AN ABSTRACT OF THE THESIS OF
GRANT LOUIS SCHOENHARD
(Name)
in
FOOD SCIENCE
(Major)
Title: AFLATOXIN B
for the
DOCTOR OF PHILOSOPHY
(Degree)
presented on
March 15, 1974
(Date)
METABOLISM BY RAINBOW TROUT
(SALMO GAIRDNERI)
■-*'
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■
J'SV
Abstract approved:
^
'
J-' Jf
5^5^
„ . „ .
Donald jyLee T
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■~~ T^ " r*"r.
Aflatoxins B,,
Q, and aflatoxicol, R F, but not B- were acti-<
1
l
o
L
vated in the presence of NADPH by the 105, 000 x g pellet from rainbow
trout (Salmo gairdner-i), Mt, Shasta strain, liver to products lethal to
Bacillus subtilis GSY 1057 (metB4, hisAl, uvr-1).
Electronmicros-
copy confirmed the microsomal fraction primarily contained smooth
endoplasmic reticulum.
An NADPH mixed function oxidase with neo-
tetrazolium reductase and aldrin epoxidase activity was present in the
microsonaes.
The activity of the oxidase was reduced parallel to a
decrease in lethal factor production by piperor^yl butoxide, NADPH
deprivation, heat treatment and certain diets.
microsomes with
14
After incubation of
C labeled B , the activity was found in unaltered
B
and three extremely polar metabolites designated NM (3. 6-5. 3%),
B
A (1-5%) and M^A (< 1%).
They were eliminated or reduced along
with microbial lethality when cytosine and cysteine were added to the
incubation media.
The structural requirement for the vinyl ether of B,, R F and
1
o
Q and the nature of the enzymatic reaction were consistent with the
hypothesis that the conapounds were metabolized to highly reactive and
unstable electrophilic products which bound to nucleophiles such as
cytosine and were lethal to B^. subtilis.
Aflatoxicol, R F, was isolated from liver homogenates and was
o
apparently fornaed by an NADPH-dependent soluble enzyme of the
105, 000 x g supernatant from rainbow trout.
R F and its diastereoo
mer, R R, were prepared by chemical reduction of B .
identity was confirmed by UV and mass spe chrome try.
Their
The ten-day
LD50 value was 0. 66 mg/kg for R F compared to 0. 46 mg/kg for B..
No mortality was observed, from R R at equivalent doses.
o
Similar
gross and microscopic pathological changes were observed for B ,
R F and R R.
The degree of damage paralleled the LD50 values.
After eight months of feeding, 20 ppb of B
and R F and 36. 6 ppb
R R gave 56. 3%, 26. 2% and 0%. incidence of hepatoma.
Addition of
50 ppm of cyclopropenoid fatty acids increased the incidence to 96. 3%,
93. 8% and 55% for Bn1, R o F and R o R,
-
■•'/".'.,
It was concluded that aflatoxicol was not an effective means of
detoxication of B , but instead extended the presence of a potentially
toxic and carcinogenic compound.
Aflatoxin B
Metabolism by Rainbow Trout
(Salmo gairdneri)
by
Grant Louis Schoenhard
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
June 1974
APPROVED:
Associate Professor of Foo^Sciencpgand Technology
in charge of major
Head of Departnnent of Food Science and Technology
,1-
f - ,»-■■-, ,
„■,- w
i ^r,
i^<l9,y
ly
Dean of Grarauate School
Date thesis is presented
Typed by Mary Jo Stratton for
March 15, 1974
Grant Louis Schoenhard
ACKNOWLEDGMENT
The author wishes to express his gratitude to Dr. Donald J. Lee
for guidance and assistance during this investigation.
The author
expresses appreciation to Professor Russell O. Sinnhuber for his
interest and support.
The author i$ grateful to Dr. P. E. Bishop for assistance in
establishing the microbial assay, Dr. J, S. Lee and Dr. D. J. Reed
for use of their laboratories, Dr. N. E. Pawlowski for the rpethod and
assistance with the preparation of the crude diastereomers of aflatoxicol, Dr. L. C. Terriere for the analysis of dieldrin, and Professor
J. H. Wales for guidance in interpretation and photomicrographs of
histological preparations.
The author thanks Mr. S. E. Howell, Mr. P, Kwan, Mrs. L. J.
Hunter, Dr. L. M. Libbey, Mr. T. Will, Mr, R. A. Foster and Miss
L. K. Moreland foi; technical assistance on various aspects of the study.
Most of all, the author would like to thank his wife, Alice, for
her complete support, technical aid with the acute toxicity trial and
assistance in the preparation of the manuscript.
This research was supported by Public Health Service Training
Grant FD00010 and by Public Health Service Research Grants
ES00541 and ES00256.
The author expresses his gratitude to this
agency for making the study possible.
TABLE OF CONTENTS
.Page
INTRODUCTION
1
LITERATURE REVIEW
2
Aflatoxin
Toxicity and Carcinogenicity of Aflatoxin Bj
Biochemical Effects of Aflatoxin Bj
DNA Binding and Metabolism
Mitochondrial Respiration
Protein
RNA
Aflatoxin Bj Metabolism
Species Comparison
Activation
Xenobiotic Metabolism by Fish
Chemical Initiation of Cancer
Carcinogens as Mutagens
EXPERIMENTAL
Metabolism Studies
Centrifugation Fractions of Liver Homogenates
Preparation of Incubation Mixtures
Calculation of Mierobial Viability
Isolation and Identification of Aflatoxicol
Aflatoxicol from Trout Preparations
Aflatoxicol Diastereomers from Chemical
Reduction of Bi
Extraction, Purification and Identification of
MjA, B2aA and NM
Acute Toxicity Trial
Chronic Toxicity and Carcinogenicity Trial
RESULTS AND DISCUSSION
Mierobial Assay
Enzymatic Conditions for Lethal Factor Production
Metabolism of Aflatoxin
Effect of Inhibitors
Extraction of Metabolites
Lethality and Identification of Metabolites
2
2
6
6
6
7
9
10
10
14
16
20
22
25
25
25
28
30
30
30
31
32
34
36
37
37
39
50
50
52
56
-Page
Acute Toxicity Trial
Mortality and General Observations
Histological Observations
Liver
Kidney
Chronic Toxicity and Carcinogenicity Trial
Liver
Chronic Toxicity
Hepatoma
Kidney
Chronic Toxicity
Significance of Metabolism of B.
1
Areas for Future Study
65
65
68
68
68
74
74
74
77
77
77
77
86
SUMMARY AND CONCLUSIONS
88
BIBLIOGRAPHY
90
APPENDICES
Appendix I,
Appendix H.
Appendix III.
Appendix IV.
Appendix V.
Appendix VI.
Appendix VII.
Appendix VIII.
Appendix
Appendix
Appendix
Appendix
IX.
X.
XI,
XII.
Microbial Assay
Bacillus subtilis GSY 1057 (metB4,
hisAl, uvr-1); Partial Confirmation
of Phenotype
Krebs Ringer's Solution Adjusted
for Fish
NADPH Generating Systems
Quantitation of Aflatoxins Bi, B2,
RQF, ROR and Qj
Thin Layer Chromatography:
Preparative and Analytical
Purification of Aflatoxicol Diastereomers RrtF and R R by Column
o
o
Chromatography
Mass Spectral Analysis of Aflatoxicol
Diastereomers R.FandR R
Neotetrazolium Reductase Assay
Aldrin Epoxidation Assay
Diets
Liquid Scintillation Counting
Fluor Solutions
120
122
123
124
125
127
129
131
132
133
134
135
LIST OF TABLES
Table
Page
1
Species Comparison of B
2
Cornparative Microsomal Activity.
19
3
Carcinogens as Mutagens.
23
4
Lethal Factor Production.
38
5
Effect of Diet a^nd Feeding on Lethal Factor
Production by 20, 000 x g Supernatant.
42
Effect of Incubation Temperature and Prior
Heat Treatment.
44
Effect of 20, 000 x g Supernatant of
Acetone Powder Preparation.
44
Lethal Factor Production and NT Reductase
Activity of 20, 000 x g Supernatant and
Ca++ Precipitated Microsomes.
46
Lethal Factor Production and. NT Reductase
Activity of 105, 000 x g Fractions.
47
Effect of Piperonyl Butoxide on Aldrin
Epoxidation and Lethal Factor Production.
49
Effect of Selected Nucleophiles on Lethal
Factor Production.
53
12
Metabolites by TLC.
55
13
Microbial Lethality of Unactivated and
Activated Aflatpxicol Diastereomers.
60
Comparative Microbial Lethality of B ,
Q and B,,
60
6
7
8
9
10
11
14
15
Metabolites.
Mortality in Rainbow Trout 10 Days after
Intraperitoneal Administration of Aflatoxins
B,, R F and R R.
1
o
o
11
66
Table
16
Page
Incidence of Hepatoma at Four and Eight
Months from Start of Feeding Trial.
78
LIST OF FIGURES
Figure
Page
1
Aflatoxin structures.
2
Flow diagram of liver fractionation by
centrifugation.
26
3
Ca
27
4
Incubation mixtures.
29
5
Calculation of miqrobial viability.
30
6
Loss of viability as a function of microbial
incubation time.
40
Loss of viability as a function of B
concentration.
41
Top layer 105, 000 x g pellet composed
primarily of smooth endoplasmic reticulum.
51
Hypothetical Schiff base formation of amino
acids with one of the resonance forms of
the phenolate ion.
62
11
Liver from trout dosed with 0. 4 mg/kg B .
69
12
Liver from trout dosed with 0. 56 mg/kg R F.
69
13
Liver from trout dosed with 1. 27 mg/kg R R.
70
14
Normal trout liver.
71
15
Normal trout kidney.
71
16
Kidney from trout dosed with 0. 4 mg/kg B .
72
17
Kidney from trout dosed with 1. 27 mg /kg R R.
72
18
Kidney from trout dosed with 0. 85 mg/kg R F.
73
19
Kidney from trout dosed with 0. 56 mg/kg R R.
73
7
8-9
10
precipitation of liver microsornes.
3
Figure
20
21
22
23
24
25
26-27
28
29
30
31
32
33
34
Page
Liver from trout fed 20 ppb B
four months.
for
75
Liver from trout fed 20 ppb R F for
four months.
75
Liver from trout fed 36. 6 ppb R R for
four months.
76
Liver from trout fed 50 ppm CPFA for
four months.
76
Liver from trout fed 20 ppb B
CPFA for four months.
79
and 50 ppm
Liver from trout fed 36. 6 ppb R R and
50 ppm CPFA for four months. 0
79
Liver from trout fed 20 ppb R F ^rad
50 ppm CPFA for four months?
80
Kidney from trout fed 20 ppb R F for
four months.
81
Kidney from trout fed 36. 6 ppb R R for
four months.
81
Kidney from trout fed 50 ppm CPFA for
four months.
82
Kidney from trout fed 20 ppb B. and
50 ppm CPFA for four months.
82
Kidney frona trout fed 20 ppb R F and
50 ppm CPFA for four months.
83
Kidney from trout fed 36. 6 ppb R R and
0
50 ppm CPFA for four months,
83
Hypothetical pathways of B
metabolism.
84
and R F
AFLATOXIN Bl METABOLISM BY RAINBOW
TROUT (SALMO GAIRDNERI)
INTRODUCTION
Aflatoxin B.. is extremely toxic and among the most potent
carcinogens.
It has been found in many foods consumed by man and
domestic animals.
Various treatments of food have been tried to
reduce the toxic and carcinogenic hazard.
These efforts could be
aided by knowledge of the structural requirements foj: the ultimate
biological activity of aflatoxin B..
As indicated in the literature review, B. has not been shown to
inhibit DNA template activity, DNA dependent RNA polymerase or
malignantly transform cell cultures in vitro without the presence of
an active microsomal NADPH-dependent mixed function oxidase.
Hypophysectomy and various diets protect rats against B
hepatoma.
B
induced
is activated to a product lethal and mutagenic to
Salmonella typhimurium in the presence of an active microsomal
preparation.
Various workers have hypothesized that B
is meta-
bolically activated to a highly reactive and unstable rnetabolite as the
ultimate toxin and/or carcinogen.
The objectives of this study were to determine whether rainbow
trout (Salmo gairdneri), Mt. Shasta strain, activated B
and to evalu-
ate the structure-'activity relationship of the metabolites for toxicity
and carcinogenicity.
LITERATURE REVIEW
Aflatoxin
Well over 1, 000 publications have appeared on aflatoxin in the
last decade.
Several comprehensive reviews including the areas not
reviewed below, such as etiology, biosynthesis and chemistry, are
available (57, 104, 121, 260).
Toxicity and Carcinogenicity of Aflatoxin B 1
Many of the early investigations with aflatoxin B
(Figure 1)
were concerned with its toxicity and carcinogenicity in a wide variety
of species (57, 121),
A hyman death.occurred after ingestion of
aflatoxin B1 contaminated food.
Toxin was isolated and identified by
spectral analysis from the severely necrotic liver (33).
The acute LDSOs for two species of macaque monkey were 2.2
mg/kg (Macaca irus) and 7. 8 rng/Kg (Macaca fascicularis) accompanied by massive liver damage (240, 265).
Toxic hepatitis similar to
that found in man in regions of the world with B
contamination of
food was found in African monkeys (Cereopithecus aethiops) administered 0. 01-0. 1 mg daily until death (8),
Initial attempts to induce carcinoma in nonhuman primates were
unsuccessful (73, 205).
after prolonged B
Recently primary liver carcinoma was induced
administration (3, 123).
_A
OH
iX
^ o '^ CK^^ OCH -.
•OCH3
Aflatoxin Mj
Aflatoxin B j
o
A
o
\n
ua
^o-^-o-^s^OH
Aflatoxin Pj
Aflatoxin Qj
o
o
OH
J
o
Sj^o-^-^OCH-
^OCH3
Aflatoxin B2
Aflatoxicol (R0)
^o' ^o- ^-^ "OCH3
Tetrahydrodeoxoaflatoxin Bj (THDBj)
o
o
r-r—fV"
HO'N5Ao'^<^oCH3
Aflatoxin B'Za
HO
HO
o
Aflatoxin B, -2, 3-oxide
o
X0X0XJ< OCH,
2, S-dihydro-Z, 3-dihydroxy-aflatoxin B.
Figure 1.
Aflatoxin structures.
4
Typical LDSOs for mature rats were 7 mg/kg (47) and 5 mg/kg
(302).
Toxicity was markedly reduced or absent in prepubertal,
castrated (245) or hypophysectomized (122) male rats and in nonpregnant females (50),
The same treatments resulted in a reduction in
hepatoma.
A dietary level of 15 ppb produced a 100% incidence of hepatoma
in rats (302).
Renal neoplasms also occurred (156, 188).
B1 or a
metabolite reaching the embryo of rats through the placenta produced
inflammatory, hyperplastic and neoplastic liver changes (124).
Mature mice were highly resistant to toxicity (88, 224, 288).
Hepatoma (202, 288) and subcutaneous carcinoma (86) have been
induced.
Newborn nnice were rnore prone, in contrast to females, to
lethal effects and hepatoma (288).
Early reports (79) suggested sheep, like mice, were resistant
to the toxic effects.
More recently an
reported for wethers (15).
1JD50
of 20 mg/kg was
Pretreatment with DDT and B
the sensitivity of rams to LD50 doees (14).
decreased
Neoplasms have been
found in three sheep over a five-year trial (164).
For rainbow trout, a crude preparation of B
gave an LD50 of
0. 5-1. 0-mg/kg- (131) while an LD50 of 0. 8 mg/kg was obtained for
pure B
(28).
