposed rearrangement accompanying internal
oxidation and reduction reactions resulting in the
introduction of one atom of molecular oxygen into
the organic substrate in the form of a hydroxyl
group concomitant with the release of the other
atom of oxygen as water;
6.
The dissociation of the hydroxylated product from
ferric cytochrome P-430 with.the regeneration of a
low spin form of ferric cytochrome P-430.
It has been demonstrated by several researchers that an
iron-sulfur protein ■plays a role in the two electron transfer
steps required for bacteria and adrenal cortex cytochrome P-430
function.
This has not been well documented with microsomal
bound cytochrome P-430.
A proposed pathway for electron
transfer and resulting substrate oxidation is shown in
Figure 8,
Early evidence suggested there
cytochrome P-430.
was more than one form of
This indication was obtained by visible
spectroscopy and electron paramagnetic resonance spectroscopy
by observing changes involved in the binding of different
ligands to cytochrome- P-450.
This indicated two forms that
were spectrally and enzymatically distinct, cytochrome P-450
and P448 (P^SO^).
Further evidence was obtained by combined
potentiometric and electron paramagnetic resonance titrations
of cytochrome P-450 from phenobarbital-treated rats indicating
three forms of cytochrome P-450.
One was a high spin ■
24
P-450-S
NADHn /P-FAD
PP-(FeS)
• Es
NAD
S-OH
P-FADHVx EP-(FeS)
Fe • 0
Fe
Figure 8»
0
Proposed pathway for,electron transfer and
substrate o x i d a t i o n # :
hemoprotein, referring to the iron, and the other two were
low spin hemoproteins#' Comai and Gaylor^ identified three
forms of cytochrome P-450 qualitatively and quantitatively by
visible spectral changes that occurred when combining with
'
various ligands#
The forms were separated on a Whatman DE-52
ion exchange column orum a Sigmadiethylaminoethylcellulose
column.
Thq forms had different binding affinities for the
different ligands.
%
The ligands used were cyanide, carbon
monoxide and octylamine#
Various pretreatments altered the relative amounts of the
three forms#
3-methylcholanthrene pretreatment increased
25
Form IIIs Form II was increased by phenobarbetal pretreatment
and pretreatment with ethyl alcohol preferentially induced
Form I0
Walton and Aust
/4.6
later resolved three forms of cytochrome
P-450 from rat liver microsomes by SDS-polyacrylamide gel
electrophonesiSe
hemoproteins
44,OOOo
They obtained three bands representing
having molecular weights of 53 ,000 , 50,000, and
Pretreatment with 3-methylcholanthrene increased the
level of the 53,000 molecular weight species (Form III),
phenobarbital pretreatment induced the level of the 44,000
molecular weight species (Form II), and the 30,000 molecular
weight species (Form I) was the major hemoprotein in the
controls*
As suggested above a variety of chemicals affect the
characteristics of the mixed function oxidase enzyme system*
Some of. these compounds are:
(I) p h e n o b a r b i t a l ^ * ^
alters mixed function oxidase activity; (2) 3-methylcholanthren.e^*^'49,50,51 ^pters mixed function oxidase activity;
(3) s u b s t r a t e s , s u c h as, aminopyrine., ethylmorphine, and
other polycyclic hydrocarbons, alter the metabolic rate of
the mixed function oxidase system; (4 ) SKF-525A and its
54
congeners^ inhibit mixed function oxidase activity by
binding to cytochrome P-450; (5) methylenedioxybenzenes^
inhibit the activity of the mixed function oxidase system by
26
binding to cytochrome P-450; (6) cobaltous chloride56 '
administration inhibits the synthesis of mixed function oxid57
ases; and (?) l-arylimidaxolesv' have recently been shown to
'
/
be inhibitors of mixed function oxidase activity.
From the discussion of cytochrome P-450 multiplicity and
the discussion of effectors of mixed function activity, it
is apparent that phenobarbital and 3-methylcholahthrene not
only induce different spectral^®5"*" and catalytic^"6 ®
5^
forms of cytochrome P-450 but also induce different cytochrome
P-450 hemoproteinse
Chemicals are not the only modifiers of mixed function
oxidase activity»
It has been shown that improper storage
58
can reduce the "in vitro" activity of this enzyme system,
bitterest, et al.,56 isolated microsomal pellets and froze
them for various periods of time, 24 hours up to 20 days.
There was no significant lose of activity up to 10 days.
Preparation of the pellet for freezing can alter the stability
and activity of this system.55
Burke and Bridges55 reported
that the best storage method was a microsomal pellet overlaid
with buffer.
If the microsomes were resuspended and then
frozen some stability was lost.
Varying the "in. vitro" assay, conditions for cytochrome
P-450 can affect the stability and-quantitation measurements.60
The best measurement of cytochrome'P-450 activity was
27
achieved when the protein content was held at approximately
2,0 to 2o5mg/ml in OelM phosphate or tris buffer at a pH
range of 6,6 to 7»0 and without KCl being present.
In 1964, Omura and Sat o ^ established a procedure for
measuring the cytochrome P-450 content of liver microsomes ■
spectroscopically0^ This method is commonly used today.
In this procedure, microsomes are isolated and suspended in
a OelM phosphate buffer at a protein concentration of 2mg/ml,
The microsomal preparation is placed in a reference and a
sample cuvette.
The microsomes in the sample cuvette are
reduced by adding sodium dithionite and CO is bubbled through
the .sample for approximately 20 seconds.
The difference
spectra are run on the microsomal preparation resulting in
a reproducible peak at 450nm,
The cytochrome P-450 concen­
tration is obtained by taking the difference in absorbance
between 490nm and 450nm and using the molar extinction
coefficient of 91 Cm-^mM
The spectral properties of reduced microsomes may be
influenced by pretreating the animals with various compounds,
such as, phenobarbital^1 ’^5? 64 or 3-rnethylcholanthrene,
This pretreatment cah change the location of the peak, ie.
450nm to 448nm, and usually results in an increase in the
concentration of cytochrome P-450,
Different spectral properties of cytochrome P-450 are
28
reflected in the binding characteristics of non-reduced
microsomes with a variety of compounds.
In 1966, Bemmer,
et alo, ^ reported that when various substrates were added to
liver microsomes two types of spectral changes resulted.
One
spectral change, termed Type I, was characterized by a trough
at 420nm and a peak at 385nm,
The other spectral change,
termed Type II, has a peak at 430nm and a trough at 390nm.
In Table q 66,6?,68
a
of compounds that cause such
spectral changes.
Table q 65,66,67
Compounds Causing Type I or II Spectral Changes
•Type I
Type II
Hexobarbital
Aniline.
Phenobarbital
DPEA
SKF-525A
Nicbtine
Piperonyl butoxide
Nicotinamide
Aminopyrine
Pyridine
Amobarbital
DDTd
p-aminophenol
*
Cortisol
Chlorpromazine
Coricosterone
N,N-dimethylaniline
Acetanilide
Testosterone
Ethylisocyanide
*
*
4
These compounds give a modified Type II
spectral change, characterized by a shifting
of the 430nm peak.
29
Narasimhulu6^. studied spectra changes in relation to the
steroid C-21 hydroxylation system*
He found that the Type I
spectral change was rapid and proceeded hydroxylation*
He
concluded that a Type I spectral change reflected the amount
of cytochrome P-450 activated for redox reactions*
Soliman, et al* ,^investigated the inter-relation of the
Type I and Type II binding sites*
By observing.various
displacements, caused by one Type on the other, he concluded
the sites were inter-related.
Drug binding affected both
sites and the extent of mutual displacement was not
dependent on spectrum produced but on the dissociation
constants, of the drugs*
Another way the. liver handles foreign compounds,
I
principally the lipid soluble compounds, is to conjugate
them with glutathione or UDP-glucuronic acid.
This gives a
more water soluble product and can take place before or
after metabolism through the mixed function oxidase system.
The conjugated product is usually considered to be non- or
less toxic than the nonconjugated form.^O;?!,72
conjugation
shernes for the two conjugators are presented in Figures 9
and 10,.
Pretreating with cysteine or diethylmaleate changes the
effective concentration of glutathione available for
conjugation.
Cysteine is an enhancer of the synthesis of
30
R*X + HSCH_CHC0NHCH_C0_H
2 I
2
2
N h c o CH-CH-CHCO0H
CL
I
C-
NH2
Glutathione S-transferase
s/
R'SCH0CHCONHCH0CO0H
^I
d c.
NHCOCH0CH0CHCO0H
I
2
NH0
^-Glutamyltransferase
n/
R*SCH2CHCONHCH2CO2H
NH2
Cysteinylglycinase
R*SCH0CHCO0H
2I
Acetylase
NH
Figure
^
R«SCH CHCO0H
I
2
2
NHCOCH=
9.
Conjugation scheme for glutathione.^
31
COOH
Uridine diphosphate
•x-D-glucuronic acid
(or glucosiduronic
acid)
UDP Oil
phenol or alcohol
O-glucuronide
COOH
Carboxylic acid
S -glu cu ron id e
Knol
COCH
OH ^
NH.
hydroxamic acid or
hydroxylamine
F ig u re 1 0 .
C on ju gation
N-glucuronide
schem e
for
U D P -glu cosid u ron ic
a c i d . 76
32
glutathione.
Figure 11.
73
It is the middle amino acid of glutathione,
Diethylmaleate is a binding inhibitor of
glutathione.^
NH
0
CH2-SH
I
l
/
COOH-CH-(CH^).-C-NH-CH-C-NH-CH^-COOH
2 ^
H
2
0
2
Figure 11.
Structure of glutathione
Salicylamide is a binding inhibitor of UDP-glucuronic
another compound.
Taking into consideration the previous information on
how the liver handles foreign compounds, what effects can be
seen from hepatotoxins, and how these effects can be altered
by various pretreatments study was conducted on the
hepatotoxin, tetradymol.
RESULTS AND DISCUSSION
Before "in vivo" or "in vitro" research with tetradymol
could begin an accurate method of quantitation the toxin had
to be devised.
The usual approach to this problem is to
radiolabel the compound with either tritium or 1^C-Carbon.
After labeling tetradymol with tritium the resulting
^H-tetradymol proved to be too intractable to isolate.
Ehrlicks reagent was tested due to the reaction of this
reagent with furan rings77(Figure 12).78
Figure 12.
The reaction of
Reaction of tetradymol with Ehrlicks reagent78
34
tetradymol with this reagent was very sensitive to small
concentrations of the toxin.
With, this reaction 2mg/ml of
tetradymol could be measured in solution with an accuracy of-.
±0,5uge
This sensitivity would be beneficial when measuring
levels of toxin found in animal organs after poisoning.
After the accumulated data were run through the least squares
program in the computer to obtain, a standard curve, an
equation of a line was obtained:y = 66x - 4,7
y '= concentration, x = 6,D,
Ehrlicks reagent arid this equation were used in all experiments
where measurements of tetradymol concentration were necessary.
Having an accurate and sensitive method of quantitating
tetradymol allowed research to proceed to substantiate the
assumption that tetradymol was an hepatotoxin and to
illucidate the fate of tetradymol in the system.
After a
compound is introduced to the animal system via the stomach,
it is absorbed into the blood stream from the small intestine.