Over a 12-month period 4. 0 ppb B
toma incidence (269).
gave a 15% hepa-
Tumor growth was greatly enhanced by
sterculic acid (157, 158, 269, 270).
Wunder (304) observed that
sexually m,ature male rainbow trout were resistant to hepatoma compared to mature females and immature fish.
Many microorganisms were sensitive to B
(42, 46, 79, 160).
Their response was genetic, i. e. , DNA inhibition, phage induction, or
nongenetic expressed as growth inhibition.
The minimal effective
concentration was approximately 1 |j.g/ml for genetic responses and in
excess of 30 (xg/ml for growth inhibition.
Genetic effects including mitotic inhibition, DNA inhibition and
chromosome aberrations and nongenetic effects including growth
inhibition, cell degeneration and cell destruction were observed at less
than litg/nrtloCBj. for a variety of cell cultures (79, 89, 98, 160, 162,
179, 206, 207, 208).
Cell cultures of rat liver have been malignantly
transformed in vitro and successfully back-trans planted in newborn
rats producing carcinoma (284).
Many of the species discussed with respect to their sensitivity to
B
have been considered for bioassays of the toxin (80, .160).
The
chick embryo assay (56, 133, 225, 286, 287) has been widely used
because it produces characteristic lesions, has a reliable doseresponse curve, and is sensitive, fast and simple.
The structure-activity relationship of aflatoxin for carcinoma
has been studied with analogs in trout (17), rats (299) and cell
cultures (99).
In each case carcinoma required the unsaturation of the
dihydrofurofuran moiety of B
and was dependent on the degree of
unsaturation and substituents on the lactone moiety.
Biochemical Effects of Aflatoxin Bj
DNA Binding and Metabolism.
Wogan (298) hypothesized that
direct interaction of B.. with DNA was the initial and critical event for
expression of toxicity and carcinogenicity.
B1 was bound in vitro to
native double-strand helical calf-thymus DNA and to a lesser extent
to heat-denatured single- strand
calf-thymus DNA, but not to enzy-
matically hydrolyzed calf-thymus DNA (31, 58, 59, 60, 61, 273).
in vitro DNA-B
The
complex was easily broken by Sephadex G-50 chroma-
tography (60) suggesting a weak non-covalent interaction.
Fluorescence
polarization studies confirmed that the interaction was weak (234).
charge-transfer interaction between B
A
and DNA was postulated (203).
It was suggested that DNA-strand breakage of HeLa cells was a direct
action of the B
with 3 HB
present (285).
(167) and with 14 CB
In vivo binding to rat DNA was found
to trout nucleic acids (16).
Inhibition of DNA synthesis was reported for regenerating rat
liver after partial hepatectomy (106, 119) and for non-regenerating
rat liver (74, 75, 249).
Mitochondrial Respiration.
the major site of B
toxicity.
Mitochondria have been proposed as
The B1 mediated decrease in mito-
chondrial dehydrogenases and electron transfer catalysts in ducklings
7
and chickens was suggested as the toxic event in mitochondria (39, 40).
B
administered to rats produced swelling of mitochondria from liver
and kidney, but not in those from heart or testes (18).
Oxygen con-
sumption and phosphorylation were depressed in mitochondrial preparations from rats administered 0. 45 mg B /kg body weight (278).
In
contrast, other workers (58) reported no change in respiratory capacity
and P/O ratios with mitochondria from rats dosed with 7 mg B •■ /kg
body weight.
Aflatoxin B ■, inhibited succinate dehydrogenase of rat
liver mitochondria (276).
Whole homogenates and mitochondria from
livers of several animal s pecies were examined for the hi vitro and
in vivo effects of B, (135).
It inhibited oxygen uptake in vivo but not
in vitro in each species studied except weanling rats.
in vitro effect on P/O ratios from male mice.
tions of 2. 5-4. 8 x 10"
There was no
Aflatoxin B, concentra-
M resulted in a 25-44% inhibition of electron
transport inactively respiring rat liver mitochondria (92, 93).
A
maximum of 63% inhibition occurred with submitochondrial particles
(SMP ) obtained by drastic sonication of liver mitochondria.
The
major site of inhibition was between cytochromes b and c^ (or c).
Mitochondria and SMP s of protein deprived animals (5% casein semipurified diet) had similar respiratory control ratios to normal animals
(20% casein diet).
The protein deprived animals had a 30-50% reduc-
tion in B. mediated inhibition.
Protein.
After B, was mix$d with calf-thymus histones in vitro,
a shift in the B, UV spectrum (273) and an increase in viscosity of the
mixture (31) was observed suggesting B-y was bound to the histones.
Injection of rainbow trout with 400 jig B /kg body weight resulted in a
65% loss of histone and acidic proteins relative to DNA within 12 hours
(55).
Administration of B i to rats produced within 15 minutes an
increase in the rate of deacetylation of histone fractions F2A1 and F3 (96).
In vitro inhibition of amino acid incorporation by B, was observed
in rat liver slices (58, 59, 272), duckling liver slices (272) and HeLa
cell cultures (134).
In vivo incorporation of
C leucine into total
liver protein was inhibited by administration of B-p 50% in monkeys
(Macaca irus) (239), but no inhibition was seen in rats (58, 263, 297).
Several semi-purified protein preparations isolated from rats
after Bi treatment were inhibited:
(1) tryptophan pyrrolase (58, 59,
110, 301), (2) zoxazolamine hydroxylase (226), and (3) prothrombin (24).
The inhibition of protein synthesis was due in cell cultures to a direct
action oh the polysomes (116).
The stimulation of microsomal hydrox-
ylase synthesis ar^d activation was similar to benzo[a] pyrene but of
smaller magnitude due to protein inhibition (129).
Nuclear DNAase II, assayed in vitro, was activated in the rat but
not in the mouse (220, 221, 223, 257) after in vivo administration of
B, and no interaction was seen by direct addition of B 1 to the enzyme
assay (220, 221).
The specific activity of liver lysosomal acid DNAase
was markedly decreased by B.. in vitro (222).
Ribonuclease activity
from treated rats was not affected (97).
Interaction of B. with acetate thiokinase was suggested to explain
the large reduction of 1-^C-acetate incorporation into skin total lipids
(172).
In chickens, uridine diphosphate glucose-rglycogen
9
transglucosylase, glycogen synthetase and glycogen phosphorylase
activity was reduced, but the hexose monophosphate shunt dehydrogenase activities were elevated (266).
A shift in tissue-specific lactic
dehydrogenases was found in the rat (5, 159) while no change was
observed in rainbow trout (281).
The early appearance of a-fetoprotein with B
induced hepatoma
(149, 150) has been used to assess the incidence of hepatoma among
human populations exposed to dietary B
RNA.
Administration of B
(238).
to rats resulted in a dramatic
decrease in RNA synthesis in regenerating liver (151, 152) and in
intact liver (5 9, 194, 273).
B
Inhibition occurred within 15 minutes of
administration and remained up to five days (110, 111).
effect was seen with B
The same
added to incubating rat liver slices (59, 61).
It was hypothesized that the decrease in RNA synthesis resulted
from inhibition of DNA dependent RNA polymerase.
found in nuclei isolated from B
dosed rats (61, 111, 118).
inhibition was time and dose dependent (227).
255).
Lack of inhibition when B
enantiomer of B
The polymerase
was not inhibited (201).
inhibited with mouse liver slices incubated with B .
tion of RNA polymerase with B
The
Precursor incorporation
into 28S and 18S nuclear RNA was suppressed (112).
isolated from mice dosed with B
Inhibition was
But, it was
In vitro incuba-
was not inhibitory (146, 198, 250,
was replaced with the synthetic
(229), B_ or tetrahydrodeoxoaflatoxin
B1 (282)
10
demonstrated an obligate structural requirement.
cluded that in vivo inhibition with B
Wogan (2 96) con-
was possible because metabblic
transformation was feasible while neither was true in vitro.
It was suspected, because of the marked effect on nuclear RNA
synthesis, that cytoplasmic RNA would also be altered.
Polysome
disaggregation was demonstrated (228, 250, 252, 294).
This was
reported to occur because B
blocked the steriod-dependent ribosome
binding sites on endoplasmic reticulum (32, 293).
Subribosomal
particle formation was also blocked in rat liver (193) and housefly
ovaries (6).
Aflatoxin B
Metabolism
Species Comparison.
Aflatoxin B
metabolism was examined in
a variety of species and several metabolites were identified (Table 1).
There were striking differences between in vivo and in vitro metabolism and the metabolites found among species.
found two significant differences in
mice.
14
CB
Wogan (264, 298, 300)
metabolism in rats and
First, the mouse excreted totally more toxin than the rat (8 9. 9%
vs.. 80. 1%). The reduced urinary excretion of 14
?.C by the rat accounted
for the difference.
Second, the rat retained more toxin in the liver
than the mouse (7. 57% vs. 1. 51%).
As indicated in Table 1, both the
rat and the mouse form M, and B^ in vitro but M, was reported only
1
2a1
11
Table 1.
Species Comparison of B Metabolites.
Species
Rat
Mouse
M.
in vivo
1, 48, 71, 212, 236,
237, 267, 274
71
in vitro
27, 109, 212, 214,
231, 232, 258, 259
259
274
in vivo
in vitro
215, 231, 232, 274
in vivo
67, 69, 70, 267
in vitro
185
Golden hamster in vitro
25
Monkey
Guinea pig
in vivo
212^ 267
in vitro
25, 212, 215
Rabbit
in vitro
Cow
in vivo
72,.. 184
Calf
in vitro
212b
Sheep
in vivo
in vitro
25
Dog
in vitro
25
Chicken
in vivo
in vitro
Duckling
in vitro
Numbers refer to references.
Conjugated metabolite.
185
215
216, 217
25, 212
212
Chick
67? 69? 70b
7, 141, 184., 196
in vivo
in vitro
215
25
in vitro
Goat
2a
Metabolites
R
175? 176b
•
216
215
215
213
216
213, 216, 217
12
once from mice in vivo.
As reviewed earlier, female rats and both
sexes of mice were more resistant to B
The mouse absorbed B
(274).
toxicity and carcinogenicity.
more quickly from the stomach than the rat
Female rats absorbed the same (237) or slightly less toxin from
their stonaachs than males. [They.metabolized it toM^ at.the same rate as
castrated males and much faster than normal males (236).
liver cells in tissue slices took up B
Mouse
more slowly and metabolized it
faster (231, 232).
The rate of B
metabolism was correlated with the basal aniline
hydroxylase level for chick, guinea pig, calf, sheep, goat and mouse,
but not for duckling, pig and rat.
There was no trend in sheep, goat,
rat, mouse and chick, which were more resistant to B
toxicity,
towards a higher microsomal hydroxylase level or greater rates of
metabolism (212).
B
like the mouse.
The highly sensitive duckling rapidly metabolized
In contrast to the mouse, the major identified
product of the duckling was aflatoxicol (R ) not M
(26, 211, 218),
R
was formed by the 105, 000 x g supernatant and not by the microsomal
pellet which produced M
in the mouse, although both transformations
required NADPH (210).
The pattern of metabolism in the monkey varied dramatically
w ith
route of in vivo administration of
tion with liver homogenates.
14.
CB
Aflatoxin P
and upon in vitro incuba-
as the glucuronide was the
major urinary metabolite, 20% of dose, after i. p. injection and M
13
represented only 2. 3% of the dose (69).
14
CB , M
After oral administration of
accounted for 20% of the label and P , 5% of the dose, as
the glucuronide and sulfate (67).
Four days after i. p. injection 5. 6%
was retained in the liver bound to proteins and an appreciable quantity
bound to serum albumin.
The urine and blood of monkeys retained
detectable activity five weeks after oral toxin administration.
sharp contrast, no P
30-50% of the B
was found after in vitro incubation.
was converted to Q
In rainbow trout 50% of the
in the urine in 12 hours.
14
and about 1%, to M
CB
In
Instead,
(68, 185).
administered i. p. was excreted
Half of that was unaltered B .
The remainder
was removed through the gall bladder into the lower gut or became
bound to the nuclear and microsomal fractions of the liver (16).
The LD50 for M. was 16 fig/40 g duckling compared to 12 (ig/
animal for B
(235).
It was carcinogenic to rainbow trout (268).
had anti-microbial activity comparable to B , but B
B_
was 200 times
as toxic using the duckling bile duct hyperplasia assay (170).
Accord-
ing to the same assay, aflatoxiqol from fungal reduction of B
was 18
times less toxic than B
(76, 77).
found from the reduction of B
Diastereomers of aflatoxicol were
by fungi (62).
It was not presented
whether they were separated for the duckling assay.
No significant
change in embryo viability or teratogenic effect was attributed to Pj
uqder conditions yielding a positive chick embryo assay, with B, (275). At
14
doses of P
10 to 20.times greater than the B
LD50 of 9. 50 mg/kg,
only two mortalities: were observed in young mice (41).
Activation.
Several types of evidence have been reported which
suggest aflatoxin B
must be metabolized, that is, activated, to exert
certain biological changes.
Hypophysectomized rats fed 4 (jig B /g
diet developed no liver tumors as compared to a 100% incidence in
intact animals (1Z2).
A marked inhibition of RNA synthesis was
observed with intact rats receiving dietary B, but was not evident
after hypophysectomy.
The surgery or SKF 525-A decreased in
vitro, micros omal drug activity resulting in an increase of a less polar
metabolite of B
produced by the 105, 000 x g supernatant and a
decrease of the microsomally produced polar metabolite.
Without
surgery there was a decrease in the less polar metabolite which was
further metabolized microsomally (108).
Increased cytotoxicity of B
to cell cultures was observed for
those cell lines, including liver, able to metabolize the toxin (256).
It was not shown whether the inhibitory product was a metabolite of B
or arose from the cells themselves as a result of exposure to B .
7, 8-benzoflavone, a competitive inhibitor of micros omal oxidation,
protected against the cytotoxicity of B
and a host of other potential
carcinogens suspected of requiring activation (262).
Similarly,
malignant transformation of cell cultures was demonstrated for B
But
15
and numerous other carcinogens preferentially in cell lines capable
of microsomal oxidation (295).
As presented earlier in this review DNA template activity, RNA
synthesis and DNA dependent RNA polymerase were inhibited in vivo
but not in vitro.
Moule and Frayssinet (195) were able to inhibit RNA
polymerase in vitro with B
in the presence of an active microsomal
oxidative system or the extractible microsomal metabolites of B
exclusive of M .
Neal (199, 200) did not observe an inhibition of the
polymerase but a decreased template activity.
Template activity was
reduced when DNA was present with a microsomal preparation actively
metabolizing B .
After removal of the microsomal system no inhibi-
tory activity was observed in the remaining supernatant.
The meta-
bolite was either unstable or lost in the removal of the microsomes.
Butler and Neal (49) proposed that the significance for carcinogenicity
of B
activation could be assessed with animals on a marginally
choline-deficient diet which was protective against B
toxicity but not
RNA synthesis inhibition or tumor induction.
Garner and others (114, 115) found male and female rat, guinea
pig, mouse, hamster and human liver microsomes converted B
to a
reactive derivative toxic to Salmonella typhimurium TA 1530 and
TA 15 31.
B
activation was dependent on oxygen and NADPH and
inhibited by aniline, SKF 525-A and carbon monoxide.
Microbial
strains C207 and G46 were insensitive, presumably because they had
16
a viable DNA excision repair system which the sensitive strains lacked.
Aflatoxins M , R
and P. produced derivatives of lesser toxicity and
B_ and B_ were not transformed.
2
2a
No active metabolite was isolated.
The common structure-activity relationship of the aflatoxins tested
was the terminal double bond of the furofuran moiety.