The blood delivers the compound to the liver where it may act
upon and be acted upon by various metabolizing systems,
A
lipid soluble compound such as tetradymol may be transformed
or conjugated into a more water soluble form and will then
be eliminated in the urine.
Water soluble compounds do not
35
require transformation to be eliminated in the urine.
Early.experiments' indicated that tetradymol- was relatively
unstable with respect to light, heat, and acidic conditions.
Since the stomach is rather acidic (pH I,0-2,0) it was important
to test the stability under those conditions.
Results given
in Table 9» proved tetradymol to be unstable in concentrated
HCl but stable in IM HCl, pH I, and O 0OlM phosphate buffer,
pH 7,4,
The pH of IM HCl is close to the pH of the stomach.
Thus experiments indicated that tetradymol would probably be
stable in the stomach.
Table 9
Acid Lability of Tetradymol
Conditions
Cone, HCl
% recovered
■ El
-1,08
0
IM HCl .
1,00
96
O 6OlM .Phosphate
buffer
7.40
.
93
The next step was to test tetradymol directly in the
stomach.
10,
The results of this experiment are shown in Table
Tetradymol was quantitatively recoverable over this, time
period, allowing for increasing absorption from the stomach
and solubilizing into the stomach wall due to the lipophilicity
of the compound.
Prom these results it was concluded that
'tetradymol was eliminated from the stomach unchanged.
36
Table 10
Tetradymol Stability in Stomach
Time
(min)
mg recovered
% recovered
0.98*0.014
98
15
0.95*0.046
95
30
0.93*0.070
93
0.78*0.006
78
O
.
45
■
Absorption from the small intestine would not be a problem
since ,tetradymol was stable at pH 7 .4 which is close to the
pH in the small intestine (pH^aO),
Tetradymol is a lipid-like compound and consequently
should be lipid soluble*
When studying lipid soluble compounds
it is to be expected that the compound will be located
throughout the body after administration and it will be
fairly evenly distributed through the body.
The distri­
bution of tetradymol was studied by poisoning mice with a
lethal dose (350mg/kg) of. tetradymol, sacrificing three mice
every two hours, extracting various organs, and evaluating the
toxin concentration.
The organs examined were brain, heart,•
lung, stomach, kidney, pancreas, liver, and upper and lower
intestine.
This gave the concentration of tetradymol per
organ over an eight hour time period.
shown in Table 11.
These results are
37
Table 11
Tetradymol Recovery, from Organs
Time
(hr)
stomach
.
(%)
■MjQ
Tetradymol recovered from
other
U,lnt/P L 0Int , p .Liver ^ organs
(%)
(%)
(%)
(%)
Total'
(%)
21,0
6,9
1,4
2.62
27.1
2 .
57.6
4
63.0 \
8,6
3.9
1.3
4,24.
18.9
6
49.1
7.7
11,7
3.9
5.52
14,7
8
41.1
26,9
13.9
3.8
2,94
11.7
I * °* total recovered
corrected by subtracting furan level of control organs from
level of poisoned organs
00 % recovered from total given
As can be seen from Table 11, tetradymol recovery went
down oyer the eight hour period which would be expected due to
increasing absorption into the tissue and biotransformation
into metabolite(s)0
Since the toxin was given directly into
the stomach, the highest level of tetradymol recovered was in
this organ and decreases over the time period due to
elimination from and absorption by the stomach.
The remainder
of the recovered toxin was located in all the organs examined
and these organs had approximately the same amount of toxin
present.
Attempts were made to release the toxin from the
tissues by treating the organs with various tissue solubilizers;
this did not result in recovery of any more tetradymol.
When
tetradymol was treated under the same conditions as the.tissues
had been, it could no longer be detected by the Ehrlicks
reagent indicating the loss of the furan ring.
These results did not indicate that tetradymol directly
affected any. particular organ. . However, it was noticed, on
removal of the various organs from the animal, that the liver
appeared discolored with yellow areas and was friable.
After being absorbed into the lipid layers lipid soluble .
compounds will slowly diffuse out, be metabolized, and continue
to appear in the feces and urine of animals given such
79
compounds. 7
A sublethal dose (lOOmg/kg) of tetradymol was
given to the mice and the urine and feces were collected for '
seven twenty-four hour periods (Table 12.)
On the seventh day the toxin was not detected in the urine
and feces and the experimented was terminated at this point.
The amount recovered per day varied due to the food
consumption, physical activity ahd eliminations of the mouse.
Day one was.the highest since the mouse would be eliminating
whatever
toxin had not been absorbed. ' This indicated that
tetradymol could survive in the animal system for an extended ■
period and that tetradymol was being solubilized by the
tissues and was then slowly released.
•
-
Although it was not known that tetradymol survived the
animal system and was distributed throughout the system, the .
39
Table 12
Dag.
Elimination of Tetradymol
*
' mg recovered
% recovered'
I
.091*0.070
3.03
2
.028*0.006
0.93
3
•.028*0.012
0.93
4
.006*0.005
0.20
5
.013*0.007
0.43
6
=016*0.005
0.53
0
7
0
Total
6.10
.182*0.015
*
corrected by subtracting control urine and
pieces furan level from value after poisoning
% recovered was based on total amount given
question remained as to which organ(s),if any, did tetradymol
affect.
The answer to this question was found in doing
histology on the various organs that had been looked at.
previously in distribution.
Mice were poisoned with a lethal
dose (350mg/kg) of tetradymol and every two hours three were
sacrificed, the organs removed, histology slides prepared and
stained with hematoxylin-eosin.
In viewing the resulting
slides under a light mibroscope, it was found that, only the
liver reflected any damage and the other organs appeared
essentially normal.
The initial suggestion of hepatic damage
was apparent after only two hours and was reflected in the
40
swelling of the cells in the centralobular area*
Swelling
was uniform and accompanied.by some slight, fine cytoplasmic
vacoular degeneration.
Figures 13, .14, 15, and 16 are liver
slides taken over a four to ten hour time period,
'In these
figures it can be seen that hepatic damage became more severe
as time progressed.
Figure I3 shows a uniform, mild cyto­
plasmic vacoular degeneration which spreads through the central
region and into the midzonal area.
Six hours after poisoning,
Figure 14, the cytoplasmic vacoular degeneration is more
severe, the nuclei are becoming irregular, and some nuclei are
missing,.
After eight hours, Figure IS9 the centralobular
degeneration is severe and necrosis is apparent.
The nuclei
are small in the damaged area indicating pyknosis and dilated
blood filled sinusoids are present.
Glycogen depletion'is
apparent as seen by the large holes in the tissue where the
glycogen has left.
Further support for glycogen depletion was
obtained by doing a periodic acid-shift stain on liver slices
over the same time period.
This stain is specific for glycogen
At the various time periods the stain became lighter indicating
a loss of glycogen.
is very severe,
apparent®
Kayloysis, pyknosis, and karyorrhexis are
There is a loss of cells and pools of red blood
cells are present.
time.
After ten hours, Figure 16, the necrosis
Glycogen depletion is more apparent at this
The mice used for the eight and ten hour slides were on
41
Figure 13. Tetradymol
poisoning, mouse liver,
four hours, 100X.
Figure 15. Tetradymol
poisoning, mouse liver
eight hours, 100X.
Figure 16. Tetradymol
poisoning, mouse liver,
ten hours, 100X.
42
^sgiii
"-•I
Iffl
Z':> .
r»: >
±
W
'
Figure 17.
Figure 18.
^
: •> .
I
SHSIi
.
!v-
Normal mouse liver, IOOX
Mouse liver after sublethal tetradymol
poisoning, 100X.
43
the verge of death when sacrificed.
These observations
strongly indicated that, of the organs tested, the liver was
the target organ for tetradymol,
A sublethal dose (150mg/kg) of tetradymol was administered
to mice to see if the hepatic damage was dose dependent.
This
was done over a time period of eight hours sacrificing three
. mice every two hours,.
Hepatic damage was not as severe in
these livers and damage was not as apparent in the first four
hours as it had been with tetradymol at a lethal dose.
Figure
18 is a slide of mouse liver after the sublethal dose at an
eight hour period after poisoning.
Comparing this to Figure
15, which is eight hours after a lethal dose, the damage is
less severe showing only cytoplasmic vacoular degeneration and
some dilated blood filled sinusoids but not complete-necrosis.
The next objective was to see if tetradymol was a binder
of cytochrome P-450,
This was accomplished by spectral binding
studies.
Liver microsomes were isolated from mice pretreated with
phenobarbital,
Hexobarbital was used.as a standard to
represent Type I binding and aniline as a standard for Type II
binding.
Results from these experiments are represented by
the difference spectra in Figures- 19 and 20,
Hexobarbital
and aniline gave excellent difference spectra representing the
two Types of binders,
As can bee seen in Figure 19, tetradymol
H e x o b a r b i t a l HmM
□
A n i l i n e 5mM
A
T c t r a d y m o l 2mM
A A bsorbance
O
360
?70 380
3 9 0 /,00 4 1 0
420
4 3 0 4 4 0 450 460
( w avelen gth )
F i g u r e 19 .
Sp ectral
a n ilin e,
change ob served w ith h e x o b a r b ita l,
and t e t r a d y m o l .
48O
45
.10
O
T e t r a d y m o l I . 50mM
□
T e t r a d y m o l I . 07mY
A
T e t r a d y m o l 0 .64mM
.0 8
.06
.0 4
.02
A A bsorbance
O
-.02
-.0 4
-.0 6
-.0 8
-.10
-.1 2 *
360
370 380
390 400 410
420
430 440
450 46 0 470
( w avelen gth )
F ig u re 2 0 .
S p ectra l change observed
co n cen tra tio n s.
v a ry in g
tetradym ol
46
follows the hexobarbital spectra indicating it to be of Type
I character*
Varying the concentration of tetradymol used
from 2e0mM to O e64mM altered the difference spectrae
The
result of this alteration was seen mostly as a shallowing in
the 420nm trough*
An interesting aspect of the difference
spectra was the Inflection point which appeared at approximately
430nm and disappeared as the tetradymol concentration was
lowered.
At higher concentrations it is possible for compounds
O0
to have both Type I and Type II binding characteristicS0
Therefore, tetradymol may have some Type II character which'
was reflected in this point which could be a peak appearing at
430nm peak in Type II binding compounds.
This proved that
tetradymol does bind to cytochrome P-450 with Type I
characteristics*
It has been suggested that the magnitude of
the difference spectra for Type I binding was a reflection of
the amount.of cytochrome P-450 activated for the hydroxylating
reaction.^* 81
Therefore, the results indicate that tetradymol
is a substrate for this hydroxylating. system and would be
metabolized via this system.
To see what effect altering liver enzyme conditions had
on tetradymol toxicity, death time studies were undertaken and
histology studies were preformed.
The death time studies were undertaken by pretreating
mice with compounds that changed the effective concentration
47
of the mixed function oxidase enzymes or the effective
concentration of the conjugative enzyme systems, either
glutathione or UDP-glucuronic acid.
After pretreating with
the appropriate compounds the mice were poisoned with a lethal
dose (350mg/kg) of tetradymol, allowed to die and death times
recorded (Table 13)»
Table 13
Death Time
Pretreatment
'Death
(hrs)
Control
7.48±1.67
Phenobarbital
5.5361.70
3-methylcholanthrene
5.1761.08
SKF-525A
13»55±lo86
Piperonyi butoxide .. '
17.9062.90
Cysteine
9.18±0.48
Diethylmaleate
6.3260.71
Salicylamide
ll.2363.0i
Olive oil
7.0363.01
Ethanol
9.7361.02
Pretreating with phenobarbital or 3-methylcholanthrene,
induces the synthesis of the mixed function oxidase
enzymes 47,48,49,50,31
This increase in enzymes should cause
48
an increase in the metabolism of tetradymol resulting in the
production of more metabolite(s)e
Increasing the metabolism
of tetradymol shortened the death time, . The shortened death
time indicated that the metabolite is more toxic than
tetradymol,
Pretreating with compounds that bind to .cytochrome
P-450 and inhibit metabolism; such as, SKF-5P5A and piperonyl
butoxide'^$yv reduce the amount of metabolite formed.
Inhibiting metabolism allowed the mice to live longer.
Since
death time is increased by inhibition of this reaction, it is
reasonable to conclude that the oxidized toxin is more toxic
than tetradymol,
To test how conjugation effected tetradymol poisoning
the mice were pretreated with appropriate compounds to. alter
the conjugation with glutathione or UDP-glucuronic acid.
Pretreating with cysteine, which.allowed for more conjugation.
73
due to an increase in glutathione present,
to live slightly longer,
Diethylmaleate pretreatment, which
74
inhibits glutathione conjugation,
slightly.
allowed the mice
shortened the death time
This difference in death times indicated that
tetradymol or a metabolite is conjugated by glutathione.
This
change is not great which led to the conclusion that
glutathione conjugation did not afford much protection to the
animal or the extent of conjugation was minimal.
Pretreatment with salicylamide allowed an increase in
■■
.
49
the death time.
This was peculiar, since blocking conjugation
by UDP-glucuronic acid and allowing all toxin and metabolite
to be present in the system should have resulted in a shorter
death time.
The longer death time would suggest that
salicylamide was effecting the system in another way other
than simply a binding inhibitor of conjugation.
The
possibility that salicylamide affected the mixed function
oxidase system was pursued by testing to see if it was a Type
I or Type II binder of cytochrome P-450.
The result of this
is shown in Figure 22 and indicates that salicylamide is a
Type I binder of cytochrome P-450.
Levy and Matsuzawa®^
showed the product of salicylamide hydroxylation via
cytochrome P-450 is gentisamide shown in Figure 21.
0
0
Il
C-NH
it
C-NH
salicylamide
Figure 21.
gentisamide
Hydroxylation of salicylamide.^2
Therefore, salicylamide may be a binding inhibitor of
cytochrome P-450.
This conclusion was further supported by
the results in the histology studies, vida infra.
50
O
Hexobarbitnl ^mM
O
Aniline 5mM
<n
o
§
rQ
In
C
Cl
«3i
—e
560
570 580 590 400
410 4 20 450 440
450 460 470
(wavelength)
Figure ?2 .
Spectral changed observed with hexobarbital,
aniline, and salicylamide.
51
The mice were .pretreated with the vehicles, olive oil
and ethanol to see if these had any effect on the death times,
Olive oil pretreatment had no effect on death time, hut
ethanol lengthened death time slightly.
Ethanol affects the
mixed function oxidase system by inducing enzyme synthesis
83
much the same as phenobarbital, ^
It has been shown that
ethanol preferentially induces a different form of cytochrome
P-450 than does phenobarbital or 3-methylcholanthrene,^
Considering this, ethanol induction of mixed function oxidase
.enzymes could lead to an alteration of the pathway by which
tetradymol would be metabolized which was not as toxic as the
metabolite formed from normal or phenobarbital pretreated
mice, .
'
The mice were pretreated with the same compounds used in
the death time study for histology studies.
Pretreating the mice with phenobarbital moved the damage
to the peripheral region of the lobule and increased the
severity.
Examples are shown in Figures 23 and 24 where
slides of livers at four and six hours after poisoning with
350mg/kg tetradymol are displayed.
very severe at both times.
The damage was uniform and
If these figures are compared t o ■
Figures 13 and 14» which are slides at four and six hours after
tetradymol poisoning only, it indicates the hepatic damage was
potentiated.
This is additional evidence that the metabolite ■
Figure 23. Phenobarbital
pretreatment, tetradymol
poisoning, mouse liver,
four hours, 100X.
Figure 24. Phenobarbital
pretreatment, tetradymol
poisoning, mouse liver,
six hours, 100X.
Figure 25. 3-methylchoL
anthrene pretreatment
tetradymol poisoning,
mouse liver, six hours
100X.
Figure 26. SKF-525A
pretreatment, tetradymol
poisoning, mouse liver,
six hours, 100X.
53
formed .from mixed function oxidase action is more toxic as far
as causing hepatic damage*
Pretreatment with 3-methylchol-
anthrene did not move'the necrotic damage from centralobular
but did appear to afford some protection against hepatic
damage*
At two and four hours after poisoning with 350mg/kg
tetradymol no damage was apparent*
Six hours after poisoning
Figure 25, damage was starting to appear in the central region*
This damage was a mild, cytoplasmic vacoular degeneration.
This along with the result obtained from the death time
study indicated that 3-methylcholanthrene pretreatment altered
the pathway for tetradymol metabolism.
This alteration
resulted in a metabolite that was more toxic in killing the
animal but did not produce as much hepatic damage *
SKF-525A pretreatment protects against hepatic damage*
Figure 26 is representative of the SKF-525A groupdfor. two
through, ten hours*
The hepatic damage was the same for the
complete time period being in the peripheral area, uniform, ..
mild, fine cytoplasmic vacoular degeneration.
Pretreatment
with piperonyl butoxide also protected against damage.
This
compound is more effective as a protector than SKF-525A*
There' is no damage apparent in the slides.for two, four, or
six hours.
Some slight damage became apparent at eight and
ten hours which is reflected in the peripheral region as fine
cytoplasmic vacoular degeneration, . This is illustrated in
5k
Figure 2?»
This data indicated that the metabolite was not
only more toxic in killing the animal but was the agent
that.caused hepatic damage,
Salicylamide pretreatment protected against hepatic
damage almost as effectively as piperonyl butoxide.
There is
no damage apparent at two or four hours after poisoning.
Figure-28 is a representative of the six through ten hour
groups, . This shows some swelling and mild, fine cytoplasmic
vacoular degeneration in the eentralobular region.
Taking
this into consideration with the results of the spectral
binding study, which showed salicylamide to be a Type I
binder, and the death time study, it was concluded that
•salicylamide is a binding inhibitor of cytochrome P-450-and
could protect against hepatic damage and death through this
meahanism,
Salicylamide must; therefore, play a dual role in
the sustem upon pretreatment:
(I) a binding inhibitor of
UDP-glucuronic acid, which allowed all of the tetradymol to
remain free or unconjugated in the system to excert whatever
effects it may have; and (2) a binding inhibitor of cytochrome
P-450, prohibiting.the formation of metabolite and allowing
the animal to live longer.
Cysteine pretreatment completely protected against
hepatic damage as can be seen in Figure 29,
This indicated
that glutathione conjugated the necrosis causing compound.
55
Figure 27. Piperonyl butoxide pretreatment,
tetradymol poisoning,
mouse liver, eight hours,
100X.
Figure 29. Cysteine
pretreatment, tetradymol
poisoning, mouse liver
six hours, 100X.
Figure 28. Salicylamide
pretreatment, tetradymol
poisoning, mouse liver,
six hours, 100X.
Figure 30. Diethylmaleate
pretreatment, tetradymol
poisoning, mouse liver,
eight hours, 100X.
56
This showed that glutathione conjugation was very important
as far as hepatic damage was concerned; although, inducing
conjugation does not effect the death time that greatly.
It
could be concluded that glutathione must be depleted before
necrosis takes place but this was not a requirement for death.
Pretreatment with diethylmaleate resulted in moderate, uniform,
centralobular cytoplasmic vacoular degeneration and dilated
blood filled sinusoids eight hours after poisoning.
The
mice sacrificed at this time were on the verge of death.
The
sections before this reflected some- damage but it was not as
apparent.
Death time after diethylmaleate pretreatment is
shortened but inhibiting conjugation did not result in
potentiating hepatic damage as expected.
This would indicate
that diethylmaleate may have another function relating possibly
to the mixed function oxidase enzymes as in the case of
salicylamide.
Pretreating with the vehicles, olive oil and ethanol, did
not change the hepatic■damage caused by tetradymol poisoning.
Figures 31 and 32 are slides eight hours after poisoning
showing uniform, moderate cytoplasmic vacoular degeneration in
the centralobular region which was comparable to the damage
at eight hours after lethal tetradymol administration only.
Since it was indicated that the metabolite was more
toxic than tetradymol it was important to see if it could be
57
Figure 31» Olive oil pretreatment,
tetradymol poisoning, mouse liver,
eight hours, 100X.
Figure 32. Ethanol pretreatment,
tetradymol poisoning, mouse liver,
eight hours, 100X.
58
shovm that tetradymol was toxic*
SKF-525A 'pretreatment doses were varied to see if a
maximum inhibition point could be obtained (Table I^Je
Table 14
Effect of Varying SKF-525A on Death Time
Group
SKF-525A '
(mg/kg)
Tetradymol
(mg/kg)
Death
(hrs)
I '
.0
350
7.48*1,61
2
10
. 350
11.70*1;04
' 3.
20
350
11.40*1*92
4
40
35P
13.75*0.60
5
60
350
14.62*1.70
6
80
350
15.85*1.66
350
14.79*1.50
7 •
100
8
120
.
'
350
.
15.69*1.70
'As can be seen from this data the maximum inhibition by SKF525A is at 80mg/kg.
At SKF-525A■doses above 120mg/kg, 140mg/kg
and I60mg/kg# resulted in death to the mice from the SKF-525A
alone*
The mice in these two groups died in less than forty- •
five minutes.
The data in Table 14 is plotted in Figure 33»
The leveling of the graph represents the maximum inhibition,
. Using 80mg/kg as the SKF-525A dose of maximum inhibition,
the tetradymol dose was varied to.see what effect this had on
V
Figure 33.
10
20
30
Plot of death time j£s 5KF-525A dose
40
50
60
70
80
SKF dose (mg/kgj
90
100 H O
120
60
death time (Table 15)„
Table 15
Varying Tgtradymol Dose
Group
SKF-525A
(mg/kg)
Tetradymol
(mg/kg)
Death
(hrs)
I
80
,250
2
80
350
16.0060.40
3
80
450
14.41±0.78
4
80
550
14.68*1.20
5
80
650
14.08*0.61
19.25*1.50
This data is represented graphically as a double
reciprocal plot in Figure 54»
As the dose of tetradymol is
increased from 250mg/kg to approximately 450mg/kg the death
time changes relatively quickly.
The slope of this area of
the graph reflects the rate of death caused by an increasing
amount of metabolite being present.