They proposed,
as Schoental (261) had, that the active derivative was a highly reactive
and unstable epoxide of the furofuran moiety.
Xenobiotlc Metabolism by Fish
Xenoblotics include all substances foreign to living organisms
(87).
They are generally lipophilic, facilitating entry to the organism
across lipoidal membranes.
To be removed from the organism they
are metabolized to more hydrophilic compounds.
They are meta-
bolized by enzymes located primarily in the smooth endoplasmic
reticulum referred to as microsomes after isolation, enzymes of
intermediary metabolism in the cytoplasm called soluble fraction on
isolation.and enzymes in the mitochondria.
Microsomal reactions
include oxidations, such as hydroxylation and epoxidation, and reductions of nitro and azo groups.
tion alsp occurs.
Nonmicrosomal oxidation and reduc-
After metabolism by the above phase I reactions
many xenobiotics are made more hydrophilic by conjugation to polar
compounds such as glucuronic acid and glutathione (105, 209).
17
Gaudette, Maickel and Brodie (117) and Brodie and Maickel (36)
reported fish lack the microsomal enzymes for xenobiotic metabolism.
They suggested fish disposed of lipoidal compounds by direct diffusion
through the gills and skin without initial metabolism.
Baker,
Struempler and Chaykin (20) reported that trimethylamine was microsomally oxidized.
Since then microsomal metabolism has been
reported for hydroxylation of biphenyl (64), 2-acetylaminofluorene
(173), acetanilide and aniline (44, 81, 82), O-dealkylation of alkoxy
biphenyls (63) and phenacetin (44), epoxidation of aldrin (53, 290),
desulfuration of parathion (233), N-demethylation of anainopyrine (44,
81, 84), azo reduction (4), numerous nitro reductions (4, 43, 45,
139) and glucuronide formation (82, 95),
The comparative metabolism
between fish species was reported by Adamson (2), Dewaide (82), and
Smith (271).
Microsomal oxidation requiring NADPH and molecular oxygen
was defined as a naixed function oxidase (182).
The system was
comprised of FAD-containing flavoprotein, NADPH cytochrorne c
reductase, NADPH cytochrorne P-450 reductase and cytochrorne P-450
(181).
b
5
Associated with it was another flavoprotein, iNADH. cytochrorne
reductase and cytochrorne bc (35, 100, 120).
b
The same compo-
nents were found for fish microsomes as other vertebrate systems
(44, 45, 82).
18
Several conditions for measurement of in vitro microsomal
oxidation were markedly different for fish (2, 44, 82, 83, 85).
Little
if any activity was found at the mammalian preparation incubation
temperature of 37
Maickel (36).
mately 25
in agreement with the earlier report of Brodie and
Instead the optima were, for temperature, approxi-
, , and for pH, near 8. 0.
Higher NADPH levels and shorter
incubation times were required.
Dewaide (82) (see Table 2) found for p-hydroxylation of aniline
the activity on a mg liver protein basis was higher for one fish species,
roach, than for rat.
Buhler and Rasmussen (44) reported the K
m
from Linewea-ver-Burk plots approximated mammalian species
although Dewaide (82) always found greater values of K .
m
Dewaide (82) concluded that fish have an active microsomal
system.
Previous lack of evidence was due to using the same con-
ditions,-, particularly temperature, for fish as for mammals.
He
argued that the contention of Brodie and Reid (37) that lipophilic
xenobiotics do not require metabolism because they readily diffused
through the lipoidal menabranes and skin was not a fact.
Further,
the consequences of metabolism also favored aquatic animals because
water-soluble products would be restricted to the extracellular phase
and in this way would be available in higher concentration for excretion
through the gill, kidney and skin.
Table 2.
Comparative Micros omal Activity.
Species
a
N-demethylation of aminopyrine /p-hydroxylation of
aniline activity per
g fresh liver
mg liver protein
mg liver DNA
100 g body wt.
Mouse
(Mus musculus)
19
/5. 8
0.11 /0. 034
9.0 /2. 7
90.
/28
Rat
(Rattus norvegicus)
15
/l. 3
0.09 /0. 008
7.4 /0. 65
59
/ 5. 0
Roach
(Leuciscus rutilas)
4.81/1.23
0.042/0.0103
2.17/0.52
13.2/3.40
Rainbow trout
(Salmo irideus)
l;10/0.35
0.009/0.0028
0.61/0.19
1.7/0.53
a
From Dewaide (82, Table 17).
vO
20
Chemical Initiation of Cancer
Farber (101) summarized two current hypotheses:
(1) the
chemical, or a derivative, reacts with a cellular target inducing
cancer directly, and (2) the process of initiation, through interaction
of the chemical with cellular metabolism, induces an altered but not
neoplastic cell that evolves into cancer.
The Millers (190, 191) proposed that most chemicals needed to
be metabolized to strong electrophilic reactants, the active products,
to initiate cancer.
Boyland (34) hypothesized epoxLdes were the
active forms of polycycllc aromatic hydrocarbons.
Dipple, Lawley
and Brookes (90) presented the theory that the activity of the carcinogen
and the site of action correlated with the stability of the epoxide.
The
hypothesis was extended by Jerina jt al.; (143) to other classes of
compounds.
As alternatives, activated methyl groups (90), free
radicals (197) and others (291) were postulated as the activated or
ultimate carcinpgen.
Evidence for the possible biological significance
of activated compounds included covalent binding of the synthetic
compound to DNA (126, 241) and protein in vitro (23, 126); DNA, RNA
and protein of cell cultures (125); DNA of mouse skin (51, 91, 242);
and histones of rat liver (144).
In the presence of hamster liver
microsomes the epoxide of benzo[a]pyrene was formed and bound to
DNA (28 9).
Inhibition of aryl hydrocarbon hydroxylase prevented the
21
binding of the carcinogenic species to DNA, RNA and protein, and
tumor formation in mouse skin (147).
The transformation products
of epoxides, phenols and dihydrodiols,were not active (204, 279).
Farber (101) stated that DNA was often cited as the target
because (1) activated carcinogens bound to it, (2) it. correlated to
mutagenicity, (3) it was not turned over during the latent period before
neoplasia first appears, and (4) it was the simplest hypothesis and did
not require a complex of interlocking events.
For mammals DNA
alterations have also included single and double strand breaks (165,
166).
With the latter the frequency of repair was dramatically
decreased (101).
Lijinsky et al. ,(168) and Baird et al. (19) have
reported that all atternpts have failed to find the same products of the
active carcinogen and DNA in vivo or in transformed cell cultures that
were found with in vitro incubation of DNA.
It was suggested that the
number of interactions with DNA necessary for transformation was
below the methods of chemical detection employed.. Consistent with
this interpretation was the observation that the native DNA structure
promoted a specific reaction with the amino groups of guanine which
lay in the narrow groove of the DNA helix (241).
Alternatively,
changes in levels of glutathione, regulatory proteins (102, 103),
NADH and NADPH (54), for example, have been reported to explain
the initiation of cancer through metabolism of chemicals.
22
Farber (101) proposed that hyperplasia and cytotoxicity were
required, for neoplastic change. Cytotoxlcity would promote the evolution
(selection) of neoplastic cells.
Through a series of experiments with cell cultures, Heidelberger
(137) found that (1) individual cells were directly transformed by
activated carcinogens as opposed to selection of pre-existing neoplastic
cells, (2) transformation did not require the presence of a viral
oncogene and (3) after activation most carcinogens were mutagens.
Carcinogens as Mutagens
Activated carcinogens were m.utagenic to mammalian cell
cultures (22, 38), Neurospora crassa (161), Salmonella typhimurium
(10) and Bacillus subtilis (178, 230).
Ames, Lee and Durston (9)
developed a series of tester strains of Salmonella typhimurium to
assess the mutagenicity of potential carcinogens.
Carcinogens,
which failed to demonstrate mutagenicity themselves, were activated
in the presence of an active mammalian mixed function oxidase (11,
12, 65, 66, 177) to mutagens.
Table 3 illustrates the correlation
among carcinogenicity, microbial mutagenicity and cell culture
malignant transformation for chemicals in the presence of a viable
activation system.
Lethality to S^. typhimurium (114, 180) and B.
subtilis (94) was found to correlate with the carcinogenicity of activated compounds in the same manner as mutagenicity in the same
strains.
Table 3.
Carcinogens as Mutagens (11, 107, 192, 230, 244, 295).
Compound for activation
Carcinogenicity
Microbial
mutagenicity
Cell culture
trans formation
2-aminoanthracene
2 - am inof luor e ne
2-acetylaminofluorene
benzidine
4-aminobiphenyl
+
+
+
+
+
+
+
+
+
+
4-amino-trans-stilbene
4 - dimethylamino-trans-stilbene
p-(phenylazo)-aniline
4-(o-tolylazo)-o-toluidine
NpN-dimethyl-p- (m-tolylazo)-aniline;
+
+
+
+
+
+
+
+
+
+
2 - naphthylamine
1-aminopyrene
6-aminochrysene
benzofajpyrene
3-n^ethylcholanthrene
+
+
+
+
+
+
+
+
+
+
+
+
7, 12-dimethyl-benz [a]anthracene
aflatoxin B i
sterigmatocystin
dinaethylnitrosamine
N-methyl-N'-nitro-N-nitrosoguanidine
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
OJ
24
Ames (11) proposed that all the compounds tested acted through
intercalation in the DNA base-pair stack since they were planar and,
after activation, were franaeshift mutagen? and had an active side
group that reacted covalently to DNA.
pairing in bases of repetitive sequence.
This would stabilize a shifted
During DNA replication or
repair this would result in an addition or deletion of base pairs in the
DNA sequence.
25
EXPERIMENTAL
Metabolism Studies
Rainbow trout (Salmo gairdneri), Mt. Shasta strain, used for
liver preparations were kept in fiber glass tanks with a water flow rate
of 4 gal/min at a temperature of 12
9. 5 ppm.
with an oxygen content of 8. 5 to
Beginning two weeks before livers were taken, the fish were
fed one of the diets (Appendix XI) to satiety at the same time of day
seven days a week.
Some were starved an additional two weeks after
the same intensive feeding regime.
Centrifugatjon Fractions of Liver Homogenates
The trout were stunned, the liver quickly excised and perfused
with ice-cold Krebs solution adjusted for fish (Appendix III).
The fish
weight, liver weight, sex and volume of bile were recorded.
All sub-
sequent operations were conducted in a cold room at 2-4 .
Centrifuga-
tion fractions were prepared from a resuspended acetone powder of
liver or a whole liver homogenate according to Figures 2 and 3.
If an
acetone power was used for centrifugation, it was prepared (52) by
homogenizing each g of liver in 10 volumes of -15
blender.
funnel.
dry acetone in a
A precipitate was collected on filter paper in a Buchner
It was resuspended in 10 volumes of -15
dry acetone and
Kenmore Blender Model 600, Sears, Roebuck and Co. , Chicago,
Illinois.
26
g whole liver or 0. 19 g.acetone powder
2 v phosphate buffer (Appendix III)
homogenize
20,000 x g for 10 min
supernatant
supernatant
105,000 x gfor 90 njin
20, 000 x g for IS min
supernatant 1 v to 10 v
105,000 x g for 90 min
supernatant
top layer
microsomal pellet
bottom layer
microsomal pellet
rinse and resuspend
in v equal to original
10 v supernatant
i
I
rinse and resuspend
in v equal to original
10 v supernatant
105,000 x g for 90 min
105,000 x g for 90 min
i
^"
supernatant
Figure 2.
\layer
top
microsomal pellet
supernatant
Flow diagram of liver fractionation by centrifugation.
bottom layer
microsomal pellet
27
g whole liver
2 volumes 0. 25 M sucrose
homogenize
12, 000 x g for 10 min
i
decant supernatant and measure volume
I
add 4 x v of 0. 0125 M sucrose
with 8 mM CaCl pH 7. 2
stir a few seconds
600 x g for 10 min
++
k
Ca
precipitated
microsomal pellet
Figure 3.
••j
supernatant
Ca.++ precipitation of liver microsomes (145).
28
collected as before.
The precipitate was washed with 10 volumes of
peroxide-free diethyl ether, placed in an evaporating dish and dried
overnight in a vacuum desiccator.
The dry powder was ground to a
fine powder with a mortar and pestle before resuspension in phosphate
buffer.
For whole liver preparations the liver was homogenized with
12 up-and-down strokes in a glass Potter-Elvehjem homogenizer tube
and a loosely fitting teflon pestle.
Preparation of Incubation Mixtures
Lethal factor production was determined with one of the preparations shown in Figure 4.
cate controls,
All incubations were in duplicate with dupli-
Each experiment was repeated at least once.
As an
alternative to direct addition of the toxin to the incubation mixture,
trout were removed from their tanks, quickly stunned leaving the heart
to continue functioning, and placed, ventral side up on a V-shaped
restraining board.
The peritoneal cavity was opened and the toxin
was infused into the hepatic portal vein for one min and circulation to
the liver was continued an additional 4 min.
and perfused with Krebs solution for fish.
assay are given in Appendix I,
The liver was excised
The details of the microbial
The method of partial characterization
of the phenotype of the strain of B_. subtjlis used appears in Appendix
II.
Neotetrazolium reductase activity and aldrin epoxidation were
29
3 ml of desired liver centrifugation fraction (CF)
or control media (CM) in 25 ml Erlenmeyer flask
or
1 ml CF or CM + 1 ml NADPH generating system
+ 1 ml MgCl2 (Appendix IV)
■</
0. 1 ml DMSO, toxicant (Appendix V) in DMSO,
toxicant in acetone (evaporate, add
0. 1 ml phosphate buffer),
and/or
piperonyl butoxide or cytosine or cysteine or lysine
or glutathione (sometimes pre incubated before
toxin addition for 5 min at 25° in shaker bath)
(standard method:
incubate
10 min, 25
in shaker bath)
add 0. 1 ml J}. subtilis (Appendix I)
incubate
(standard method: 20 min, 25
Figure 4.
in shaker bath)
Incubation naixtures.
30
determined according to the methods In Appendices IX and X.
Protein
concentration was determined by the method of Lowry (174).
Calculation of Microbial Viability
The number of microorganisms for each treatment was determined from the triplicate plate count.
The two methods used to
express the results are illustrated in Figure 5.
I,
no. viable bacteria
.nn
— '
''(treated)
— ' — ■' x 100
no. viable bacteria (control)
no. viable bacteria
II.
% reduction in viability = [l -
Figure 5.
(treated)—_ j
no. viable bacteria
(control)
100%
Calculation of microbial viability.
Isolation and Identification of Aflatoxicol
Aflatoxicol from Trout Preparations
Aflatoxin B
was incubated with a 13, 500 x g supernatant plus
0. 5 M glucose or a 20, 000 x g or 105, 000 x g supernatant plus an
NADPH generating system in Erlenmeyer flasks with an air atmosphere
for 30 min to 1 hr at 25
on a shaking water bath.
tion 1. 5-2. 5 volumes of chloroform were added.
was drawn off in a separatory funnel.
At the end of incubaThe chloroform layer
The incubation medium was
31
re-extracted with 1. 5-2. 5 volumes of chloroform:water (3:1).
Emulsions were broken by centrifugation at 2, 000 rpm for a few
minutes.
The chloroform extract was dried over Na_SO..
2 4
Aflatoxicol
was isolated by repeated preparative TLC on MN-silica gel G-HR
2
with benzene:acetone:ethyl acetate (50:6:12) as the solvent or by
chromatography on silica gel H
3
columns eluted stepwise with hexane,
hexane:diethyl ether (7:3), (6:4) and (1:1).