The metabolite
concentration will increase until a saturating amount of
tetradymol is reached (approximately 450mg/kg) then the
metabolite concentration will remain constant even though
the tetradymol dose may go up.
slope of the graph changes.
At this point ■(450mg/kg) the
Now the death time is not
changing as quickly but is still slowly going down.
Since
the metabolite concentration is remaining constant in this
area any change in the death time or slope of the graph is
Figure 34.
Double reciprocal plot of death time vs tetradymol dose.
8.0
(hours)
7.0
I/death time xlO
6.0
5.0.
4.0-
_|_______ Lj_______I_______ I_______ I_______ I_______ i_______ j_
0.5
l.o
1.5
2.0
2.5
I/ CtetradymoiJ xlO-^ (mg/kg)
3.0
3.5
4.0
62
attributed, to the toxidity of tetradymole
Although this is not conclusive proof that tetraidymol is
toxicj more points are needed to further support the conclusions
84
drawn, in independent experiments Holian
has shown tetradymol
to be an uncoupler of oxidative, phosphorylation.
By using two groups of phenobarbital pretreated mice, a
control group and measuring the cytochrome P-450 concentration
in all groups a plot was obtained that gave the approximate .
metabolite death time.
The death times and cytochrome P-450
concentrations are given in Table 16«
Table 16
Death Times and Cyt0 P-450 Concentrations
Group
Phenobarbital
Death
(hrs)
'
(Cyt« P-450)
(mM)
5»53*1»70
2.27*0.92
Phenobarbital-diethylmaleate 5»73*2.31
2.58*0.67,
Control
1.49*0.91
7.48*1.61
*
Diethylmaleate pretreatment. will be. explained shortly.
The data in Table I6 is illustrated in Figure 35 as a
plot of death time vs the reciprocal of the cytochrome P-450
concentration.
The death rate was inversely porportional to
cytochrome P-450 concentration.
Consequently, the plot can
be extrapolated to maximum cytochrome P-450 concentration or
63
— —
l/(C y t.
nM
F i i -Urv
35*
H o t . o f d o n tli t i m e
co n cen tra tio n .
r-450)
vc recip ro ca l
of
Cyt .
P -450
. 6 4
essentially complete transformation of tetradymol' to metabolite
and the metabolite death time will fall between 5o53 to 3„2
hours.
It was not possible to obtain more points for this
graph due to the inability to obtain the microsomes in a half
induced state.
Therefore, only control and full induced mice
can be. used,
In the experiment where SKF-525A dose was varied-and in
one phenobarbital pretreatment group the mice were also
pretreated with dlethylmaleate.
It was interesting to notice
that diethylmaleate pretreatment seemed to have no effect on
the death times when used in conjunction with the other
compounds,
SKF-525A pretreatment by itself gave a death time
of 13o55 hours with diethylmaleate pretreatment the death time
was 13*73 hours.
The death time with phenobarbital
pretreatment alone was 5*33 hours, with diethylmaleate
pretreatment the death time was 3*73 hours.
This was further
support for the.earlier assumption that glutathione conjugation
was not effective in protecting the animal against death or
the extent of conjugation of the killing compound was minimal.
Summary - Tetradymol is an hepatotoxin of moderate toxicity
having an LD^q of approximately 273mg/kg, This research has
I
shown that after poisoning with tetradymol the toxin was evenly
distributed through the various organs examined in the body.
It is recoverable from the urine and feces for at least seven
65
dayS0
After poisoning mice with a lethal- dose of tetradymol
hepatic damage was apparent in the centralobular. region of the
lobule and progressed in severity from two to ten hours*
The
necrosis was dose dependent as shown by the fact that the
damage was not as severe when a sublethal dose was administered
to mice*
Tetradymol was shown to be a Type I binder to
s
cytochrome P-450 which indicated that it may be metabolized
through the mixed function oxidase system.
Further support
for this metabolism pathway was obtained from pretreatirig
experiments, . If the mixed function oxidase enzymes were,
induced the resulting death time for tetradymol poisoning was
decreased; if inhibited the death time was increased.
Furthermore, pretreatment altered the hepatic damage,
Pretreatment either potentiated or prevented necrosis.
Pretreatment also indicated the metabolite was more toxic than
tetradymol.
By inducing metabolism enzymes and producing more
metabolite, the death time was shortened.
Conversely,
inhibiting these enzymes and not allowing production of
metabolite, the death time was lengthened.
It has been
indicated that tetradymol is toxic but not as toxic as its
metabolite.
Glutathione conjugation did not play a significant
role in protecting against death from tetradymol poisoning.
Increasing the amount of this conjugator did protect against
hepatic damage.
Inhibiting the conjugation with diethyl-
66
maleate also protected against hepatic damage which would
indicate a role for this compound other than simply a binding
inhibitor of glutathione.
Pretreating with salicylamide to
see the effect of U DP-glucuronic acid conjugation resulted in
an unexpected lengthening of the death time.
Pursuing this
peculiarity it was found that salicylamide was a Type I binder
to cytochrome P-450. Taking this into consideration with the
histology results, it was indicated that salicylamide was a
binding inhibitor of cytochrome P-450.
From this information two models can be drawn, one
representing death and the other representing hepatic damage.
Both' models indicate there is more than one metabolite
formed from the biotransformation of tetradymol.
Death Model
Hepatic Damage Model
T-tetradymol; D-death, N-necrosis; P-phenobarbital; 3-MC3-Methylcholanthrene; S-SKF-525A, PB-piperonyl butoxide;
Sal-salicylamide; Cys-cysteine; DFM-diethylmaleate
Tetradymol poisoning leads to death presumably through
the metabolite formed by the action of the mixed function
oxidase enzymes.
This action is potentiated with phenobarbital
and 3-methylc.holanthrene increasing the rate of death &nd
67
inhibited by SKF-5E^A51 piperonyl butoxide, and salicylamide
decreasing the death rate*
Tetradymol poisoning leads to hepatic necrosis*
This
necrosis is potentiated by phenobarbital, protected against
by SKF-525A, piperonyl butoxide, and salicylamide, protected
against by 3-methylcholanthrene probably by some alteration,
in the pathway of metabolism, and protected against with ■
cysteine inducing the conjugation of the necrotic species and
diethylmaleate by some unknown method*
■EXPERIMENTAL SECTION
Ie. Reagents
All reagents were used as obtained from the company
without further purification*
■N-hexane (Northwest) was used as solvent in feeding
experiments, for column.and thin layer chromatography, and
for extracting tissues.
Anhydrous diethyl ether (Baker) was
also used as solvent for column, and thin layer chromatography,
■95% ethanol (US Industrial Chemical) was used as an
injection vehicle and as solvent for various solutions used
in spectroscopic studies,
Olive oil (Pompeian, Inc,) was
used as an injection vehicle,
Ehrlicks reagent was prepared by dissolving $0 mg of
p-.dimethylaminobenzaldehyde in Iml of 95% ethanol with 2
drops of concentrated H Cl,
77
-
Potassium monophosphate and potassium diphosphate (Baker)
were used for preparing various buffer solutions,
Phenobarbital (Merck Se Co,), 3-methylcholanthrene .
(Calbiochem), piperonyl butoxide (K&K), SKF-525A graciously
supplied by Dr, David Burke, cysteine (Calbiochem), diethyl- .
maleate (Eastman), and salicylamide (Chem Service) were used
for the pretreatment of mice,
Hexobarbital (Winthrop), aniline (Mallinckrodt) and
salicylamide were used in spectral experiments.
69
Activated, neutral alumina oxide (Ventron) was used
for column packing and was not pretreated before use.
The
alumina was deactivated with 2ml methanol per 100 grs alumina
/
in an anhydrous diethyl ether slurry when preparing columns.
For thin layer chromatography'Silica Gel IB (Baker) was
used and for preparative thin layer chromatography, plates
were prepared, with Adsorbosil-J (Applied Science Laboratories)
poured as a water slurry with a Desaga spreader.
The plates ■
were activated for one hour at I20°C and stored in an air­
tight dessicator until used.
For the development of the thin layer chromatography
plates the sprays were either a sulfuric-dichromate spray
solution of Chromerge (Manostate Corp,) or a solution of ■
p-dimethylaminobenzaldehyde$ 2 grs in 20ml concentrated HCl
and 80ml of 95% ethanol.,^^
Scintillation counting solutions were prepared using
.4% PPO (ICN) and 4% Cab-O-Sil (RPI). in toluene.
Tetradymol was isolated in the laboratory following the
proceedure described by Dr. Reeder,
5
New England Nuclear
labeled tetradymol with tritium by the Wilzbach method.
II.
Instruments
Spectral measurements for cytochrome P-450 concentration,
binding studies, and tetradymol concentration were obtained
•on either the Cary .14 made by Varian or the Varian Techtron
■70
Model 635 using matched quartz cells.
The Sorvall.EC2-B centrifuge and the Beckman Model L-2
ultracentrifuge were used, for the isolation of microsomes.
The Beckman LS-IOO scintillation counter was used for
tritium detection.
The Potter-Elvehjem homogenizer was used in homogenizing
all liver tissue.
III0 Jttce
Dub/ICN mice were obtained from Flow Laboratories.
colony was started and maintained.
A
The mice were housed in
plywood and metal cages, normally six mice per cage, and were
maintained on Purina Lab Chow and water "ad libitum".
For
elimination experiments the mice were housed in metabolic
cages, one mouse per cage, made of steel wire and glass petri
dishes.
An illustration of the metabolic cages is given in
Figure 36.
The mice were starved for twelve to fifteen hours before
feeding experiments; tetradymol was administered in hexane
.(4mg/.lml) via stomach tube.
For isolation of microsomes the
livers from non-starved mice were used.
IV.
Pretreatment of mice
• Mice were pretreated with.compounds that changed the
effective concentration of the mixed function Oxidase system
71
water bottle
hinged door
wire cage
food holder
petri dish
Figure 36.
Metabolic cage
or cytochrome P-450 specifically.
Phenobarbital is an enhancer of the synthesis of the
mixed function oxidase enzymes^’
and was administered
as an intraperitoneal injection at 80mg/kg in water for three
days prior to poisoning with tetradymol.
Another enhancer of
the synthesis of this system is 3-methylcholanthrene 47,48,49,
which was administered intraperitoneally as an olive oil
suspension at 20mg/kg for two days prior to poisoning.
The
mice were poisoned forty-eight hours after the last phenobarbital injection and twenty-four hours after the last
3-methylcholanthrene injection.
SKF-525A (2-diethylaminoethyl-2,2diphenyl valerate)
and piperonyl butoxide (o<.-(2-(2- butoxyethoxy)ethoxy)-4,5methylene dioxy-2-propyl toluene) are binding inhibitors of
cytochrome P-450.;l,^>'? SKF-525A was administered intra-
72
peritoneally in water at ZfOragAgs forty-five to sixty ■minutes ■
'prior to poisoning and piperonyl butoxide was administered as
'
an intraperitoneal injection, in 95% ethanol at JZfO m g A g s
thirty minutes prior to poisoning*
Mice were pretreated with compounds that modified the
effective concentration of conjugator systems such as
glutathione or U DP-glucuronic acid.
Cysteine is an enhancer of the synthesis of glutathione.^
It was administered intraperitoneally in.water at 150mg/kgs
five minutes before ahd twenty.minutes after poisoning,
'7k ■was
Diethylmaleates a binding inhibitor of glutathiones
administered intraperitoneally in olive oil at . J m l A g s thirty
minutes prior to poisoning.
of U DP-glucuronic acid
75
Salicylamide is a binding inhibitor
and was administered intraperitoneally
as an olive oil suspension at ZOOmgAg thirty minutes prior to
poisoning.
Salicylamide is eliminated very quickly as the
conjugated product;
therefore, it was necessary to continue .
to inject the mice every hour and a half at a dose of IOOmgAge
Vo
Histology slides
After sacrifice by decapitation the various organs were
removed from the mice and placed in 10% neutral formalin
obtained from the Bozeman Deaconess Hospital laboratory.
The organs were then placed in paraffin .blocks, cut, mounted.
73
and stained with hem'atoxylin-eosin.
All histology slides
were prepared by Gayle Callis.
Dre James Inhelder, DVM, read the slides to identify
kind, amount, and location of any necrosis that was present.
He viewed the slides at various microscope powers and
photographic slides were made of certain histology slides at
.
IOOX microscope power by Don Frittse
VI.
Isolation of microsomes
After decapitation the livers were removed from the mice
and homogenized in a 10 fold dilution of either cold 0.1M
phosphate buffer at pH 7.4 or cold 0.05M phosphate buffer in .
1.13% KCl at pH 7,4.
Elvehjem homogenizer.
The livers were homogenized by a PotterThe homogenate was centrifuged at
9000xG for twenty minutes which spun out cell debri, nuclei,
and mitochondria.
The supernant was saved and centrifuged at
103,OOOxG for one hour and a reddish microsomal pellet was
obtained.
The supernant was discarded and the pellet was
washed with either 0.1M phosphate buffer or O.05M phosphate
buffer in .1.13% KCi.
The microsomes were recentrifuged after
Washing at 105,OOOxG for fifty minutes and the supernant was
discarded.
The microsomal pellet was then stored as a pellet
or as a suspension in either 0.1M phosphate buffer or 0.05M
phosphate buffer for up to two weeks.
74
VII.
Experiments
Quantitation of tetradymol - 250mg of tetradymol was sent to
New England Nuclear to be labeled with tritium by the Wilzbach
method.
In the Wilzbach method the sample is placed in a
sealed gas chamber which contains 3 Ci of tritium gas and
allows for exchange of labile hydrogens.
The tritiated sample
was purified by the following procedure.
^H-tetradymol was placed on a neutral alumina column,
deactivated with 2% methanol, and eluted with 6ml fractions
of hexane-ether-methanol.
2ml fractions were collected and
these were spotted on TLCi
The elution pattern is given in
■Table 17.
Table 17
Elution Pattern of Alumina Column
Hexane
70
:
Ether
.
30
60
40
. 50
50
40
60
30
70
20
.
:
Methanol
' '•
80
.10
9.0
0
100
99.5
0.5
75
3
^H-tetradymol came off the column in fractions 15 through
19 although tritium activity, which was monitored'by liquid
scintillation counting, was found in all fractions.
Preparative TLC.was run on the ^H-tetradymql fractions.
The
■^H-tetradymol collected from this procedure was checked with
TLC for purity and with the scintillation counter for activity.
The compound appeared to be pure but activity was found
throughout all sections of the plate,
3
^H-tetradymo.l
was then
sublimated at 50-60°C, 0,01mm Hg and checked again with TLC
and scintillation counting.
3
•
The ^H-tetradymol appeared to be
pure but there was still activity throughout the TLC plate,
3
Finally H-tetradymol' mixed with cold tetradymol was sub­
limated at 50-60°C, OeOlmm Hg,
The results from this was the
same as after the first sublimation,.
A solution containing tetradymol was mixed 1:1 with
Ehrlicks reagent and a blue color appeared from the reaction
. 7 7
with the furan ring.
The blue color appeared after 45
minutes and was stable for one hour,
SeVeral standard
concentration curves ranging.from 10 to IOOug were made with
the Cary 14»
With the aid of Dr, David Smith and the computer,
the data was averaged and put into a least squares program
which resulted in an equation which was used for tetradymol
measurements.
The equation is:
y = 66x - 4«7
y = concentration; x = 0» D,
76
Stability of tetradymol - Tetradymol.was placed in concentrated
HCl (Ph -I0OS), IM HCl (pH I), and O 0OlM phosphate buffer
(pH 7»4)«
These solutions were allowed to stand for 4 to 5
hours and were then extracted with several volumes of hexane.
The tetradymol concentrations was checked with Ehrlicks reagent.
Tetradymol was recovered from IM HCl and 0.01M phosphate
buffer but not from concentrated HCl0
Results are on page 35,
Table 9$ Results and Discussion section.
To test the stability of tetradymol in the stomach live
mi.ce were used.
The mice were given an injection of pheno-
barbital to aid in keeping them asleep then they were
anesthized with ether*
The mice were opened to expose the
stomach which was tied off at the esophagus and small intestine
to minimize elimination of any material from the stomach,
Img
of tetradymol in hexane was injected directly into the stomach
and every 15 minutes three stomachs were removed.
The
stomachs were ground with a mortar and pestle'and extracted
with several volumes of hexane.
one hour.
This procedure continued for
The sample solutions were then checked for tetradymol
concentration with Ehrlicks reagent.
Results' are shown in
Table 18 ,
Distribution study - Starved, control mice were sacrificed by
decapitation and the blood and nine organs were removed.
The
nine organs were brain, heart, lung, stomach, kidney, pancreas,
77
Table 18
Tetradymol Recovered from Stomach
Time
% recovered
0
102.6
0
92.2
0
98.3
15 •
79.9
15
97.3
15
108.2
30
79.9
30
118.8
30
82.0
45
75.8.
45
.79.9
45
79.9
liver, and upper and lower intestine#
Ave %
98
95
93
78
The organs were ground
with mortar and pestle and the blood and organs were extracted
with several volumes of hexane#
These solutions were checked
for furan level with Ehrlicks reagent and the values obtained
were subtracted from the levels obtained after poisoning.
Starved mice were poisoned with 350mg/kg tetradymol.
Every
two hours three mice were sacrificed and the blood and the
aforementioned nine organs were removed.
The organs were
78
ground with mortar and pestle and the blood and organs were
extracted with several volumes of hexane.
The tetradymol
concentration was checked with Ehrlicks reagent and a full
visible spectra was run on the Ehrlicks-tetradymol solutions
to further verify the compound .present as tetradymol
(Table 19),
Elimination - Control mice were placed in metabolic cages and
their urine and feces were collected for 24 hours.
The urine
and feces were then ground together with a mortar and pestle
and the mixture was extracted with several volumes of hexane.
The solutions were checked for furan level with Ehrlicks
•
reagent and the values obtained were subtrated from the levels
obtained after poisoning.
Each mouse was given 3mg of
tetradymol, placed in a metabolic cage and allowed food and
water "ad libitum".
The urine and feces were collected every
24 hours, ground together with a mortar and pestle and
extracted with several volumes of hexane.
The tetradymol
concentration was checked with Ehrlicks reagent.
procedure continued for seven days,
This
A full visible spectra
was run on the solutions to verify the compound present as
tetradymol (Table 20),
Histology studies - Starved mice were poisoned with either
350mg/kg tetradymol or a sublethal dose of 190mg/kg
•79
Table 19
Tetradymol Recovered from.Organs
Time
(hr)
Stomach
(mg)
2
1.105
0.403
0.133.
4
1.270
0.150
O.IO3
0.022
■6
0.531
0.083
0.125
0.042
8
0.485
0.310
0.163
0.045
U0 Intestine
(mg)
L. Intestine
(mg)
Liver
(mg)
■ 0.026
Time
(hr)
Lung
(mg)
Heart
(mg)
Brain
(mg)
2
. 0.073
0.030
0.055
0.058
0.026
0.012
4 . ' ■0.042
0.024
0.015
0.031
0.037
0.053 .
0.045
0.037
0.030
0.025
0.032
0.013
'6
0.060
0.077
0.046
8
0.044
0.017
0.035
Pancreas
(mg)
.
Kidney
(mg)
Blood
■ (mg)
Table 20
Elimination of Tgtradymol
Mouse ■ ■ I
(mg)
2
(mg)
(mg)
Day;- '
_L_
(mg)
(mg)
6
(mg)
.020
.035
' .021 ■
0
. .005
.010
.025
0
I
.038
.020
.022 .
2
.038
.028
.010
'3 .
■.060
.037
.037 '
.005
.010.
.235
.026
.043
.011
.015
4 ' .
7.
(mg)
• died
.017
0
80
tetradymole
For the 350mg/kg dose three mice were sacrificed
by decapitation every two hours for ten hours and the same
nine organs were removed as in the Distribution Study,
Histology slides were preared on all the organs and examined
by Dr.- Hill0
The slides revealed necrotic damage only in the
liver and no other organ.
For the sublethal dose of tetradymol
three mice were sacrificed every two hours and the livers
were removed.
This continued for ten hours.
Histology slides
were prepared of the livers and examined by Dr. Inhelder.
Mice were pretreated with effectors of the mixed function
oxidase system, phenobarbital, 3-niethylcholanthrene, SKF-525A,
and piperonyl butoxide, as described in 'Pretreatment of mice'.
The mice were starved and then poisoned with 350mg/kg
tetradymol."
Three mice were sacrificed by decapitation every
two hours and the livers were removed.
ten hours or until death.
This continued for
Histology slides were prepared /from
the livers and examined by Dr. Inhelder.
Mice were pretreated with effectors of glutathione or
U DP-glucuronic acid conjugation systems, cysteine, diethylmaleate or salicylamide, as described in 'Pretreatment of
mice'.
The- mice were starved and poisoned with 350mg/kg
tetradymol.
Three mice were sacrificed every two hours and
the livers were removed.
until death.
This continued for ten hours or
Histology slides of the livers were prepared
81
and examined by Dr0 Inhelder0
Mice were pretreated with injection vehicles, olive oil
at a dose equivalent to that given with diethylmaleate and
ethanol at a dose equivalent to that given with piperonyl
butoxlde. ' The mice were starved and.poisoned with 350mg/kg
tetradymol,.
Three mice were sacrificed at four and eight
hours and the livers removed.
Histology slides were prepared
of these livers and examined by Dr, Inhelder0
■All histology slides were compared as to amount, kind,
and location of damage.
The results of these investigations
are given in Appendix B and pages 41-42, and•52-55 in the
Results and Discussion,
Death time study - Starved mice were poisoned with-350mg/kg
tetradymol and placed in cages without food and water.
Mice
would not eat or drink even if food and water were provided, ■
The mice were allowed to die and the death times recorded.
Mice were also pretreated with effectors of the mixed,
function oxidase system, phenobarbital, 3-methylcholanthrene,
SKF-525A, and piperonyl butoxide, as described in 1Pre­
treatment of mice1.
After pretreatment these mice were starved
and poisoned with 350mg/kg tetradymol,
They were placed in
cages without food and water, allowed to die and the death•
times recorded.