The remaining aflatoxin B
and unknown metabolites were removed with chloroform:acetone:
isopropanol (87:10:3).
The column fractions containing aflatoxicol
were located by TLC with MN-silica gel G-HR developed with benzene;
acetone:ethyl acetate (50:6:12) or chloroform:acetone:isopropanol
(87:10:3).
Aflatoxicol Diastereomers from
Chemical Reduction of B
B
was chemically reduced to the diastereomers of aflatoxicol by
the method of Pawlowski (219)and purified by.column'chroma.tography with
neutral alumina (Appendix VII).
The diastereomers as well as R
o
from fish were identified by UV and mass spectra (Appendices V and
VIII).
2
3
Brinkman Instruments, Inc. , Westbury, New York.
Brinkman Instruments, Inc. , Westbury, New York.
32
Extraction, Purification and Identification
of M-.A, B0 A and NM
1
2a
After incubation, the preparation, containing an appropriately
treated centrifugation fraction of trout liver, toxicant and/or other
compounds (Figure 4) in a 24 ml Erlenmeyer flask was transferred to
a 250 ml Erlenmeyer flask.
The smaller flask was rinsed with 96 ml
of chloroform:acetone (58:38 or 38:58) and the rinse was added to the
3. 2 ml of media in the 250 ml flask.
shaking on a water bath for 1 hr.
at -2 3 .
Extraction was continued by
The extracts were stored overnight
The volume of extract was reduced on a flash evaporator and
re-extracted with hexane.
The remaining fraction was re-extracted
with either acetone:chloroform:water (58:38:4 or 38:58:4), acetone or
chloroform.
The amount of label in the extracts was determined by
scintillation counting (Appendix XII).
Fluorescent metabolites were
observed by exposure to 365 nm UV light on silica gel TLC plates
(Appendix VI).
Purification of MA, B9 A and NM was first tried by preparative TLC on MN-silica gel G-HR developed with chloroforrruacetone
(3:1) and column chromatography with silica gel 60
4
eluted with
benzene:acetone: ethyl acetate (50:6:2) followed by stripping with
acetone.
4
Separation of the metabolites was also attempted by high
Brinkman Instruments, Inc. , Westbury, New York.
33
5
speed liquid chromatography on a 2.1 x 500 mm stainless steel column
packed with Vydac and eluted with chloroform.
Crude separation was achieved on 5 g of silica gel H packed with
hexane on a 30 x 300 mm column.
7
The column was washed with
hexane and the sample applied in acetone or chloroform.
Lipid
soluble material was removed with hexane and hexane:diethyl ether
(50:50).
The compounds were separated with sequential 50 ml addi-
tions of chloroform, chloroform: isopropanol (99:1, 98:2, 97:3, 96:4)
eluted with approximately 0. 5 psig nitrogen.
The fractions containing predominantly B
Li a.
A and NM were
separated on 8 g of activated silica gel H (110 for 2 hr) in a 20 x 500
g
mm column.
The compounds were eluted with a gradient of chloroform to chloroform:isopropanol (8:2) under pressure with a metering
pump
9
at 60 ml per hr.
Final purification of M A, B_ A and NM was accomplished by
X
Ct a.
repeated chromatography on 4 g silica gel (0. 05-0. 2 mm)
5
in a
Model 3100, Chromatronix, Inc., Berkeley, California.
Chromatronix, Inc. , Berkeley, California.
7
o
9
Chromaflex column K-420540, Kontes Glass Co. , Vineland, New
Jersey.
Jacketed Chromaflex extender, K-422430, Kontes Glass Co. ,
Vineland, New Jersey.
Cheminert metering pump, Model CMP-1, Chromatronix, Inc.,
Berkeley, California.
Brinkman Instruments, Inc. , Westbury, New York.
34
30 x 300 mm column.
The support was washed with 25 ml chloroform,
an increasing gradient of 50 ml chloroform to chloroform: isopropanol
(50:50) followed by decreasing the gradient over 50 ml to chloroform,
and 50 ml chloroform.
The samples were applied in 0. 25 ml chloro-
form followed by five 0. 25 ml washes of the sample container.
Frac-
tions were collected with 100-300 ml chloroform and, where necessary,
followed by 1% increases in isopropanol every 50 ml.
After each column the fractions containing metabolites were
located by their fluorescence with 365 nm UV light on TLC plates of
MN-silica gel G-HR developed with chloroform:-acetone:isopropanol
(87:10:3) and benzene:acetone:ethyl acetate (50:6:12).
Non-fluorescing
material was observed by iodine-staining, coumarin spray (Appendix
VI), 254 nm UV light absorbance on MN-silica gel G-HR F..,,
or by
^54
12
locating the label with a radiochromatogram scanner.
Similar
fractions were combined and concentrated on a flash evaporator.
UV
and mass spectra were taken.
Acute Toxicity Trial
Nine-month-old rainbow trout, fasted 48 hr and weighing
approximately 60 g, were given a single intraperitoneal injection of
Brinkman Instruments, Inc. , Westbury, New York.
12
Varian Aerograph, Walnut Creek, California.
35
the toxicants.
Immediately before injection, each fish was anesthetized
with MS-222 (tricaine methane sulfonate
13
) and weighed.
They were
injected at a point just anterior and dorsal to the left pelvic fin.
toxicants were dissolved in distilled dimethyl formamide (DMF
administered in a volume of 1 jil DMF/g of body weight.
are indicated in Table 15. (p.
Aflatoxin B
14
The
) and
Dose levels
66).
was produced by Aapergillus flavus (ATCC 15517)
cultures grown on rice, isolated and purified according to the method
of Ayres (16).
The diastereomers of aflatoxicol were prepared as
described earlier.
The feeding response was determined by presenting a small
amount of diet each day to the fish.
At the same time their physical
appearance and behavior was observed.
Mortalities during the 10-day
trial were recorded and the fish autopsied.. At the end of day 10, the
surviving fish were sacrificed.
The LD50 was determined by the
metho'd of Litchfield and Wilcoxon (171).
During the autopsies gross
abnormalities were recorded and the livers and kidneys were removed
and preserved in Bouin's fixative.
These tissues were sectioned at
4 |j. with a microtome and stained with hematoxylin and eosin for light
microscope examination.
13
14
Sando, Inc. , Hanover. New Jersey.
Merck Co. , Inc. , Rahway, New Jersey.
36
Chronic Toxicity and Carcinogenicity Trial
The rainbow trout eggs were spawned and hatched in our laboratory facility and the fry held on control diet 3 (Appetidix XI)4 months before
initiation of experimental diets.
randomly selected.
Groups of 120 fingerlings were
Each group was held in a 6 gal plastic bucket,
with many holes for water flow and an effective rearing area of 0. 5
cubic ft.
Two buckets for duplicate diets were suspended in 4-ft
diameter fiber glass tanks with a flow rate of 4 gal/min at a temperature of 12
with oxygen content of 8. 5-9. 5 ppm.
After 2 months,
each bucket of trout was transferred to an individual 4'-ft fiber glass
tank.
Duplicate groups of trout received diet 3 (Appendix XI) with the
toxicants indicated in Table 16 (p.
"78 ).
One ml of ethanol/100 g of
herring oil was used as the toxicant carrier and added to the control
diets.
After 4 and 8 months, 40 trout were taken from each tank.
The
trout were killed with MS-222, weighed and autopsied for gross
abnormalities.
The livers and kidneys were examined for tumors and
toxic damage with a dissecting light microscope.
They were preserved
in Bouin's fixative and were1 prepared for histological evaluation in the
same manner as the acute toxicity trial samples.
37
RESULTS AND DISCUSSION
Microbial Assay
The primary objective was to determine whether rainbow trout
(Salmo gairdneri) activated aflatoxin B .
Despite indirect evidence,
the identity of the active metabolite from any species had not been
reported.
Explanations were that it was produced in extremely small
quantity or was highly reactive and quickly bound to subcellular targets
or was unstable.
metabolite.
A microbial assay could be used to detect a fleeting
Bacillus subtilis GSY 1057 (hisAl, uvr-1, metB4),
insensitive to high levels of B
(30), was examined for the assay of the
metabolite based on the hypothesis that it would be incapable of excising
nucleic acid bases which had an active metabolite covalently bound and
cell death would result.
When cloned, the B. subtilis culture for this study required
histidine and methionine and 99% lethality was observed in the presence
of mitomycin C.
This agreed with the phenotype (140).
As shown in Table 4, there was amarked reduction in viability
of microorganisms incubated with centrifugation fractions of liver
which had been infused in vivo th.rough.the hepatic portal vein.
some lethality from unaltered B
Since
with rich media had been reported
(30), Pennassay broth was used as a control.
The reduction was
one-third of that found with a viable liver fraction.
38
Table 4.
Lethal Factor Production.
Fraction
Liver (g)/incubation
volume (ml)
Phosphate buffer
Treatment % Reduction
(MB,)
in viability10
3.5
20, 000 x g supernatant
(boiled)
15/32
lo"4
7.0
20, 000 x g supernatant
(boiled)
15/32
4
0. 3 x 10"
9.0
4
ID-
32. 6
15 /32
10-4a
99. 9
105, 000 x g pellet
1. 5/3.2
10-4a
30
20, 000 x g supernatant
1. 5/3.2
lo"4
99. 9
20, 000 x g supernatant
1. 5/3.2
4
0. 3 x ID"
99. 9
20, 000 x g supernatant
1. 5/3.2
lO"5
99. 9
Pennassay broth
105, 000 x g supernatant
Intravenous infusion in vivo.
39
Incubation of B1 with the 20, 0.00..-X g supernatant gave the same
reduction in viability as fractions from liver after in vivo infusion of
B .
Out of 10
7
cells, only 10
3
survived.
Figures 6 and 7 indicate that
a maximum response was approached with a 20 min microbial incubation and 10
-5
M B
in the media.
The same magnitude of effect
occurred if acetone wds used as the solvent for B
and was removed
by evaporation before addition of other materials.
A 0. 01 ml of
ethanol as the solvent for B
did not alter the assay.
A 0. 1 ml of
ethanol produced a 10% reduction in viability and reduced the magnitude of the lethality with B
present.
It was not determined whether
the protection by ethanol was a direct inhibition or a generalized effect
such as protein denaturation.
It was decided that B. subtilis would
serve as an effective assay.
Enzymatic Conditions for Lethal Factor Production
The next objective was to define the enzymatic conditions-l
for lethal factor production.
As seen in Table 5, the type of protein,
level of protein and feed intake clearly influenced lethality,
B
induced hepatoma (153), cyclopropenoid fatty acid damage (277) and
histologically observed liver changes (154, 155) paralleled the above
results for corresponding diets.
Restoration of some of the lethality
by NADPH addition suggested a cofactor requirement.
an enzymatic requirement for B
activation.
This supported
The temperature
40
100 -®
o
o
-H
X
^ ^
(1) o
H
-M
a 4->
In o
o
-M
10
^-4
N-^-
n)
.^
M
0)
-M
o
d
.^4
(H
(U
4->
CJ
n) nJ
Xi ^3
0)
—^
^-4
rH
rO
<U
rt
> >
Q
•
o o
c c
OJ
.-J
.»-H
0. 1
Time (min)
Figure 6.
Loss of viability as a function of microbial incubation
time. (20, 000 x g supernatant and 10"6 MB).
O
p
P>
<-r
H
P
A
o
o
p
o
Cd
O
p
n
P
1"
U1
en
CD
o
to
O
o
o
o
no. viable bacteria (treated)
x 100
no. viable bacteria (control)
o
o
*>■
42
Table 5.
Effect of Diet and Feeding on Lethal Factor Production by
20, 000 x g Supernatant. a
Liver wt.
% body wt.
Protein (mg)/
3. 2 ml incubate
% Reduction
in viability
Casein
1. 51
88.8
99
FPC1? high protein
1. 57
80.4
90
b
FPC, low protein
1. 5
90. 5
79
Starved + NADPH
0. 65
48. 1
69
Starved
0. 65
48. 1
0
D let
a
Incubation media contained: homogenate from 1. 5 g liver/3. 2 ml and
1 0"6 M B
Fish protein concentrate.
43
limitations and heat sensitivity of the system. (Table 6) agreed with
previously reported conditions for active fish enzymes.
In the early experiments little activity was. found in the 105, 000
x g pellet from homogenates of 1 g liver diluted two to three times.
Activity was retained in the concentrated 10 5, 000 x g supernatant.
Patterson and Roberts (216) had reported aflatoxicol was produced by
the soluble enzymes in the supernatant and required NADPH.
Pre>-
liminary evidence for a metabolite with similar TLC characteristics
had been found in this laboratory.
Perhaps it was the lethal factor.
For this reason, it was imperative to determine which fraction produced the lethal factor.
It was possible that the lethal activity was
associated with microsomes retained in the 105, 000 x g supernatant
because the horn ogenate was too concentrated, therefore, retarding the
sedirnentation.
The supernatant would contain the necessary NADPH
as found in a 20, 000 x g supernatant.
At 10
MB, which gave more than 99% lethality with a fresh
20, 000 x g supernatant, no lethality was observed with a supernatant
from an acetone powder extract of liver (Table 7).
fold increase in B
After a hundred-
concentration, the lethality increased to the level
seen earlier with a rich broth.
The enzyme did not appear to be an
acetone precipitable soluble protein, or it was inactivated by the method
of preparation.
After the liver homogenate was fractionated by centrifugation,
neotetrazolium (NT) reduction and microbial death were used as
44
Table 6.
Effect of Incubation Temperature and Prior Heat Treatment
Incubation temperature of
20, 000 x g supernatant
Protein (mg)/
3. 2 ml incubate
% Reduction
in viability
25°
66. 9
99
30
95. 4
94
37
66. 9
0
b
77. 7
14
25°
159. 9
0
25
Incubation media contained: . homogenate from 1. 5 g liver/3. 2 ml;
1. 5-1. 9% liver wt/body wt; lO-6 M B .
Supernatant heated at 80
for one min prior to addition of B .
Same as b with twice concentration of liver.
Table 7.
Effect of 20, 000 x g Supernatant of Acetone Powder
Preparation.
Livera (g)/
3. 2 ml .
incubate
Protein, (mg) / .
i.3. 2 ml:
incubate
% Reduction
in viability
1. 50
77.8
HT4
30.6
0. 75
38. 9
lO'4
28.0
38. 9
5
20. 5
0. 75
10'
6
1. 50
77.8
lO"
0
1.50b
77.8
10'6
0
1. 6% liver wt/body wt..
b
Treatment
(MBj)
NADPH generating system added.
a
45
markers of enzyme activity.
Chan (52) found some NT reductase
activity was dissociated from the 105, 000 x g microsomal pellet and
occurred in the supernatant.
The microsomes from fish had lower
epoxidative activity than from rats.
Chan's post microsomal fraction
from fish produced a greater than additive enhancement of microsomal
activity when incubated with rat microsomes.
by Ca
Microsomes prepared
precipitation (21, 145) were reported to retain the soluble
proteins lost by the above methods.
As indicated in Tabled, Ca.
NADPH-dependent NT activity.
for microbial death.
precipitated microsomes possessed
Inconsistent results were obtained
With insect microsomes (21), the Ca
reduced cytochrome P-450 levels..
Reduced activity was reported
(145) for rats because Ca++ and Mg
sites.
treatment
competed for the same binding
This could have been the case with the fish preparations.
Dewaide (82) found fish required a greater Mg
mammals.
Sucrose was also used in the Ca
concentration than
precipitation prpcedure
and has been reported to yield microsomes with reduced activity
(187).
The microsomal pellet from centrifugation was composed of a
clear gel bottona layer and a top layer like a typical rat pellet.
was true even from starved fish.