If the mice were still alive after 12 hours
82
food and water were provided but the mice would not eat or
drink i, ,
Mice were pretreated with effectors o f .glutathione or
UDP-glucuronic acid conjugation systems, cystine, diethylmaleate, and salicylamide, as described in 'Pretreatment of
mice'.
These'mice were placed in cages without food and
water, allowed to die. and the death times recorded.
If the
mice were still alive after 12 hours food and water were
X
provided.
Mice were pretreated with injection vehicles, olive oil
.at a dose equivalent to that given with diethylmaleate and
ethanol at a dose equivalent to that given with piperonyl
butoxide.
tetradymol.
The mice were starved and poisoned with 350mg/kg
They were placed in cages without food and water,
allowed to die and the death times recorded.
Mice., were pretreated with varying doses of SKF-525A,
lOmg/kg, 20mg/kg, 40mg/kg, 60mg/kg,: 80mg/kg, lOOmg/kg,
120mg/kg, 140mg/kg, and l60mg/kg, all given 43 minutes before
■poisoning with 350mg/kg tetradymol.
The mice were also pre­
treated with 0 .3ml/kg of diethylmaleate thirty minutes before
poisoning.
After poisoning, the mice were placed in cages
without food and water, allowed to die and the death times
recorded.
If the mice were alive after I'd hours food and
, water were provided.
The mice given the. 140mg/kg and I60mg/kg
83
doses of SKf'-525A died of the SKF injection.
Mice were pretreated with phenobarbital and diethylmaleate, as described, and were poisoned with 350mg/kg
tetradymol.
They were placed in cages without food and
water, allowed to die, and the death times were recorded.
Mice were pretreated with 80mg/kg SKF-525A, were starved,
and poisoned with varying doses of tetradymol, 250mg/kg,
350mg/kg, 450mg/kg,550mg/kg and 650mg/kg,
The 450mg/kg,
550mg/kg, and 650mg/kg doses were prepared in hexane at
6mg/.lml solution.
The mice wepe placed in cages without
food or water, allowed to die, and the death times recorded.
. Results for this section can be found in Appendix C.
Spectral studies - Microsomes for CO spectra for quantitating
■the cytochrome P-450 were prepared with.0.1M phosphate buffer.
After, suspending the micrbsomes, 2ml of the suspension were
placed in a sample and 2ml in a reference cuvette.
The '
microsomes in the.reference cuvette were reduced with sodium
dithionite and CO was bubbled through for about 20 seconds.
The difference spectra was run and the concentration of
cytochrome P-450 was obtained by taking the difference of the
O.D. at 450nm and 490nm and using the extinction coefficient
of '91cm*"^mM
^
Microsomes for cytochrome P-450.spectral binding studies
were prepared with 0.05M phosphate buffer in 1.15% KCl and
84
O e05M phosphate buffer.
After suspending the microsomes,
3ml of the suspension were placed in a sample cuvette and ^ml
in a reference cuvette.
Appropriate compounds were mixed with
the microsomal suspension in the reference cuvette to
represent the two types of binding.
Hexobarbital (5mM) was
used as a standard for Type I binding and aniline (5mM) was
used as a standard for Type II binding.
Tetradymol was mixed
at various concentrations, 2.OmM, l.jjmM, 1.07mM, 0.86mM, and
Oo64mM.
Salicylamide was tested using a concentration of 2mM.
The data may be found on pages 44» 45» and 50 in the
Results and Discussion.
APPENDIX A
Structures of Pretreatment Compounds
86
P h on ob arb ital
3 -m eth y lch o la n th ren e
Salicylamide
C^-C-(CHp)P-O-CHp
CH^-CHp-CH
P ip ero n y l
Nn
I 2
SH-CHp-CH-COOH
C ystein e
b u t ox:! d<
Si
Si
CvH 7j- C H p - O - C-CvH — CH— C—0 —C H p CII
Diethylmaleate
APPENDIX B .
Table of Histology Slides
.HISTOLOGY- SLIDES
Slide
Treatment
Tetr'adymol.
(mg/kg)
Time
(hr)
Lesion
Location
8
.None
350
"2
C
Extent
U
.17
None .
350
2
26
None
35P
2
35
None
350
: 4
44
None
350
4
Not Remarkable .
53
None
350
4
C to Mz
.62-
None
350
71
None
. 350
6
80
None
350
6
89
None
350
8
98
None
350
’107
None
350
'
6
Severity
Mi
FCVD
'Mi
CVD ■
Not R&markable
•
Not Remarkable
Not Remarkable
,
C' to. Mz
.U
IT.
Mo
CVD
SI .
CVD
■Not Remarkable -
.F
Mz .
F
Mi
FCVD
;8
C to Mz
U
Sv
DBS,
NEC,
NC
8
C to Mz
U
Sv
DBS,
NEC,
NC
'10
C to Mz
U
Mo.
NEC,
DBS,
NC
.G
. ■
116
None
350.
Nature
S lid e
T reatm ent
T e.trad ym ol
(m g/kg)
Tim e
(hr)
Lesion
L o ca tio n
125
-134
'
E xtent
S ev erity
Sv
None
330
10
C t o Mz '
U
None .
350
.10
C t o Mz
u
300
2
C
•
N ature
'NEC,
'DBS ,
..NC
Sv
• NEC,
BBS,
NC
. U
SL
VFCVD
196
. .None
197
None
300'
2-
C
U
SL
FCVD
198
Non e
300
2
C
U
SL
FCVD
199
None'-
300
4
P
U
. SL
FCVD
200
None
300
4
. C
U
SV
CVD,
DBS
201
None
300.
4
C t o Mz
U
Sv
CVD
202
.None
300
6
C
U
SV
CVD,
DBS,'
NEC
203
None
300
6
. C
U
Mo
'
204
None
300
205
None
150
'
CVD,
DBS,
NEC
6'
C
TL ' •
Mo
CVD
2
G
U
Mi
FCVD
S lid e
■
T reatm ent
T etradym ol
(rng/kg) .
Time
(hr)
Lesion
L o ca tio n
E xtent
S ev erity
N ature
■t
J
Mi
. S w ellin g
U
Mi
S w ellin g
Mi
S w ellin g
Mi .
S w ellin g
206
None
150
2
C
20?
None
150
2
c-
208
■N o n e
150 '
4
-C-
u
209
None
150
/4.
C
■F
210
■ None
150
4
C -
U
Mi
S w ellin g
211
None
150
6
C
U'
Mo
CVD
212.
None
150
6
C
■ U
Mo
CVD .
213
. None
150
6 •
C
U
Mo
CVD
214
None
1 5 0 ■■
8 - ..
C
U
Sv
CVD , '
DBS
215.
None
150' .
8
C
U
Sv
CVD-,
DBS
216
None
150 ’
8
c.
Sv
CVD,DBS
244
SK F-5 2 5 A
: 350
2
■245
SK F-525A
.350
246
SKF-525A.
247
SK F-525A
.
-
•
'
.
-
.U ■
P
U
■
Mi
FCVD .
2
'P
• 'U
.
Mi
FCVD
350
2 ■
.p - ‘
- Mi
FCVD
350
4
-
.P
U .
U
Mi
FCVD .
^
O
T reatm ent
S lid e
T etradym ol
(m g/kg)
Time
(hr)
-■ Lesion
L ocation
E xtent
P-
U
P -
U
248
SKF-929A
330
4'
249
SK F-9 2 9 A
390
'4' .
2$0
SK F-329A
390
6
251
SK F-929A
330
6'
252
SK F-9 2 9 A
330
"6
293
SKF-929A
390
8
N ot R em arkable
25.4
SK F-9 2 9 A
390
8
P
255
SK F-525A
390
8
P •
296
SK F-329A
390
10.
297
SKF-929A
350
10
'P
298
SK F-9 2 9 A
330
10
P
FGVD
Mi
FCVD
F-
SI
FCVD
P '
F
SL-
FCVD'
U
Mi
FCVD
■ U.
Mi
FCVD
U
Mi
FCVD
' U
Mi
FCVD
M i'
FCVD
'
P.
•'
U
390
2
N ot R em ark able
P ip ero n y l
b u to x id e
390
2
N ot R em arkable
303 . P i p e r o n y l b u t o x i d e
390
2
N ot R em arkable
304
P ip ero n y l b u to x id e
390
4 .
N ot R em arkable
309
P ip ero n y l
b u to x id e
330
4'
N ot R em arkable
306
P ip er o n y l b u to x id e
390
4
N ot R em arkable
302
Mi
P
b u to x id e
.
•
N ature
■ N ot R em arkable
P ip ero n y l
301
S everity
T reatm ent
S lid e
T etradym ol
(m g/kg)
Time
(hr)
■Lesion
L o ca tio n
•
P ip ero n y l b u to x id e
311
P ip ero n y i b u to x id e
350
,6
P ip ero n y l b u to x id e
350
6
313
P ip ero n y l b u to x id e
350
S .­
P
314
P ip ero n y l b u to x id e
350
'S .
P
313
P ip ero n y l b u to x id e
.350
8
P ip ero n y l b u to x id e
350
318
F ip ero n y l b u to x id e
30?
■
350
5
S everity
M ature
N ot R em arkable
31-0
312
■
E xtent
N ot R em arkable
■ Not R em ark ab le'
U
si
FCVD
U
. SI
FCVD
P
U
SI
FCVD
10
P
U
si
FCVD
350
IO
B
N
SI
FCVD
E thanol
.350
4
Not R em ark able
308
E thanol
350
4
Not R em ark able
309
E thanol
350
4
.C t o Mz
U
Mo
CVD
316
E thanol
350
8
C t o Mz
U
Mo
CVD
229
P h en o b a rb ita l,
350
2
P
U
Mo
CVD
FI
230
P h en ob arb ital
350
2
P
U
Mo
CVD
FI
231
P h en ob arb ital
350
2
P-
U
Mo
CVD
FI
317
•
■ .
,
.
Slide.
Treatment
T etradym oi
(mg/kg)
Time(hr)
Lesion
L o ca tio n
E xtent
S ev erity
N ature
U
Sv
CVD
232
T h en ob arb ital
350
4
P
233
P h en ob arb itai
330
4
P
U ”
Sv
CVD
234
P h en e-b arb ital
330
4
P
U
Sv
CVD
235
P h en ob arb ital
330
6
P
U
SV
CVD
NC
236
P h en ob arb ital
330
6
P '
U
Sv
CVD
NC
292
3-m ethylch o la n th ren e
330
2
N ot R em arkable
293
. 3 -m eth y lch olan th ren e
330
2.
N ot R em arkable
Mo
CVD
'
'
.
294
3 -m eth ylc h o lanth r e n e 1
330
2
N ot R em arkable
298
3 -m eth y lch o la n th ren e
330
4
C
299 . 3 - me t h y I c h 01 a n t h r e n e
330.
4
N ot R em arkable
300
3 -m eth y lch o la n th ren e
330
4
N ot R em arkable
295
3 -m eth y lc h o lanthrene
330
6
' C
U
Mi
CVD
296.
3 -m eth y lch o la n th ren e
330
6
C
U
Mi
CVD
297
3 -m e th y ic h o la n threne-
' 33C
6
C
Mi
CVD
240
O liv e
o il
330
4
N ot R em arkable
241
O liv e
o il
330
4
.U
.
- C to Mz
■ u"
F
Mo t o - CVD
Sv
Slide
T reatm ent
T ptradyaol
(m g/kg)
•L e s io n
Time
(hr)
L o ca tio n
E xtent
S ev erity
N ature
242
G liV e o i l
350.
4
l\To t R e m a r k a b l e
237
O liv e
o il
35C
6
C‘
U
Mo
CVD
238
O liv e
o il
350
6-
C
U -
Mo
CVD
239
O liv e
o il
350
6
C
F
Mi ■
CVD
259
C ystein e
350
2
N ot R em ark able
260
C ystein e
350 .
2
N ot R em arkable
261
C ystein e
390
■2
N ot-R em arkable
262
C ystein e
350
4
No t R e m a r k a b l e
263
C ystein e
350
4
C t o Mz
264
C ystein e
330
4 '
N ot R em arkable
263
C ystein e
6
N o t R em arkable
266
C ystein e
350
6
N ot R em arkable
267
C ystein e
350
6
C
268
D ie th ylm aleate
350
2
■ N o t R em arkable"
269
D iet hyIm ale a t e
350
2.
Not- R e m a r k a b le
270
D ieth y lm a lea te
350
2 .
N o t Re m a r k a b l e
271
D ie th y lm aleate
350
4
N o t "R em arkable
35C
•
U
U
Mi
.
FCVD
SI
FCVD
S lid e
T reatm ent
T etradym ol
(m g/kg)
Tinie
(hr)
L esion
L o ca tio n
E xtent
272'
D i e t h y I m a l e ate
- 350
4
N at R em arkable
273
D ieth y lm a lea te
350
4
No t R e m a r k a b l e
2?4
D ie t h y Im a lea te
350
6
N ot R em arkable
275
•' D i e t h y l m a l e a t e
350
■6
N o t R em arkable
276'
D ieth y lm a lea te
350
6
277
D ie t h y lm a l e a t e
350
8
C-
278
' D ieth y lm a lea te
350
8
279
D ieth y lm a lea te
350
8
319
S alicylam id e
350
2
N ot R em arkable
320
S alicyiam id e
/350
2
Not R em arkable
321
S a licy la a id e -
350
2
N ot R em arkable
322
S a licylam id e
350
4
C
323
S alicylam id e
350
U
N ot R em arkable
324
. S a licy la m i.d e
4
N ot R em arkable
325
S alicylam id e
■
350
'
350
.
6
•. P
.
-
F
S ev erity
N ature
Mo -
CVD
' U
Mo
CVD
DBS
C
U.
Mo
CVD ,
DBS
' C
U
Mo
’ CVD
DBS
Mi
CVD
C
U .
'U •
Mi
.FCVD
S lid e
T reatm ent
T etradym ol
(-iA y )
Tim e
(hr)
I e s i on
h n cation
Txtent-
S everity
C
U
Mi
FSVj
6'
C
U
Mi
FGVD
550
a
C
U .
Si'
FCVD
S a l i c y l am ide
3-50
S
C
U
SI
FGVD
550
S alicylam id e
550
8
C
U
SI
FGVD-
331
S a licylam id e
550
10
C
U
SI
FCVD.
332
S alicylam id e
550
10
U'
Mi
FGVD
550 ;
10
U
Mi
FCVD
J 26
*
S o l i e y l am ide
350
6
J2V ■
S a l i c y l a m i d e '•
550
J 2c
S a l i c y I am ide
52?
335 ' ' ’ S a lic y la n id e
C
'
•
M akxrs
KEY FOR LESION DESCRIPTION
Location in lobule
C = Central
Mz = Midzonal
P = Peripheral
Extent in section
U = Uniform, every lobule
F = Foral, some lobules, not all
Severity of changes
SI =. Slight
Mi = Mild
Mo = Moderate
Sv = Severe
Nature
NR = Not remarkable, no lesion
CVD = Cytoplasmic vacoular degeneration
FCVD = Fine cytoplasmic vacoular degeneration
NC = Nuclear change (karyolysis, pyknosis)
NEC = necrosis
DBS = Dilated blood filled sinusoids
APPENDIX C
Death Times
Death Times
Mouse
I
Pretreatment
Tetradymol
(mg/kg)
Dpath time
(hrs)
none
350
5.92
none
350
10.42
. none
350
8.42
. 4 .
none
350
5.17
5
none
350
8.60
6
none
350
6*36
7
phenobarbital
350
2.88 ./
8
phenobarbetal
350
9
phenobarbital
350
7.38
10
phenobarbital
350
5.13.
11
phenobarbital
350
12
phenobarbital
350
6.30 .
13
phenobarbital
350
4.76
14 .
3-methylcholanthrene
350
4.52
15
3-methylcholanthrene
. 350
3.52
16
3r-methylcholanthrene
350
4.77
' 17
3-methylcholanthrene
350
'5.27
18
3-methylcholanthrene
350
19
SKF-525A
330
11.25
20.
SKF-525A .
350
16.25
21
SKF-525A
350
' 12.75
22
SKF-525A
350
15.5P
23
SKF-525A
350
12.00
2
.
3
.
8.88
. 3.38
.
7.77 .
.
100
Mouse
Pretreatment
Tetradymol
.(mg/kg)
D eath tim e
(hrs)
24
piperonyi butoxide
350
16.25,
25
piperonyl butoxide
350
16.17 "
26
piperonyi butoxide
350
' 16.42
27
piperonyl butoxide.
350
15.50
28
piperonyi butoxide
350
25.00
29
cysteine
350
9.33
30
cysteine
350
8.08.
31
cysteine
350
9.58
32
cysteine
350
9.83
33
cysteine
350
9.08
350 .
7.00 .
34
. diethylmaleate
35
diethylmaleate
350
6.58
36
diethylmaleate
350
■ 7.00
37 .
diethylmaleate
350
5.25
38
diethylmaleate
.350
5.75
39'
salicylamide
350
8.33
40
salicylamide
350
41
salicylamide
350
10.50
42
salicylamide
350
14.33
salicylamide
. *350
15.67
43
■
44
ethanol.
350
45
ethanol \
350
.
7.33
. 10.58 '
.
8.83
101
Mouse
Pretreatment
Tetradyraol
(mg/kg)
Death Time
(hrs)
46
ethanol
350
. 10.58
47
ethanol
350
''10.58
48
ethanol
350
8.08
49
olive oil
■ 350
5.50
50
■olive oil
350
&.25
51
olive oil
350
5.75
52
olive oil
350
10.75
53
olive oil
350
. 5.00
54
SKF-5 25A (lOmg/kg)*
350
13.50
55
SKF-525A(10mg/kg)*
350
11.50
56
. SKF-525A(10mg/kg)*
350
. 11.25
9.75
57
SKF-525A(10mg/kg)ft
350
58
SKF-525A(10mg/kg)*
350
. 12.50.
59
SKF-5'25A(20mg/kg)*
350
11.50
60
SKF-525A(20mg/kg)*
. 350
’ 11.50
61
' SKF-5.25A(20mg/kg)#
. 350
10.92 .
(>2
SKF-925A (20mg/kg)" '
350
11.50
63
SKF-925A(80mg/kg)*
350
11.58
64
SKF-325A(40mg/kg)#
350
. I^oOS
65
SKF-525A(40mg/kg)*
350
13.17
66
SKF-325A(40mg/kg)#
350
18 =33
350
15=42
SKF-525A(40mg/kg)*
67
*
Pretreatment with diethylmaleate'
102
Mouse
Pretreatment
68'
SKF-525A(40mg/kg)• ■
69
SKF-5 25A (60mg/kg)
70.
SKF-525A(60mg/kg)#
71
72 .
'Tetradymol
(mg/kg)
I
•Death Time
(hrs)
350 .
13.75
350
16.67
350' ,
15.25 .
■ SKF-525A(60mg/kg)*
350
14.17 ' ■
SKF-525A(60mg/kg)*
350
12.50
350.
14.50
73
. SKF-525A(60mg/kg)*
74
SKF-525A(80mg/kg)*
75
SKF-525A(80mg/kg)*
76
..
350
. 19.53
350
14.50
SKF-525A(80mg/kg)*
350
15.40'
77
SKF-525A (80mg/kg)*
350
. 13.50
78
SKF-525A(80mg/kg)*
350
16.32
79
SKF»525A(100mg/kg)
... 350
SKF-525A(100mg/kg)
350
13.00
350
16.50 .
350
16.16,
80
.
81'
.82
83
84 '
SKF-525A(100mg/kg)
■
.
SKF- 5.25A (lOOmg/kg)
■ SICF-525A( lOOmg/kg)
13.50 ;
350
■SKF-525A(120mg/kg) '
350
12.75
350
15.50
85 . .
SKF-525A(:i20mg/kg)
86
SKF-525A(120mg/kg)
350
19,00
87
SKF-525A (I20mg/kg)
350
; 15.50
68
SKF-525A( 120mg/kg) ..
350 .
phenobarbital*
89
*■
■Pretreated, with diethylmaleate.
'
350
:
5.33
■
■
103
Mouse ■
90
91
'
Pretreatment
*
phenobarbital
'*
phenobarbital .
92
■phenobarbital
93
phenobarbital
phenobarbital
99
96
phenobarbital
97
phenobarbital
Death Time
(hrs)
350
7.33
350
7^75
350
4.33
350 . . '
7.92
35P
8.33
350
4.25
350
3.12
350
, 8.00
#
phenobarbital
94
Tetradymol
(mg/kg)
*
*
*
98
phenobarbital
. 350
3.33
99
*
phenobarbital .
350
3.33
100
SKF-525A(80mg/kg)
250
16.25
101
SKF-525A
"
250
19.75
102
SKF-525A
"
250
20.25
103
SKF-525A
"
250
"
350
'15,00
350
16.00
- 16.00
104
;
■ SKF-525A
105
'
SK F-525A
"
106
SKF-525A
"
350
10?'
SKF-525A
"
350
108
SKF-525A •
"
350
109
SKF-525A
" .
SKF-525A
"
LlO
.
Pretreatment with diethylmaleate
.
16.00 '
. 17.0 0 . ■
14.50
4 50
450
. 20.75 ■
.
15.50
■
104
M ouse
Pretreatment
Tetradymol
(mg/kg)
Death Time
(hrs)
111
SKF-52.5k (8 0 m g / k g )
450
13.25
112
SK F-5 2 5 A
"
550
.12.25.
113
SK F-525A
»
550
15.50
114
SK F-5 2 5 A ■
"
550
16.00
115
SK F-525A
»
550
15.00
116
SK F-5 2 5 A
» '
650
13.50
117
SK F-525A
"
650
13.45
118
SK F-5 2 5 A
"
650
15.00
LITERATURE CITED
Io
C. Ee Fleming, M 0 Re Miller and Le Re Vawter, The Unive
of Nevada Agricultural Experiment Station Bulletin, 104,
September, 1922,
2,
Je Me M 9 Brown, J e S0 Afr, Veta Medo Ass,, 30, 403'? 1959?