This
As seen in Table 9, rinsing and
recentrifugation reduced the naicrobial activity of the bottpm layer,
although protein and NT activity were still detectable.
The specific
46
Table 8.
Lethal Factor Production and NT Reductase Activity of
20, 000 x g Supernatant and Ca++ Precipitated Microsomes.
Incubation
conditions
ProteirV(mg) /
• 3; 2 ml
iticubate
NT
reductase
% Reduction in
viability
20, 000 x g supernatant
88.8
76. 2
78
6. 69
^ ++
• -^ i. ,
Ca
precipitated
microsomes
No NADPH
15. 0
0
3
No;NADPH
22. 7
0
8
Low NADPH
22. 3
6. 17
37
High. NADPH
22. 7
1. 98
0
High NADPH
15. 0
8. 17
66
Incubation media contained: homogenate from 1. 5 g liver/3. 2 ml;
1. 9-2. 3% liver wt/body wt; lO"6 M B .
47
Table 9.
Lethal Factor Production and NT Reductase Activity of
105, 000 x g Fractions. a
Protein,'(rag) /
:3. 2 mliricubate
Incubation
conditions
NT
reductase
% Reduction
in viability
105, 000 x g supernatant
98. 1
94. 2
Fen^ale
Male
4. 9
8. 6
Bottom layer microsomal pellet
1. 5
1. 5
1, 36
NADPH
Starve db + NADPH
trace
trace
0
11
88
0
Bottom layer microsomal pellet (rinsed)
1. 32
1. 14
Female + NADPH
Male + NADPH
0. 76
0. 88
6. 3
0
Complete microsomal pellet
12.7
NADPH
7.23
61
Top layer microsomal pellet (rinsed)
No NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
14. 3
14. 3
11. 1
12. 0
1.2C
0. 12d
--9. 9
15.0
9.6
4.2
0
99
95
99
99
41
Incubation media contained; homogenate from 1. 5 g liver /3. 2 ml;
1, 5-1. 9% liver wt/body wt; 10"6 M Bj.
0. 63% liver wt/body wt.
"Homogenate from 0. 15 g liver /3. 2 ml.
Homogenate from 0. 015 g liver/3. 2 ml.
48
activity of NT reductase and microbial lethality markedly increased on
a mg protein basis for the top layer compared to the post-mitrochondrial
fraction and complete microsomes.
Neither NT reductase nor micro-
bial death was evident with boiled controls.
Since adjustments were
not made for variations in turbidity between fractions for the NT
reductase assay they should be interpreted as trends and not compared
as absolute values.
The conversion of aldrin to dieldrin, a known microsomal
epoxidation requiring cytochrome P-450 in fish (52), was examined in
the post-mitochondrial fraction.
Piperonyl butoxide (PB) presumably
bound to cytochrome P-450 and inhibited epoxidation (132).
incubation of PB with the preparations before aldrin or B
"Without
addition
only a slight reduction occurred in epoxidation and none in microbial
death (Table 10).
After preincubation with higher levels of PB there
was a marked inhibition of lethality, that is, an average of 2. 3 x 10
cells /ml out of 2. 3 x 10
cells /ml with B
alone.
7
cells /ml survived compared to 1. 6 x 10
5
Epoxidation was inhibited to a much greater
extent.
Again, as the system was refined the specific activity increased
as seen with the top layer.
8 x 10
3 x 10
5
3
In the presence of PB an average of
cells/ml out of 2. 6 x 10
cells/ml with B
alone.
7
cells/ml survived compared to
The incomplete inhibition of lethal
factor formation agreed with the results of Garner, Miller and
49
Table 10.
Effect of Piperonyl Butoxide (PB) on Aldrin Epoxidation
and Lethal Factor Production, a
m
Protein (mg)/;
o -.
.
1.3.-2 ml?
iticuba-te
,.
Treatment
pmoles dieldriu/ „, _ ,
i-- • \ i
■
% Reduction
mm mg ■
in viability
proteiii)
20, 000 x g supernatant
Aldrin
5
Aldrin + 10"
10"
M PB
M B
10"6 M B
+ 10"5 M PB
Aldrin
3
Aldrin + 10"
b
M PB
10"6 M B
10~6 M B
+ 10^3 M PBb
79.2
2.79
79.2
2.59
79. 2
--
99. 0
79.2
--
99.0
88.8
3.04
88.8
0.12
88. 8
--
98. 0
88.8
--
74.0
Top layer microsomal pellet
Aldrin
11.4
Aldrin + 10"3 M PB
11.4
0.65
11. 4
--
97.0
11.4
--
91.7
6
10"
M B
10"6 M B
3.
+ 10"3 M PB
Incubation media contained:
1. 6-1. 8% liver wt/body wt.
21.4
homogenate from 1. 5 g liver/3.2 ml;
PB incubation before addition of aldrin or B .
50
Miller (114) that B
rapid.
conversion to the toxic product was extremely
Aldrin epoxidation which was slower was inhibited 96%.
Boil-
ing completely inactivated the system.
The electron micrographs (Figures 8 and 9) indicate the top
microsomal layer was composed of vesicles of smooth endoplasnaic
reticulum extremely free of other subcellular components.
the data suggested that the microbial assay indicated B
Together,
conversion by
an enzyme system associated with the snaooth endoplasnaic reticulum
with properties of the NADPH-dependent mixed function oxidase
reported for trout.
Metabolism of Aflatoxin
The purpose of establishing the microbial assay and the enzynaatic conditions which mediated microbial death was to identify new
biologically active metabolites of B .
Effect of Inhibitors
The hypothesis was that the ultimate biologically active metabolite was unstable and had a short half life.
epoxide of B
(Figure 1).
An example would be the
As an electrophilic derivative it could
readily react with nucleophilic compounds added to the incubation
medium.
It was postulated in this manner the covalently bound
derivative of the ultimate metabolite could be trapped for isolation
51
m
,»■
* i
»
Figatie 8.
<}
Top layer 105, 000 x g pellet composed primarily of smooth
endoplasmic reticulum. Electron micrograph of pellet
fixed in 5% gluteraldehyde, stained with 1% OsO^ and
section embedded in araldite-epon mixture. X41, 200.
i
1
K'
*
.
,,
Figure 9.
V'r:
,
nm
Same as Figure 8 (above).
■ 1
Ik
X105, 000.
1
52
and identification.
The microbial assay could be used to determine
the success of a nucleophile as a trap.
Microbial lethality would be
less for better traps.
In order to reduce the amount of lethality to a level that the
effectiveness of a trap would be readily apparent, 20, 000 x g supernatant fraction from two-week starved fish were used.
of lethal factor production 10
B
MB
was itself inhibitory but 10
gave the expected reduction (Table 11).
50 x 10
-5
-3
-3
-6
M
At either level of B
M cytosine reduced the lethal effect.
tion of cytosine to 50 x 10
10
-5
At that level
Raising the concentra-
M produced lethality without B .
Lysine,
M, was an ineffective trap.
Since the top portion of the microsomal pellet in the presence
of an NADPH generating system was responsible for lethal factor
production, a 1:100 dilution of it was tried.
Cytosine at 10
not lethal but it reduced the lethality in the presence of B
-3
M was
by 60%.
Increasing the enzyme concentration enhanced microbial death.
Addition of cysteine, although somewhat toxic, reduced lethality by
50% while glutathione was ineffective even if soluble fraction was
added back.
Extraction of Metabolites
The microbial assay, the enzymatic requirement and the effect
of added nucleophiles were evidence for a metabolite(s) of B
In
53
Table 11,
Effect of Selected Nucleophiles on Lethal Factor Production.
% Reduction in
viability
Treatment
20, 000 x g supernatant
a
10"5 M B1
10"5 M Bj +
50 x 10
M cytosine
50 x 10"5 M cytosine
19. 7
0
0
20, 000 x g supernatant
6
10"
M Bj
10"6 M Bj +
50 x 10" M cytosine
50 x 10"5 M cytosine
97
0
92.8
Top layer microsomal pellet
lO"6 M B.1
_ -i
10"6 M Bl +
10
M cytosine
10"3 M cytosine
15
0
6
Top layer microsomal pellet
10"6 M B
10"
M B
+
lO"6 M Bj +
glutathione (reduced)
glutathione (reduced)
lO'3 M cysteine
10''3 M cysteine
64
16
58-67
20
29-31
Incubation media contained: homogenate fronn 1. 5 g starved liver/
3.2 ml; 1.0% liver wt/body wt; 15 9 mg protein/3.2 ml.
Incubation media contained: homogenate from 1. 5 g starved liver/
3.2 ml; 1.0% liver wt/body wt ; 100 mg protein/3.2 ml.
Incubation media contained: homogenate from 0. 015 g liver/3. 2 ml;
1. 5% liver wt /body wt; 0. 118 mg protein/3. 2 ml.
Incubation media contained: homogenate from 0. 030 g liver/3. 2 ml;
1. 2% liver wt /body wt ; 0. 212 mg protein/3. 2 ml.
54
order to isolate the metabolite(s) various extraction procedures were
examined.
The results of extraction, after incubation of-various trout
liver preparations, with chloroform:acetone:water (58:38:4 or 38:58:4)
are given in Table 12.
The designations of suspected metabolites
were based on the proximity of R
(TLC) plates to standards.
's on thin layer chromatography
That is, suspected metabolites with an
R_ equal to aflatoxicoj. were called R F and compounds with R 's
F
o
F
similar to B„ or M, were designated
B0 A or M.A.
e
2a
1
2a
1
Little R F was isolated from 105, 000 x g supernatant from
o
dilute homogenates of fed or starved fish.
Appreciable R F was
formed after addition of an NADPH generating system to the preparation of fed trout.
Less was found from starved fish.
A new metabolite, NM, was observed near the origin of TLC
plates.
Like all the metabolites, it fluoresced on exposure to UV
light with a 365 nm wavelength.
polar than M, and B_ .
1
2a
In all TLC systenns used it was more
The most NM occurred with the same condi-
tions which produced maximum microbial lethality,
Addition of two
nucleophiles, cytosine and cysteine, decreased the amount of NM and
microbial death.
NM occurred.
In parallel, a new fluorescent spot more polar than
Addition of three times the amount of B
as substrate
or doubling the incubation time increased the yield of metabolites
nonlinearly.
« of the label
After incubation with 14 C labeled B , 99+%
w?as found in the metabolites and unaltered B .
None of the suspected
Table 12.
Metabolites by TL.C.
a
Fraction
Metabolites
Incubation conditions
20, 000 x g supernatant (starved)
20, 000 x g supernatant (fed)
RoF> BZaA
105, 000 x g supernatant (starved) + NADPH
R0Fb
105, 000 x g supernatant (fed) + NADPH
RoFb
Top layer microsomal pellet (starved) + NADPH
Glutathione (reduced)
Cysteine
Cytosine
NM
B
2aAC
-o^'
B2aA' NM
B
2aAC'
M
AC
I
'
^M^
NM
tra
P
d
NM , NM trap
Top layer microsomal pellet (fed) + NADPH
B2aA, MjA, NM
^CB,1
Cysteine
14
CR o Fd,
14
CB9^ia
,Ae,
14
CNMf
NMd, NM trap
Chloroforrruacetone (9:1); chloroform:acetone:isopropanol (87:10:3); benzene:acetonei
ethyl acetate (50:6:2).
R^F
o fed>> R o F starved,
< 1%,
C
e
d
f
< 0.05%.
i-5%.
U1
3. 6-5. 3%.
56
metabolites were found after incubations with phosphate buffer, without NADPH and boiled fractions.
Only a trace of R F was found from
o
20,000 x g supernatant heated to 65-85
for 1 min.
Apparently the
soluble enzyme was more stable than the mtcrosomal system.
Together, the evidence indicated that the TLC spots were enzymatic
metabolites of B .
The results suggested that R F was produced by an NADPHrequiring soluble enzyme similar to that reported by Patterson and
Roberts (215).
It did not appear to be the lethal factor since no micro-'
bial death occurred after incubation of B
fraction without microsomes.
and the soluble enzyme
B_ A, .MA and NM were postulated to
2a
1
be either the active metabolite or its degradation products,
NM trap
appeared to correspond to the postulated covalently bound products of
the active metabolite and the added nucleophiles.
Lethality and Identification
of Metabolites
i
'
' '
Y
|i
i—
Preliminary evidence for several new metabolites was seen by
TLC.
Experiments followed to assess their microbial lethality and to
identify the metabolites.
Aflatoxicol was extracted initially from post-microchondrial
preparations.
It was isolated by preparative TLC.
The UV spectrum
in ethanol or methanol gave maxima at 331, 261 and 255 nm.
The
57
maxima .and shape of the spectrum were in agreement with aflatoxicol
from B
transformed by Tetrahymena pyriformis (246, 283),
Dactylium dendroides, Absidia repens, and Mucor griseo-cyanus (76,
77, 78), and Rhizopus arrhizus (62).
The parent m/e 314 by mass
spectrometry was in agreement with the mobecular weight.
The
presence of the hydroxyl was indicated by a peak at m/e 296 from the
loss of a molecule of water.
These and the additional peaks (Appendix
VIII) were identical to those reported by other workers (62, 76, 246).
After chemical reduction of B
two compounds were obtained
by preparative TLC or from column chromatography on alunaina.
The
one corresponding to the isolate from fish preparations moved ahead
of the other in all TLC solvent systems employed and was designated
F.
Similarly, the one lagging in the rear was called R.
Both gave
UV and mass spectra identical to aflatoxicol and were designated R F
o
and R R.
Cole, Kirksey and Blankenship (62) reported an identical
pair of compounds from the degradation of B1 by Rhizopus arrhizus.
Based on UV, infrared (IR), nuclear magnetic resonance (nmr) and
mass spectrometry, they concluded the compounds were diastereomers.
In addition they reported that during purification on silica gel in the
presence of ethanol and acidic conditions the corresponding ethyl
ethers of the diastereomers were found.
For this reason, great care
was taken during all isolations of R F and R R to avoid the use of
o
o
alcohols and acids. A new column chromatography procedure
58
(Appendix VII) was developed to specifically circumvent artifact
formation.
With neutral alumina and benzene, ethyl acetate and
acetone as solvents, no compounds were isolated that corresponded
to the reported ethers.
If R F or R R was- allowed to stand a day at
o
o
'
room temperature iq. acidic methanol, compounds similar to the
reported ethers were seen.
They moved well ahead of B , R F and
1
o
R R in all TLC systems examined,
o
Several extinction coefficients (« ) have been reported for R F
o
and one for R R (Appendix V).
14, 100.
For R F they ranged from 13, 780 to
They were similar to the decrease in c from 21, 800 to
13, 900 for the complete reduction of the cyclopentenone of B
THDB,.
1
to
Because the reported « of 25, 200 for R R was unexpected,
r
o
a rough deternaination of the extinction coefficients was made.
Enough pure R F and R R were not available for an accurate reevaluation.
No difference was found.
Further, no difference in fluorescence
intensity on TLC plates was seen for a series of concentrations in
duplicate based on 13, 780 as the extinction coefficients for both R F
0
and R R.
o
If the concentration of R R was based on 25, 200 as the
o
the fluorescence intensity of R R was greater
than R F.
0
o
o
€
The results
suggested a value of 25, 200 provided too low an estimate of the
concentration.
For this reason 13, 780 was selected to compute the
concentrations of R F and R R.
o
o
,
59
R F without enzymatic activation did not produce a significant
reduction in rnicrobial viability (Table 13).
For some unknown reason
the diastereomer, R R, not isolated from fish but produced by chemio
'
cal reduction, repeatedly exhibited some toxicity.