' ibid,, 37, 203, 1966, ibid',, 37, 321, 1966,
3,
A. Be Clawson and W. T 0 Huffman, The National Wool Grower,
26, 18, 1936. .
4,
A. Be Clawson and W, T, Huffman, The National Wool Grower,
27, 13, 1937. -
5,
S, 'K. Reeder, Isolation and Identification of the Toxic
Principle from Tetradymia Glabrata^ Ph0D0 Thesis, Montana
. State University, 1970,
6,
J, Co'Hurley, The Isolation and .Characterization, of a
Toxic Principle of Tetradymia Glabrata, PhoD0 Thesis,.
Montana State University, p. 35, 1972,
7,
Se J, Reeder, ojd, cit,, p, 26,
8,
Ibid,, p, 92,
9,
Ibid,, p, 31o
10.
Arthur Ham, Histology, J 0 B, Lippincott Co,, p, 717, '1965.
11.
Arthur C. Guyton, Textbook, of Medical Physiology, W 0 B,
Saunders Co,, p, 968, 1961,
12.
Arthur Ham, ojd. cit., p. 716.
13.
Ibid., p. 718. .
14.
Ibldao p. 712.
15.
Arthur C, Guyton, ojd. cit.,. p. 969.
16.
Arthur Ham, ojd. cit,, p. 713..
17.
Arthur C, Guyton, op. cit.. p. 970,
18.
Arthur Ham, ojd. cit., p, 714.
19»
106
Arthur Co Guyton, oj>o Cit0, p e 9760
20o
Arthur Ham, 0£0 clt0, p« 715»
210
E0 A0 Newsholme and C0 Start, Regulation in Metabolism,
John Wiley and Sons, p 0 286-287, 1973»
22.
H 0 J0 Zimmerman, Ann0 New York Acade Science, 104, ^o 954
955, 1962.
25.
Victor A. Drill, Ann. New York Acad0 Science, IO4 , p« 859
1962.
24.
H0 J e Zimmerman, op. cit.
Po 956.
25.
Victor Ao Drill, opb cit,
Po 866.
r\)
crx
H 0 J 0 Zimmerman, opo cit,
P. 957.
'27.
Victor Ao Drill, Op0 Cito
Po 860O
.
.
CO
OJ
Hugh A. Edmondson and W0 ;.0 D0 Anderson,- Pathology, The
'C0 V 0 Moxby, Co0, p. 821, 196lo
290
Ibid0, Po 822«
300
Dre James Inhelder, Diagnostic Laboratory, Montana State
University, Personal Communication.
310
Hugh A. Edmondson and W0 A 0 De Anderson, OjD0- cit.« p. 826
32,
F. A. Denz and W0 G 0 Hanger, Journal of Pathology and
Bacteriology. 81, .91-99, 1961b
33.
A. A, Sewright and Bobyn M . -O 1Donahoo, Journal of
Pathology. 106, 251-259, 1971.
vPu
A0 A e Seawright and Jana Hrdlicka, British Journal 0 f
Experimental Pathology. 53, 242-252, 1972.
55»
J. P0 Mitchell, D0 J 0 Jollow, W0 Z. Potter, D0 Ce Davis,
J 0 P0 Gillette, and B0 B. Brodie, The Journal of
Pharmacology and Experimental Therapeutics. 187(1), 185.194, 1973.
10?
36 o
:
D. J. JoIlow , J. R 8 Mitchell, W8 Z8 Potter, D8 C8 Davis,
J 8 R 8 Gillette, and B8 B8 Brodier The Journal of
Pharmacology and Experimental Therapeutics. 187?!)i 195202 , 1973 .
37.
J. R8 Mitchell, D8 J8 Jollow, W8 Z8 Potter, J 8 R8 Gillette,
and B 8 B8 Brodie, The Journal of Pharmacology and
Experimental.Therapeutics, 187(1)» 211-217, 1973« •
38.
J 8 R8 Mitchell, W8 D8 Reid, B8 Christie, J 8 Moskowitz,
G 8 Krishna and B 8 B8 Brodie, Research Communications in
Chemical Pathology and Pharmacology, 2(6), 877-888,1971. ■
39«
B 8 B 8 Brodie, W8 D8 Reid, A 0 K 8 Cho, G 8 Sipes, G 8 Krishna,
and J 8 R8 Gillette, Proceedings of the National Academy of
Science. 68(1), 160-164, 1971 •
40.
N'. Zampaglione, D8 J. Jollow, J 8 R. Mitchell, B8 Stripp,
Mo Hamrick, and J 8 R8 Gillette, The Journal of Pharma­
cology and Experimental Therapeutics, 187(1), 218-227,
■ 1973.
41.
R8 W8 Estabrook, J. Baron, M8 Franklin, I8 Mason, M8
Waterman, and J. Peterson, "Cytochrome P-450 - Panacea or
Plague", Miami Winter Symposia, The Molecular Basis of
Electron Transport, Academic Press, p. 200, 1972.
42.
Ibid8. Po 206.
4Jo
Ibid8, p8 207-208«
44.
Ibid... p 8 210.
45.
K8 Comai and J8 La Gaylor, The Journal of.Biological
Chemistry, 248(14), 4 9 4 7 - 4 9 5 5 , 1 9 7 3 .
46.
A8 F0 Welton and S8 D9 Aust, Biochemical and Biophysical
Research Communications. 56(47, 898-906, 1974.
47.
A8 H8 Conney, Pharmacology Review8 19, 317-366, 19.67.
48.
A. H8 Conney, W8 Levin* M 0 Ikeda, R. Kuntzman, D. Y8
Cooper, and 0. Rosenthal, Journal of Biological Chemistry.
243, 3812-3915, 1 9 6 8 .
!
■
■
106
49 o
A. H.o Conneyj W0 Levin, M 0 Jacobson, R0 Kuntzman, D0 Y0
Cooper, and O0 Rosenthal, Microsomes and Drug- Oxidations.
Academic Press, 279-295» 1969*
50 o
A* Ye -H0 Lu, W0 Levin, S0 B 0 West, M 0 Jacosbon, D0 Ryan,
Re Kuntzman, and A* H e Conney, Journal of Biological
Chemistry. 248, 456-460, 1973,
51.
A0 Pe Alvares, G e Schilling, W0 Levin, and R, Kuntzman,
Biochemical and Biophysical Research Communications.
29(4), 521-526, 1967*
52*
A* G* .Hildebrandt, M 0 Speck, and I* Roots, NaunynSchmeideberg1S ArChieves of Pharmacology, 281, 371-382,
1974.
53 o
L 0 Ernster and S0 Orrenius, Federation Proceedings.
24, 1190-1199, 1965.
54.
M 0 W6 Anders and G 0 J0 Mannering, Molecular Pharmacology.
2, 319-327, 1966.
55.
M 0 W 0 Anders, Biochemical Pharmacology. 17, 2367-2370,
.
1968
.
;
56* ' T* R0 Tephly and P0 Hibbeln, Biochemical and Biophysical.
Research Communications, 42,- 589-595» 1971*
57*
K* Ce Leibman, and E* Ortiz, Drug Metabolism and ■
Disposition. 1(6), 775-779$ 1973.
58.
C* L0 -Li I Lerst, E6 G e Mimnaugh, R 0 L* Reagan, and' T0 E0
Gram, Biochemical Pharmacology. 23, 2391-2394, 1974*
59*
M* D0 Burke and J 0 W0 Bridges, Biochemistry Journal.
130- 71P , 1972*
60*
A* E0 Wade, B e Wu, and F 0 E 0 Greene, Toxicology and
Applied Pharmacology. 22, 503,512, 1972*
61*
T0 Omura and R* Sato, The Journal of Biological Chemistry.
239(7), 2370-2378, 1964.
62*
T* Omura and.R* Sato9 The Journal of Biological Chemistry.
239(7), 2379-2385, 1964.
63*
T0 Kamataki and H* Kitagawa, Chemical and Pharmacology
Bulletin, 22(1), 171-175, 1974*
109
64»
Tc Kinoshita and S0 Horie9 The Journal of Biochemistry,
61(1), 1967»
65»
He Remmer,' J» Schenkman, R0 W» Estabrook, Ho Sasame, J e
Gillette, S0 Narasimhulu, D0 Y0 Cooper, and O0 Rosenthal,
Molecular Pharmacology» 2, 187-190,. 1966»
66„
J o Be Schenkman, H» Remmer, and R 0 W0 Estabrook,
Molecular Pharmacology, 3, 113-126, 1967»
67»
Re M 0 Philpot and Ee Hodgson, Molecular Pharmacology, '8,
204-214, 1972»
68.
S0 Narasimhulu5 Archives of Biochemistry and Biophysics.
147,
391-404,
1971.
69»
M0 R» T0 .Soliman, H e D» Johnson, and Ao E0 Wade, Drug
Metabolism and Disposition5 2(1), 87-96, 1974»
70«,
L 0 E 0 Chasseaud, Drug Metabolism Reviews, 2(2), 185220,
71»
1973o
E» C0 Heath and J 0 V 0 Dingell, Drug Metabolism and
Disposition, 2(6), 556-565, 1974»
72» .W0 H 0 Habig, M 0 J 0 Pabst and W 0 B0 Jakoby, Journal of
Biological Chemistry, 249(22), 7130-7139, 1974»
73»
E0 Boyland and L 0 F 0 Chasseaud, Biochemical Journal, 104,
95,
1967.
74»
D0 Jollow, W0 A0 Potter, M 0 Hashimoto, D» C» Davisj and
J0 R0 Mitchell, Federation Proceedings, 31, 539, 1972»
75»
C 0 Levy, J 0 A 0 ■Procknal, Journal of Pharmaceutical
Sciences, 57(8), 1330-1335, 1968» '
76»
H0 O0.KJ.ingele, Absorption, Djstributlon, Transformation,
and Excretion of Drugs5 Charles Co Thomas, p» 102, 1972»
77»
T» Reichsteln, Helv0 Chem0 Acta, 15, 1110, 1932»
78»
Dr» Arnold C» Craig, Chemistry Department, Montana. State
University, Personal Communication0
79o
I G 0 LeFovre,- Absorption, Distribution, Transformation,
and Excretion of Drugs, Charles C0 Thomas, p» 5-38, 1972«,
O
HO
80.
J. A. Roth and Re A e Neal, Biochemistry. 11(6), 955-961,
1972.
81.
Sten Orrenuis and Lars Ernster, Molecular Mechanisms of
Oxygen Activation, Academic Press, p. 225, 1974.
82.
G. Levy and T. Matsuzawa, Journal of Pharmacology and
Experimental Therapeutics, 156(2). 285-293, 1967.
83.
P. Luoma and M0 Vorne, Acta Pharmacology et Toxicology.
33, 442-448, 1973.
84«
A. IIolian9 Chemistry Department, Montana State University,
Personal communication.
85.
H. Sprince, Journal of Chromatography, 3, 97, I960.
J
MONTANA STATE UNIVERSITY LIBRARIES
3 1762 1001 4344 3
i
/
N378
H717
cop. 2
Holian, Sandra K
Metabolic fate and
toxic effects of one of
the components of Tetradymai glabrata
/44?
I