In the presence of
20,000 x g supernatant or purified microsomes with a NADPH generating system, R F was activated to a lethal product.
o
and Miller (114) reported similar results.
Garner, Miller
They found aflatoxins M ,
P and G which contain the vinyl ether were activated whereas no
activity was associated with B_, G_ and B,, which lack the vinyl
/
2
2
2a
'
ether.
The acetyl derivatives of M,1 and R O were prepared
and no
r
t-
correlation for a change in activity was found.
In agreement with Garner's results B_ could not be activated but
Q
which has the vinyl ether was activated (Table 14).
At concentra-
tiong where a several log reduction in viability was observed with B
and R F, less than a one log decrease was observed for Q .
In this
case the presence and location of the hydroxyl group was significant.
A significant decrease in viability was not obtained with crude
extracts of B, or R F incubation media, crude column fractions with
1
o
labeled and fluorescent material and compounds B_ A, M,A and NM.
2a
1
Similarly, Garner, Miller and Miller (114) were unable to extract an
active metabolite from rat, mouse, guinea pig, hamster and human
microsomal preparations which in the presence of B
factor lethal to Salmonella typhimurium TA 1530.
produced a
Neal (199) found, in
60
Table 13.
Microbial Lethality of Unactivated and Activated
Aflatoxicol Diastereomers.
Fraction
Phosphate buffer
Phosphate buffer
Phosphate buffer
Phosphate buffer
20, 000 x g supernatant
Top layer microsomal
pellet + NADPH
Table 14.
Treatment
8. 9 x 10"5 M R F
o
5
1. 8 x 10" M R F
o
5
0. 7 x 10" M R F
o
2. 9 x 10"5 M R R
o
5
5. 0 x 10~ M R F
o
0. 7 x 10'5 M R o F
Comparative Microbial Lethality of B , Q
Fraction
Phosphate buffer
Phosphate buffer
Treatment
10"5 M B
% Reduction
in viability
0
6. 3
0
21-36
66
82
and B?
% Reduction
in viability
0
5
M Q]
0
5
ID'
20, 000 x g supernatant
10~
M Bj
99
20, 000 x g supernatant
10"4 M B-
0
Top layer microsomal
pellet + NADPH
10"5 M B
99
Top layer microsomal
pellet + NADPH
lO"13 M Q 1
22
61
the presence of an active microsomal system and B , RNA polymerase
was inhibited.
If the polymerase was added after stopping microsomal
activity, no inhibition was observed.
preparation was not inhibitory.
Likewise, an extract of the
He suggested that inhibition was
either dependent on some other microsomal function or that the
metabolite was unstable or so reactive Lt was not extractible.
In
contrast, Sarasin and Moule (253) extracted an extremely polar
compound from microsomal preparations of B
which was inhibitory
to the synthetic activity of normal rat liver polysomes in a cell free
system.
The seemingly conflicting results could be explained with the
epoxide of B
proposed by Schoental (261) and Garner, Miller and
Miller (114).
As a highly reactive electrophile it could react readily
with nucleophiles such as DNA and RNA bases.
Garner found micro-
bial lethality was inhibited by addition of RNA and DNA.
Only after
reaction in the presence of active microsomes wasB, inseparable
from RNA.by Sephadex G-'IO chTomatography.
Alternatively, a
diol could be formed by epoxide hydrase or nonenzymatically and
react with nucleophiles.
Instead, Gurtoo (127, 128) and Gurtoo and
Dave (130) proposed the hemiacetal of B , B_ , was formed directly
1
or by rearrangement of the epoxide.
<£a
They obtained spectral evidence
that in the presence
of active microsomes, B, was bound as B-, to
r
1
2a
RNA and protein and could not be separated by Sephadex G-25
62
chromatography.
Patterson and Roberts (213, 215) reported that the
hemiacetal of B,
readily opened.
The aldehydes could bind to amino
groups to give Schiff bases (Figure 10).
o
A diol could react similarly.
o
i^n-^nx^HO^o^-^-oC^
CHO CHOo*^OCH
CH
Aflatoxin B2a
^
CH
-^-OCj,
^
T
OOC E'R^'COO'
Figure 10.
Hypothetical Schiff base formation of amino acids with
one of the resonance forms of the phenolate ion.
Unfortunately, Gurtoo was not able to determine if the binding was a
natural consequence of B_
metabolism or the result of chemical
degradation in the presence of Tris-HCl.
Since Garner and Gurtoo were attempting to identify the compounds bound to RNA, efforts as described below were made to isolate
and identify B
A, M.. A and NM directly rather than continuing with the
trapped products.
Purification by partition chromatography on cellulose or
kieselguhr were unsuccessful.
chromatography supports.
The compounds absorbed to glass and
To take advantage of this property,
adsorption chromatography with various silica gels was employed.
Attempts were rnade to avoid alcohols because aflatoxicol could be a
precursor and it readily formed ethers with alcohols.
Because the
63
compounds co-extracted and co-migrated with polar lipids and
adsorbed so strongly to silica gel, it was necessary to use isopropanol
to pretreat the columns.
No alteration of R
F
on TLC indicative of
ether formation was observed.
After repeated column chromatography, fractions of each metabolite were obtained that were free of other UV fluorescing and absorbing material and I_-staining compounds^ The UV absorption maxima
of B_ A were 263 nm and 220 nm.
2a
NM had maxima at 261 nm and
255 nm like R F but had only an extremely minor absorption at 331 nm
compared to R F.
Koes, Forrester and Brown (148) extracted ametab-
olite of B,, which displayed an absorption maximum at 270 nm, from active
microsomes of guinea pig.
They employed the same extraction pro-
cedure as the present study; however, methanol was used to recover
the metabolites from silica gel.
Subsequent ether formation could
explain the non-polar behavior of the metabolite.
with authentic M
MA co-migrated
with chloroform:acetone; isopropanol (87:10:3) and
benzene:acetone:ethyl acetate (50:6:12) as solvents.
MA had a
shoulder at 262 nm and maximum, absorption at 220 nm in the UV
compared to 226 nm, 265 nm, 357 nm for M
(141).
Using mass spectrometry, no single m/e could be conclusively
assigned as the parent peaks of any of the cornpounds.
The compounds
appeared to be much more labile than the standard B
used to
establish instrumental conditions. A characteristic spectrum was
64
obtained for B
but the unknowns appeared to degrade into many
fragments of low mass.
Recently, Swenson, Miller ^nd Miller (280) reported 2, 3-dihydro2, 3-dihydroxy-aflatoxin B
(B.-diol) from acid hydrolysis of a B
derivative bound to rat liver rRNA.
The structure was confirmed by
comparison of the UV absorption in neutral and alkaline solution, the
R 's in many TLC systems and mass spectra of the acetonide and
r
diacetyl derivatives of authentic synthetic material.
The
RT-.'S
of NM
were similar to those reported for B -diol in each of the solvent
systems,
Migration of NM and the reported B ^diol on H BO -silica
gel sheets was retarded as expected for a vie-diol.
The RNA-B
adduct did not exhibit the bathochromic shift in
alkali found with the B -diol and B_ .
For this reason it was postu-
lated the covaient link to RNA was through carbon 2.
They speculated
the major base attacked was the strong nucleophile guanine.
(113) reported that the microsomal system bound B
Garner
to a greater
extent to polyriboguanylic acid than any other homopolyribonucleotide.
Determination of whether NM or one of the other metabolites is
a similar diol would be extremely interesting siace Swenson only found
the diol after acid hydrolysis of the B
product bound to rRNA.
65
Acute Toxicity Trial
Mortality and General Observations
The mortalities due to B, or R F ten days after intraperitoneal
1
o
injection are reported in Table 15.
According to the method of
Litchfield and Wilcoxon (171) the LD50 for B
95% confidence limits of 0. 35-0. 59 mg/kg.
was 0. 46 mg/kg with
The slope function was
1. 80 with 95% confidence limits of 0. 66-1.37.
For R F the LD50 was
o
0. 66 mg/kg with 95% confidence limits of 0. 46-0. 94 mg/kg.
The
slope function was 1. 78 with 95% confidence limits of 1. 19-2. 67.
slopes for B
and R F were examined for parallelism.
The
The slope
function ratio was 1. 01 with 95% confidence limits of 0. 59-1. 75.
Since the slopes were significantly parallel an estimate of the relative
difference was made.
of 0. 92-2, 22.
potency.
The ratio was 1. 43 with 95% confidence limits
The compounds were not statistically different in
No mortalities due to R R were observed.
o
Greater doses
were not tried due to lack of additional pure R R.
o
The LD50 of B , 0,46 mg/kg, was less than the LD50 of 0. 81
mg/kg previously reported from this Laboratory {2/8). .Every attempt was
made to duplicate the conditions of the original study and the explanation may be the variation in sensitivity of trout from eggs of different
females.
This variation was observed during trial experiments.
Detroy and Hesseltine (76, 77) reported that R
was 1/18 as toxic as
66
Table 15.
Mortality in Rainbow Trout 10 Days after Intraperitoneal
Administration of Aflatoxins B,, R F and R R.
loo
Total dose
(me/ke bodv wt.)
Mortality
Aflatoxin B
0 (DMF control)
0/10
0.2
2/10
0. 3
1/10
0. 4
4/10
0. 6
6/10
0.8
9/10
Aflat oxin R F
Aflatoxin R R
0 (DMF control)
0/10
0/10
0. 28
1/10
-7 T
0. 56
4/10
0/10
0. 85
5/10
—
1. 27
9/10
0/10
o
0
67
B. with the duckling bile duct hyperplasia assay.
It was not clear
whether they had separated the diastereomers of R
o
and, if they had,
which one was assayed.
The time interval between toxin administration and the first
mortality was dose-dependent for both B
and R F.
doses of both, death first occurred on day four.
mortalities were not observed until day eight.
At the higher
At the lower doses,
For B
dosed trout,
inappetence, weakness and darkening in external coloration were
observed a day or two before death.
The time interval was only a few
hours for trout given R F.
o
The severity of gross pathological changes of internal organs
observed at autopsy paralleled the dose for both B, and R F.
1
o
hemorrhaging appeared to be the cause of. death.
pale patches at the lowest dose.
Internal
The liver had a few
At mid* doses, it was almost
entirely pale yellow to white in color and at high doses it was
extremely mottled.
The liver floated in Bouin's solution at the mid and
high doses suggesting a decrease in density with increasing intoxication.
The degree of multiple hemorrhages in the fat bodies and entire
gastrointestinal tract increased with the dose.
filled with blood at mid and high doses.
visible internally at 0, 56 mg /kg R R.
patches were observed on the liver.
The intestine was
No pathological cha,nges were
At 1. 27 mg/kg only a few pale
68
Histological Observations
Liver.
The degree of liver damage paralleled the dose-response
seen with mortality and gross pathology.
A higher dose of R F was
required to give the same degree of damage as with 0. 4 mg/kg B
(Figures 11 and 12),
R R produced significantly less damage and the
dose-response was not the same (Figure 13).
At all doses of B, and R F, altered liver architecture, parenchy1
o
mal cell necrosis with cytoplasmic yaculation, irregular nuclei with
vaculation and disruption of the vascular system with hemorrhaging
were evident.
In the case of 1.27 mg/kg R R (Figure 13), more
normal liver architecture was retained as indicated by the prominent
sinusoids and muralia, or cords, which were characteristically two
cells thick.
Gross pathological changes seen on atuopsy and damage seen
histologically suggested R F was somewhat less potent than B
despite
the lack of statistical significance which was due to the large variation
in response for the number of fish tested.
Kidney.
In the kidney, the degree of damage paralleled mortality
and gross pathology for B
(Figure 16), but not R F (Figure 18).
Apparently less R R, 0. 56 mg/kg, was required to produce damage
similar to 0. 85 mg/kg R F.
o
diastereomer R F.
Trout converted Bn to the aflatoxicol
1
Perhaps the kidney was not capable of excreting
69
f** >>' "1*
-» v
- - ^
■••..
. "A"
Vte&: ^^ -.^
Figure 11.
Liver from trout dosed with 0. 4 mg/kg B . Note the hemorrhaging and complete destruction
of the cord structure and hepatocytes on the right. Some original tissue is seen on left.'
Vacuoles are abundant and nuclei are irregular. Hematoxylin and eosin. X128.
Figure 12.
Liver from trout dosed with 0. 56 mg/kg R F, Generally similar to 0. 4 mg/kg B.. In
upper left, tissue destruction is nearly complete. Nuclei are all bizarre. Bile duct in
lower right appears unaffected. Hematoxylin and eosin. XI28.
70
Figure 13.
Liver from trout dosed with 1. 27 mg/kg R0R with
enlarged, vacuolated and bizarre parenchymal cell
nuclei. Gross structure is essentially normal.
Hematoxylin and eosin. X128,
71
Figure i4.
Normal trout liver. The cords, or muralia, are two cells wide and separated by sinusoids
in which Kupfer and circulating blood cells are found. Nuclei are uniform in size and shape.
Note hepatic vein in upper left. Hematojcylin and eosin. X320.
Figure 15.
Normal trout kidney showing a cross section of a collecting duct (d), a glomerulus (g), and
many sections of renal tubules, or nephrons, embedded in a matrix of hemapoietic tissue.
Hematoxylin and eosin. X128..
72
Figure 16.
Kidney from trout dosed with 0, 4 mg/kg B . Note the enlarged, vacuolated and deformed
epithelial nuclei in the tangential section of a renal tubule. Numerous vacuoles are
present in the cytoplasm. Hematoxylin and eosin. X320.-
Figure 17.
Kidney from trout dosed with 1. 27 mg/kg R R showing the nuclei of the tangential
sections of the second proximal segments of the tubules are greatly deformed compared
to those in the first proximal segments. Hematoxylin and eosin. X320.
73
v>
>m
IOT
».^lm
-^
Figure 18.
Kidney from trout dosed with 0. 85 mg/kg R F. The only obvious sign of toxicity is the
deformity of the epithelial nuclei of the nephrons. Hematoxylin and eosin. X128,
Figure 19.
Kidney from trout dosed with 0. 56 mg/kg R R. The nuclei show the deformities similar
to those from trout receiving 0, 85 mg/kg R F (above). Hematoxylin and eosin. X128.
74
the unnatural diastereomer as well, hence the greater toxicity from
R R.
In all cases damage consisted of one or more tissue changes
including necrosis, bizarre nuclei and abnormal glomeruli and hemapoietic tissue,
Chronic Toxicity and Carcinogenicity Trial
Each of the toxins was fed at a level of 20 ppb on the basis of the
extinction coefficient (e ) reported in the literature (Appendix V).
As
discussed earlier, it was concluded that the e of 25, 200 for R R was
o
too high and that it more closely approximated 13, 780 for R F.
With
that e , R R was actually fed at 36. 6 ppb.
Liver
Chronic Toxicity.
The form of toxic injury was similar, although
less extensive, to that observed for acute administration.
B
and
R F produced similar changes (Figures 20 and 21) and R R was much
o
o
less toxic overall although severe in some cases (Figure 22),
The
addition of 50 ppm of cyclopropenoid fatty acids (CPFA) to the control
diet (Figure 23) produced typical signs of injury (277).
extensive regeneration was seen.
In many fish
The addition of CPFA to the B
diets produced much regeneration with fibers present.
This resulted
in a reduction in apparent toxic damage compared to those not receiving CPFA.
The response to R F and R R in the presence of CPFA
75
J
Figure 20.
Liver from trout fed 20 ppb Bj for four months with extensive parenchymal cell damage
and abnormal nuclei, Note the islets of darkly stained, regenerating parenchymal cells.
Blood vessel walls appear normal. Hematoxylin and eosin, X128,
msttti
.(< E»V-».
.-i '
«\ »*••**%
'£*j&tf'
tte
Figure 21.
Liver from trout fed 20 ppb R-F for four months. Injury to the parenchymal cells is as
great as with 20 ppb B (above)
/e) although the extent of regeneration is less advanced.
Hematoxylin and eosin. X128.
76
Figure 22.
Liver from trout fed 36. 6 ppb R0R for four months. The extent of nuclear abnormalities
in the parenchymal cells is nearly as great as with 20 ppb R F. There is no evidence of
regeneration. Hematoxylin and eosin. X128.
Figure 23.
Liver from trout fed 50 ppm CPFA for four months. Many parenchymal cells contain fibers
and are filled with lipid globules. The nuclei are generally normal. Hematoxylin and
eosin. X128.
77
was extremely variable fronn normal.to severe damage with regeneration.
No fibers were observed.
Hepatorna.
The incidence of hepatorna is presented in Table 16.
Histological evidence for neoplasia is presented in Figures 24-27 for
the four month sample.
The incidence and severity of hepatorna paral-
leled the degree of toxicity at four months with CPFA.
were microscopic for R F and R R.
o
o
Most nodes
At eight
months R R, the
0
o
diastereomer not produced by trout, remained significantly less potent
despite it being fed at nearly twice the level of B, and R F.
1
o
It
remains to be seen whether R R will be carcinogenic without CPFA
at 12 months.
If not, it would be interesting to know if it would at
higher levels or, i,nstead, it acts as a cocarcinogen to CPFA,
Kidney
Chronic Toxicity.
The kidney histology did not appear to follow
exactly the same pattern among,: toxins although nuclear abnormalities were the primary symptom of toxicity.
cantly increased tubule damage.
Addition of CPFA signify
Tubule damage was greatest with
R R and nearly equal for B., and R F.
o
1
o
tNoneobliastic: changes
were
0
observed in the kidneys.
Significance of Metabolism of B
Evidence has been presented that B, was metabolized to R F by
i
o
Table 16.
Incidence of Hepatoma at Four and Eight Months from Start of Feeding Trial.
Hespatoma Incidence
Diet
Dupli.cate 4 mo. sample
H epatoma/40 trout
%
Duplicate 8 mo. sample
Hepatoma/40 trout
%
Control
0
0
0
0
Q
Va
v
0
0
0
18
27
56. 3
0
0
0
11
10
26. 2
b
RoR
0
0
0
0
0
0
0
CPFA
C
0
0
0
1
1
2. 5
Bj* + CPFAC
6
3
11. 2
39
38
96. 3
R0Fa + CPFAC
3
2
h. 3
38
37
93.8
R0Rb + CPFAC
2
1
3. 8
25
19
55.0
"20 ppb
36. 6 ppb
'50 ppm
oo
79
Figure 24,
Liver from trout fed 20 ppb B. and 50 ppm CPFA for four months. The more basophilic
tissue on the left is a portion of a hepatoma. Note that the nuclei are small, oval and
uniform, and the cells contain no glycogen or lipid globules. The normal tissue on the
right is filled with glycogen vacuoles and the nuclei are more irregular in shape and size.
Hematoxylin and eosin, X128.
Figure 25.
Liver from trout fed 36. 6 ppb R R and 50 ppm CPFA for four months. Note the presence
of a few large fluid-filled vacuoles and a few bizarre nuclei in the basophilic (hepatoma)
tissue. The normal tissue (upper) contains many glycogen vacuoles. Hematoxylin and
eosin. XI28.
80
Figure 26.
Liver from trout fed 20 ppb R F and 50 ppm CPFA for four months. The small, superficial
hepatoma is typical except for the vacuolated areas. The original contents of these areas
is unknown. The scattered, basophilic areas in the normal tissue are regenerating
parenchymal cells. Hematoxylin and eosin. X20,
Figure 27.
Detail of above photo showing edge of the hepatoma. Note the typical, small, basophilic
hepatoma cells with their uniform, oval nuclei. Islets of regenerating tissue are seen in
the normal liver (right). Hematoxylin and eosin. X128.
81
Ml
[•^
««•
\ &
Figure 28.
Kidney from trout fed 20 ppb R0F for four months. Note the degenerating epithelial cells,
bizarre nuclei and atypical cytoplasm. Hematoxylin and eosin. X320.
i
Figure 29.
Kidney from trout fed 36. 6 ppb R R for four months. Much degeneration, cytoplasmic
vacuoles and bizarre nuclei are evident in the second proximal tubules whereas the first
proximal segments appear essentially normal. Hematoxylin and eosin. X320,
82
»l f>
^
^ ^
Figure 30.
Kidney from trout fed 50 ppm CPFA for four months. The abundance of vacuoles in the
epithelial cells with essentially normal nuclei are early signs of tubular degeneration,
Hematoxylin and eosin, X320,
Figure 31.
Kidney from trout fed 20 ppb Bj and 50 ppm CPFA for four months, A few of the trout
such as the one above had degeneration of the epithelial cells with cytoplasmic
degradation and bizarre nuclei, Hematoxylin and eosin. X320.
83
*** V M
y>
•^'^r
Figure 32.
Kidney from trout fed 20 ppb R F and 50 ppm CPFA for four months. The extent of
damage among trout was variable. The epithelial cells of some had much cytoplasmic
degeneration and extensive nuclear deformation as shown above. Hematoxylin and eosin.
X320.
'% f _. ^
r
. _
%
9
<?•.
Figure 33.
Kidney from trout fed 36. 6 ppb R R and 50 ppm CPFA for four months. Extensive
degeneration of kidney tubules was evident from R R fed trout. Note the bizarre nuclei
and cytoplasmic vacuoles. Hematoxylin and eosin. X320,
84
soluble enzymes of the 105, 000 x g supernatant and NADPH.
The
microbial assay, in agreement with that of others (114), indicated B
and R F were not toxic but were further metabolized to lethal products,
o
The metabolism was mediated by the microsomal fraction containing
^.n active NADPH mixed function oxidase.
The data suggested the
following pathways (Figure 34).
microsomal
^7-r--,—, ' T A" ' WTT '
oxidase + NADPH
T,
B.
1
T,
J.± -L. ,-J.
B. active metabolite
1
soluble reductase
+ NADPH
V
R F
o
microsomal
v
—7T—
, TVT A T-.T-.U '
pxidase 4- NADPH
„ _,
,_■
. , ,-^
R F active metabolite
o
or
II.
B
Figure 34.
-*
-)
R F
o
Hypothetical pathways of B
R F active metabolite
o
and R F metabolism.
Pathway II is consistent with the evidence that B
produce similar biological effects.
and R F
Pathway I reconciles the differ-
ences in magnitude of activity between B
and R F.
It must be
remembered that R F was administered extracellularly and the differo
ence in degree of activity for similar doses may represent the difference in entry of R F into the cell.
o
Patterson and Roberts (213) found
a TLC spot with an R^ corresponding to B, after incubation with R F
F
1
o
85
and proposed R o F was metabolized back to B,11
and the B, was further
metabolized.
A similar spot was observed in this study but it did not
have the UV or mass spectra of B .
R F does not represent a significant detoxication of B
whatever the pathway.
Instead, R F conserves the presence of a
potentially biologically active compound,
bolite of the duck.
by trout
R F is also the major meta-
This perhaps explains why these species are the
most sensitive to B .
Strong evidence has been reported that microsomal activation
was required for both hepatic necrosis (243) and carcinogenicity (137)
of aromatic hydrocarbons and aflatoxin.
In both cases the first step
was believed to be the transformation to the epoxide.
The highly
reactive electrophilic epoxide could covalently bind to critical cellular necleophiles or rearrange to toxic phenols.
Farber (101) proposed
that toxicity by killing many cells served as a selective pressure for
the malignantly transformed cells.
This would explain the comple-
mentary consequences of microsomal activation of potential carcinogens
and toxins.
The validity of the hypotheses remains to be proven.
It is
possible the correlation of microsomal activity of toxicity and carcinogenicity is happenstance.
Instead, the interaction of the carcinogens
and toxins with naicrosomal oxidation may involve some yet unidentified
process.
Regardless, it is noteworthy that the trout, a
86
phylogeneticaUy more primitive species and the most sensitive one to
B
induced carcinoma, produced a lethal factor with an NADPH-
dependent microsomal preparation similar to rodents and man.
Areas for Future Study
1.
Complete the identification of unknowns (metabolites).
Chemical
ionization spectrometry offers the potential of increasing the
percent ionization of metabolites too unstable for conventional
electron impact mass spectrometry.
empirical formulas might be obtained.
In this way the M. W. and
Derivatives could also be
made for conventional mass spectrometry.
Repeated computer-
mediated nmr scans of the small amount of products available
could provide structural evidence.
2.
Further characterize the enzymatic conditions for R F and other
o
metabolite fornaation.
Perhaps in this way greater quantities of
unknowns could be made and the metabolic relationships of the
metabolites better understood.
3.
Through the use of selected diets, inhibitors, age of trout or
species, determine the significance of activation and the relationship of B, and R F to each other in the sequence of toxic and
1
o
carcinogenic events.
4.
Study the influence of R F and other metabolites on cellular
o
metabolism.
87
Determine what effect CPFA has on R F and metabolite producr
o
tion and their influence on metabolism.
Determine the absolute configuration of R F and R R, for
o
o
example with X-ray diffraction, and assess why the difference
in structure is significant.
88
SUMMARY AND CONCLUSIONS
Rainbow trout, Salmo gairdneri, Mt. Shasta strain, activated
aflatoxin B., R F and Q. but not B0 to rproducts lethal to Bacillus
1
o
1
Z
subtilis GSY 1057.
Aflatoxicol, R F, was found as a metabolite.
o
It
was formed apparently by an NADPH-dependent soluble enzyme of the
105, 000 x g supernatant from liver.
Activation to products lethal to
B. subtilis required the 105, 000 x g pellet and NADPH and was
dependent on the diet.
An active mixed function oxidase with neotetra-
zolium reductase and aldrin epoxidase activity was present in the
microsom.es.
Activity was reduced with piperonyl butoxide, NADPH
deprivation and heat treatment.
After metabolism of
14
C labeled B
the label was distributed between unaltered B
and three extremely
polar products designated NM (3. 6-5. 3%), B
A (1-5%) and MA
(<1%).
Their presence paralleled microbial death and they were
eliminated or reduced along with microbial lethality when cytosine or
cysteine were added to the incubation media.
The LD50 of the diastereomer of aflatoxicol (R F) formed by
trout was 0. 66 mg/kg compared to 0. 46 mg/kg for B .
There was no
significant statistical difference in potency although gross and microscopic pathological changes were less for R F.
o
No lethality occurred
and toxic injury was much less severe for the other diastereomer
89
(R R) produced by chemical reduction even at the highest levels of
o
R F tested,
o
After eight months of feeding 20 ppb of B
and R F and 36. 6 ppb
R R, the relative incidences of hepatoma were 56. 3, 26. 2 and 0%
o
respectively.
The incidences with the addition of 50 ppnn CPFA were
96. 3, 93. 8 and 55% respectively.
It was concluded that aflatoxicol was not an effective means of
detoxication of B , but instead extended the presence of a potentially
toxic and carcinogenic compound.
It was suggested that enzymes
present in trout microsomes activate aflatoxins B , aflatoxicol and
Q to the ultimate toxins and carcinogens.
the vinyl ether and B
Since they each possess
does not, it appeared that this structural
feature was required for activation.
These findings are consistent
with, but do not prove, the hypothesis that the ultimate metabolite is
an epoxide.
90
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Stoloff, Leonard £t al, Toxicological study of aflatoxin P,
using the fertile chicken egg. Toxicology and Applied
Pharmacology 23:528-531. 1972.
276.
Strufaldi, B. , D. M. Noyueira and F. I. Pedroso. Effect of
aflatoxin B, on metabolic activity of the hepatic mitochondria!
fraction from Rattus norwegicus (rat). Revista de Farmacia
e Bioquimica da Universidade de Sao Paulo 8:1-23. 1970.
277.
Struthers, Barbara Oft. Alteration of certain physiological and
biochemical parameters in livers of rainbow trout fed methyl
sterculate. Doctoral dissertation. Corvallis, Oregon State
University, 1974. 147 numb, leaves.
278.
Svoboda, Donald, Harold J. Grady and John Higginson,
Aflatoxin B, injury in rat and monkey liver. American
Journal of Pathology 49:1023-1051. 1966.
279.
Swaisland, Alan J. , Philip L. Grover and Peter Sims. Some
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1973.
280.
Swenson, David H. , James A. Miller and Elizabeth C. Miller.
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281.
Taylor, S. L. , Mf W. Montgomery and D. J, Lee. Liver dehydrogenase levels in rainbow trout, Salmo gairdneri, fed
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282.
Terao, Kiyoshi, Mikio Yamazaki and Komei Miyaki, The
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117
in vitro from the chicken embryo. Zeitschrift fiir Krebsfor"
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283.
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284.
Toyoshima, Keijletal. In vitro malignant transformation of
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Gann 6:557-561. 1970.
285.
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286.
Verrett, M. Jacqueline, Jean-Pierre Marliac and Joseph
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287.
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288.
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289.
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290.
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291.
Wilk, Manfred and Wolfgang Girke. Reactions between
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292.
Williams, Charles H. and Henry Kamin. Microsomal triphosphopyridine nucleotide cytochrome c reductase of liver.
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118
293.
Williams, D. J. , R, P. Clark and B. R, Rabin. The effects of
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294.
Williams, D. J. and B. R. Rabin. The effects of aflatoxin B j
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295.
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296.
Wpgan, Gerald N. Effects of aflatoxins on in vivo nucleic acid
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297.
Wogan, Gerald N, Experimental toxicity and c?ircinog^nicity
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298.
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299.
Wpgan, G. N. , G. S. Edwards and P. M. Newberne. Structureactivity relationships in toxicity and carcinogenicity of
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300.
Wogan, G. N. , G. S. Edwards and R, C, Shank, Excretion and
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301.
Wogan, Gerald N. and Marvin A. FriedxrCan. Effects of
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302.
Wogan, G. N. and P, M. Newberne. Dose-response characteristics of aflatoxin B-. carcinogenesis in the rat. Cancer
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119
303.
Wogan, Gerald N. and Raymond S. Pong. Aflatoxins. Annals
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304.
Wunder, W. Der durch Aflatoxine hervorgerufene Leberkrebs
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Untersuchung und -Forschung 151:250-255. 1973.
APPENDICES
120
APPENDIX I
MICROBIAL ASSAY
Use aseptic technique for all preparations, including trout
enzymes, that are in contact with the microbial assay.
A.
First Culture
1.
Streak a fresh tryptose blood agar base
15
plate with
Bacillus subtilis GSY 1057 (metB4, hisAl, uvr-1)
and
incubate 12 hr at 37 .
2.
Add 20 ml Pennassay broth (PAB)
17
to 250 ml side arna
culture flask.
B.
3.
Inoculate flask with streak off fresh 12 hr plate,
4.
Incubate fla,sk at 37
on rotary shaker
at Z50 rpm.
Second Culture
1.
Add 10 ml of PAB for each 3 ml of cells desired to a 250
ml side arm flask.
15
2.
Add 0, 5 ml medium from first culture flask which has
19
reached an absorbance of 0. 35
at 660 nm,
3.
Incubate as first culture was.
TBAB, Difco Laboratories, Detroit, Michigan.
J. A. Hoch, Scripps Clinic and Research Fourndation, La JoUa,
California.
17
18
19
Antibiotic medium No. 3, Difco Laboratories, Detroit, Michigan.
Model G''24 environmental incubator with gyratory shaker, New
Brunswick Scientific Co. , New Brunswick, New Jersey.
Spectronic 20 spectrophotometer, Bausch and \jomb Inc. ,
Rochester, New York,
121
C.
Microbial Suspension
1.
Transfer medium from the second culture flask after it
has reached an absorbance of 0. 56-0. 60 to a centrifuge
tube.
2.
Centrifuge at room temperature a,t 8, 000 x g for 5 min.
3.
Pour off supernatant and resuspend organisms with 3 ml of
0. 9% saline for each 10 ml of second culture medium.
D.
Microbial Assay
1.
At time of inoculation of experimental medium, prepare a
serial dilution in Spizizen's (13) minimal salts medium
of the microbial suspension.
2.
E.
Plate, in triplicate 0. 1 ml of 10
dilution oqi TBAB plates.
Assay of Experimental Medium
lt
At the conclusion of incubation of experimental rr^edium,
prepare a serial dilution in Spizizen's minimal salts
medium.
2.
Plate in triplicate 0. 1 ml of 10
TBAB plates.
-2
and 10
-4
dilution on
1 22
APPENDIX U
BACILLUS SUBTILIS GSY 1057 (metB4, hisAl, uvr^l);
PARTIAL CONFIRMATION OF PHENOTYPE
Streak a fresh TBAB plate with B^ subtiljs from a second
culture (Appendix I) which has an absorbance of 0. 56-0. 60,
Incubate for 12 hr at 37 .
Prepare a TBAB master plate by inoculating 52 stab holes
(from a stab repUeator) with separate colonies from the above
freshly incubated plate.
Incubate for 12 hr at 37 .
Transfer with a stab replicatoir inoculum from the fresh 12 hr
master plate to triplicate plates with the following media:
a)
TBAB + 0. 05 \xg/rnl mitomycin C
b)
Spizizen's minimal salts + 0, 2% glucose
c)
Spizizen's minimal salts + 0, 2% glucose + 50 [xg/ml
histidine
d)
Spizizen's minimal salts + 0. 2% glucose + 50 jig /ml
methionine
e)
Spizizen's minimal salts + 0. 2% glucose + 50 (j.g/ml
histidine and methionine,
Incubate the TBAB plates 12-24 hr and the other plates 48 hr.
123
APPENDIX III
KREBS RINGER'S SOLUTION ADJUSTED FOR FISH
Component
Concentrat ion
A mourit
NaCl
0. 935%
515
KC1
1. 19%
20
2. 19%
5
MgS04- 7H20
3. 97%
5
NaHC03
1. 35%
15
Sodium phosphate bijffer (PH7. 2)
0. 02 M
90
2
4
650
Sodium phosphate buffer (pH 7. 2, 0. 02 M):
Na.HPO^
2
4
1. 47%
4 volumes
NaH2PO
1. 43%
1 volume
124
APPENDIX IV
NADPH GENERATING SYSTEMS
The following highly concentrated generating system gives
appreciable activity in trout: For each 3. 2 ml of incubation medium,
add 0. 0025 g NADP; 1. 0 nal of glucose-6-phosphate dehydrogenase
(G6PdH) plus glucose-6-phosphate (G6P) made up as 0. 5 mg G6PdH
(405 units/mg) and 43 mg G6P in 5 ml of 0. 1 M potassium phosphate
buffer pH 7. 2; and 1. 0 ml of MgCl
(3..03 g/100 ml).
Reduced activity is obtained in trout with the foliowit^g system:
For each 3. 2 ml of medium, add 0. 1 ml of the following made up in
0, 02 M sodium phosphate buffer pH 7. 2: NADP (0, 01925 g/ml),
G6P (0. 00414 g/ml) and G6PdH (0. 000148 g/ml).
APPENDIX V
12 5
QUANTITATION OF AFLATOXINS B., B0, R F, R R and Q,
1
2
o
o
1
The following data are used in calculating the concentration of
the aflatoxins:
Toxicant
M. W.
(nm)
(EtOH (3r MLtOH)
Reference
B
312
360
265
223
21, 800
12, 400
22, 100
248
B2
314
362
265
222
24, 000
12, 100
18, 600
248
R F
o
314
331
261
255
13, 700
10, 000
8, 7 90
62
R R
o
314
332
261
255
25, 200
19, 000
17, 000
62
328
366
267
223
17, 500
11, 450
19, 030
183
Q1
20
After removing all other solvents, add the selected alcohol to
give an approximate concentration of 8-10 |j,g/ml of the toxicant.
Measure the absorbance at the applicable wavelength with
a suitable recording spectrometer, BeckmanDK-l or
PB-GT.21
20
M. S, Masri, WesternRegional Research Laboratory^ Agricultural
Research Service, U;>S. Department of Agriculture, Berkeley
•keley,
California.
21
Beckman Instruments, Inc. , Fullerton, California,
126
The concentration is calculated from
/
,
|jLg/ml
=;
(A) (M. W. ) (1000)
(CF)
!
l i
' ' "' ■" '
''
- —"-*■
where A is the absorbance; M. W, is th^ molecular weight of
the toxicant; CF is the correction factor for the spectrometer
22
€ is the molar absorptivity for the absorbance at the designated
wavelength and solvent.
As a criteria of purity for B , compare the ra,tios of the
absorbances to 220/265 = 1. 77 and 362/265 p 1, 76.
22
Calculated by the method of Rodricks and Stoloff (247).
;
127
APPENDIX VI
THIN LAYER CHROMATOGRAPHY: PREPARATIVE
AND ANALYTICAL,
1.
Prepare TLC plates with one of the following absorbents as
indicated by the manufacturer:
a)
MN-silica gel G-HR23
b)
MN-silica gel G-HR F
23
£* O TC
2.
"«»
c)
MN-cellulose powder 300-HR
d)
Kieselguhr G
e)
Silica gel plastic sheet
23
23
24
Quickly spot or streak under incandescent light.
To assess
purity of the fluorescent aflatoxins spot 10 JJII, 15 |J.l and 20 |j.l
in duplicate of 200 |j.g/ml of the compounds of interest and
appropriate standards.
3.
Develop plates in solvent of choice in an unlined chromatography
tank protected from light and sudden temperature changes.
23
24
a)
Benzene:acetone:ethyl acetate (50:6:12)
b)
Chloroform:acetone;Lsopropanol (87:10:3)
c)
Chloroform:acetone:water: isopropanol (88:12:1.5:1.0)
d)
Chloroformiacetone (9:1)
Brinkman Instruments, Inc. , Westbury, New York.
, ,
Chromagram 6060, Eastman Kodak Co. , Rochester, New York,
128
4.
e)
l-butanol:water:acetic acid (25:15:7)
f)
Chloroform:methanol (9:1)
Locate fluorescent or absorbing compounds with a 365 nm and
254 nm light source.
5.
'
To determine amount of label, scrape the spots and count in
toluene gel (Appendix XII) or scan with radiochromatogram
scanner.
6.
Locate I_-staining compounds by developing plates a few min in
a TLC chamber saturated with I_ vapor.
7.
To identify compounds containing a coumarin moiety (169)
spray developed plate with 2 N NaOH, immediately heat at
100
for 5 min and spray with 0.4% £-nitrobenzenediazonium
tetrafluoroborate.
27
With a coumarin and NaOH treatment, a
deep red color is seen.
Without NaOH treatment, a very faint
yellow color is observed.
25
26
27
Chromato-vue cabinet, U. V. Model C-5, Ultraviolet Products, Inc. ,
San Gabriel, California.
Blak-Ray UVL-22, Ultraviolet Products, Inc. , San Gabriel,
California.
Eastman Kodak Co. , Rochester, New York.
129
APPENDIX VII
PURIFICATION OF AFLATOXICOL DIASTEREOMERS
R F and R R BY COLUMN CHROMATOGRAPHY
o
o
1.
Deactivate aluminum oxide G type E
28
to an activity of
Brockmann III by addition of 3% water.
Seal and shakje in a
round bottom flask with much free heads pace for 1 hr on a wrist
action shaker.
Equilibrate overnight.
Prepare fresh alumina
weekly.
2.
Slurry 35 g of alumina with approximately 200 ml hexane and
pour into a column with dimensions close to 4 x 30 c;m,
3.
Add a 2. 5-3. 0 mm layer of fine glass beads to the top of the
alumina.
4.
Wash the support with an additional 100 ml of hexane.
5.
Apply the sample in 1 ml of chloroform.
Rinse the sample
container with 0. 5 ml of chloroform several times and apply
the rinses.
6.
Elute the column with 60 ml of hexane with 0. 5 psig N_.
7.
Continue to elute with benzene:acetone:ethyl a,cetat^ (50:6:12)
i^ntil the R F band has been removed.
8.
Elute the R R with benzene:acetone:ethyl acetate (300:40:75),
(100:72:48) and (100:72:72).
Continue the last system until
R R is removed,
o
28
Brinkman Instruments, Inc. , Westbury, New York.
130
9.
Rechromatograph R F and R R until they are pure by TLC and
UV spectroscopy.
131
APPENDIX VIII
MASS SPECTRAL ANALYSIS OF AFLATOXICOL
DIASTEJREOMERS, R F AND R R
o
o
1.
Prepare sample concentration of approximately 1 (xg /(j.1 acetone
or other suitable solvent.
2.
Load solid sample cup with approximately 0. 8 ng of sample.
3.
Mass spectra are obtained with a Finnigan 1015C GC/MS using
the following operating conditions:
Solid probe temperature: 70
Ion source temperature:
Vacuum:
10
-7
Torr
lonization voltage:
Mass range:
Scan speed:
4.
100
70 ev
12-380
1 sec
The significant peaks and suggested assignments are:
m/e
Assignment
314
Molecular ion
313
Loss of H
296
Loss of HO
268
Loss of HO + CO
267
Loss of H20 + CHO
132
APPENDIX IX
NEOTETRAZOLIUM REDUCTASE ASSAY
To achieve greater activity with trout use the more concentrated
generating system in Appendix IV but replace the MgCl- solution with
Li
the following neotetrazolium (NT)-MgCl- solution.
Dissolve 30 mg
NT in a small amount of chloroforrruacetone (1:1).
Add 45 ml water.
Boil until odor of chloroform is not detected.
Add 1. 525 g MgCl and
water until 50 ml total volume is reached.
For the assay, add 0. 0025 g NADP, 1 ml G6PdH and G6P solution and 1 ml of NT-MgCl_ to a 15 ml conical centrifuge tube.
per the tube and incubate for 5 min at 37 .
centrifuge tubes in ice,
NaCl.
min.
Transfer 0. 2 ml to
Start reaction every 30 sec by adding 0. 1 ml
of the desired trout centrifugation fraction.
min.
Stop-
Incubate at 37
for 10
Stop reaction with 3 ml of acetone and add a few crystals of
Immediately shake the tube vigorously and centrifuge for 5
Read the absorbance in the UV of the supernatant against a
blank at 555 nm.
. .
Activity =
A absorbance x 1000
——-^-'—-: mg protein x min
References: Lester and Smith (163), Maze! (186) and Williams and
Kamin (2 92).
133
APPENDIX X
ALDRIN EPOXIDATION ASSAY
To assay the conversion of aldrin to dieldrin by the 20, 000 x g
supernatant, incubate 4. 8 ml of the supernatant, 0. 1 ml DMSO and
250 nmoles aldrin in 0. 1 ml of methyl cellosolve.
For inhibition
studies with piperonyl butoxide (PB), add the desired concentration
in DMSO or in acetone (followed by evaporation) and assay directly or
after incubation for 5 min before addition of the aldrin.
To measure
activity with the top layer microsomal pellet, add 1. 6 ml of the
resuspended pellet and the following a.mounts of the more actiye
generating system of Appendix IV:
and G6P and 1. 6 ml MgCl .
0. 004 g NADP, 1. 6 ml of G6PdH
Add aldrin and PB as above.
either preparation for 30 min at 25
Incubate
on a shaking water bath.
ml hexane and shake an additional hr.
Add 10
Determine the percent con-
version of aldrin to dieldrin by gas chromatographic analysis according to the method of Chan (52).
134
APPENDIX XI
DIETS
Diet 1
,(%)
Diet 2
(%)
Diet 3
(%)
49. 5
38.0
--
Casein
--
--
49. 5
Gelatin
8. 7
9. 0
8. 7
15. 6
--
15. 6
Dextrose
--
7. 0
„-
• b
Mineral, mix
2. 5
3. 0
4. 0
CMCC
1. 0
2.0
1. 0
Alpha cellulose
9. 7
18. 0
8. 2
Choline chloride (70%)
1. 0
1. 0
1. 0
Vitamin mix
2. 0
2. 0
2.0
10. 0
20. 0
10. 0
Ingredient
Fish protein concentrate (FPC)
Dextrin
a
Herring oil
Cerulose, CPC International, Inc. , Englewood Cliffs, New Jersey.
Calcium carbonate (CaCC^) (2. 100%); calcium phosphate (CaHPCV
2H2O (73. 500%); potassium phosphate (K2HP04) (8. 100%); potassium
sulfate (K2S04) (6.800%); sodium chloride (NaCl) (3. 060%); sodium
phosphate (Na2HP04- 6H20) (2. 140%); magnesium oxide (MgO)
(2.500%); ferric citrate (FeC6H507-3H20) (0. 558%); manganese
carbonate (MnCC^) (0.418%); cupric carbonate (2CuC03Cu(OH)2)
(0.034%); zinc carbonate (ZnCC^) (0.081%); potassium iodide (KI)
(0.001%); sodium fluoride (NaF) (0.002%); cobalt chloride (C0CI2)
(0. 020%); and citric acid (0. 686%). Modified from Bernhart and
Tomarelli (29) by addition of the NaF and CoC^.
Carboxymethyl cellulose, Hercules Powder Co. , San Francisco,
California.
rj
Thiamine hydrochloride (0. 3200%); riboflavin (0. 7200%); niacinamide
(2.5600%); biotin (0.0080%); Ca-pantothenate (D) (1.4400%); pyridoxine hydrochloride (0. 2400%); folic acid (0. 0960%); menadione
(0.0800%); B12 (cobalamine-3, 000 fig/g) (0.2667%); i-inositol (meso)
(12. 5000%); ascorbic acid (6. 0000%); para-amino-benzoic acid
(2.0000%); vitamin D2 (500,000 usp/g) (0.0400%); vitamin A (250,000
lU/g) (0. 5000%); DL alpha tocopherol acetate (2. 5000%); and celite
(70. 7293%).
APPENDIX XII
LIQUID SCINTILLATION COUNTING FLUOR SOLUTIONS
14_
b
C counting
Efficiency0
Background
(%)
(cpm)
Fluor
solution
components3.
Solution
volume
(ml)
Toluene
1 liter toluene
4 g PPO
40 mg POPOP
15
0-1
Organic
82
26
Toluene gel
1
4
40
40
15
0-1
TLC scrapings
82
26
Name
liter toluene
g PPO
mg POPOP
g Cab-O-Sil
Sample
Volume
(ml)
Type
PPO: 2, 5-diphenyloxazole (Scintillation grade); POPOP: (1, 4-bis-2-(5-phenyloxazolyl)-benzene)
(Scintillation grade); toluene (reagent grade); Cab-O-Sil: Godfrey L. Cabot, Inc. , Boston,
Massachusetts.
Nuclear-Chicago Liquid Scintillation Spectrometer, Nuclear-Chicago, Des Plaines, Illinois.
'Efficiency determined by optimal settings of unquenched sample.
standard used to determine efficiency of samples.
Reference:
Schoenhard (260, p. 85)
Channels ratio and internal