[CANCER RESEARCH 37, 1016-1021 , April 1977]
Comparative in Vitro Metabolism of Aflatoxicol by Liver
Preparations from Animals and Humans1
Abdulazim S. Saihab and Gordon S. Edwards
Department of Pharmacology, The George Washington University Medical Center, Washington, 0. C. 20037
SUMMARY
The metabolism of [14C]aflatoxicol by liver postmitochon
drial and microsomal fractions from humans and eight
other species was compared. A major metabolic pathway
involves the dehydrogenation of aflatoxicol yielding afla
toxin B,. Human liver preparations were more active in this
regard than preparations from any of the other species
tested. The aflatoxicol dehydrogenase activity was mainly
associated with the microsomal fraction and required a
hydrogen acceptor (e.g., nicotinamide adenine dinucleo
tide phosphate), but was not inhibited by carbon monoxide,
which implies that it was not dependent on the heme-con
taming microsomal drug-metabolizing system. It had a pH
optimum of 8.0.
Postmitochondrial liver fractions also oxidized aflatoxicol
(and/or the aflatoxin B, made from it) to at least five other
metabolites that comigrated on thin-layer chromatography
plateswithauthentic
standardsofaflatoxins
Q,,P,,H,,M,,
and B.@,.None of these oxidative metabolites were formed in
the presence of carbon monoxide.
We also report on the in vitro reduction of aflatoxin B, to
aflatoxicol by the cytosol fractions from eight species. Most
active in this regard were rabbit and trout preparations,
while this activity was almost absent in the guinea pig.
Preparations from humans and four other species were
intermed iate between these extremes.
INTRODUCTION
AFB,,2
a
product
of
the
mold
Aspergillus
species,
is
a
potent hepatotoxin and hepatocarcinogen in a variety of
animal species, including nonhuman primates (1, 10, 21).
The carcinogenicity of AFB, to man is uncertain, although
recent reports have shown an association between AFB,
contaminated diets and an increased incidence of liver can
cer in certain human populations (2, 19). Much evidence
indicates that AFB, requires metabolic activation to elicit its
I Supported
by
Contract
223-74-2165
from
the
Food
and
Drug
Administra
tion and Grant 501-RR-5359-12 from NIH.
2The abbreviations used are: AFB1, aflatoxin Bi; AFL, aflatoxicol; TLC,
thin-layer chromatography; AFM,, aflatoxin M; AFB@, aflatoxin B@; AFQI,
aflatoxin 0,; AFH,, aflatoxin H,; AFP,, aflatoxin P,; G-6-P, glucose 6-phos
phate; LD@,dose lethal to 50% of the population; S12, supernatant resulting
from centrifugation of liver homogenates at 12,000 x g for 30 mm; 5225,
supernatant resulting from centrifugation of the 12,000 x g supernatant at
225,000 x g for 60 mm; P225, sediment resulting from centrifugation of the
12,000 x g supematant at 225,000 x g for 60 mm.
Received June 23, 1976; accepted December 20, 1976.
1016
carcinogenic and toxic effects (8, 9, 18), although the ulti
mate carcinogenic form has not been firmly identified. Thus
the wide differences in susceptibility of different animal
species to aflatoxin may be, at least in part, associated with
differences in their ability to metabolize the compound (5,
14, 21).
A number of laboratories have reported that AFB, is con
verted to AFL by in vitro preparations made from the livers
of a variety of species (7, 13, 14, 17). The biological effects
of AFL are largely unknown, although McCann et a!. (11)
and Stich and Laishes (20) have shown it to be a potent
frameshift mutagen (in Salmonella typhimurium) that can
elicit unscheduled DNA synthesis in fibroblasts incubated
with a rat liver postmitochondrial supernatant fraction. AFL
also is a potent toxin in ducklings (6) and is toxic to bac
teria in the presence of trout liver microsomes (17).
We therefore have prepared pure AFL to study its toxico
logical and metabolic characteristics. This paper reports
the comparative metabolism of AFL by liver preparations
from 5 animal species (man, monkey, rat, mouse, and dog).
The metabolites stud ied and their interrelationships are il
lustrated in Chart 1. We also compare the oxidative and the
reductive metabolic pathways that are involved in the AFB,
AFL interconversion
in8 different
species.
MATERIALS AND METHODS
Chemicals. Crystalline AFB, and ring-labeled [14C]AFB,
were purchased from Makor Chemicals, Ltd. , Jerusalem,
Israel. When necessary, these compounds were repurified
to greater than 98% purity by TLC on Silica Gel GHR (Brink
man Instruments, Inc. , Westbury, N. Y.) developed with
chloroform:acetone (9:1 , v/v). [‘MC]AFL
was biosynthesized
from [14C]AFBI by the method of Salhab and Edwards (16)
and purified on TLC plates developed with anhydrous di
ethylether. AFL, AFM,, AFB@,,,AFQ,, AFHI, and AFP, stan
dards were kindly donated by D. P. H. Hsieh (University of
California, Davis, Calif.), G. N. Wogan (Massachusetts In
stitute of Technology, Cambridge, Mass.), and L. Friedman
(Food and Drug Administration, Washington, D. C.) or were
biosynthesized in this laboratory. The identity of these stan
dards and of the AFL made by trout was confirmed by mass
spectrometry and UV spectra. G-6-P, G-6-P dehydrogenase,
and NADP were purchased from Sigma Chemical Co. , St.
Louis, Mo. Reagent grade chloroform was redistilled to
remove impurities and preservatives.
Liver Preparations. Table 1 is a list of the animals and
humans used as the source of liver samples for this study.
Included in this table are LD50values taken from the litera
CANCER
RESEARCH
VOL. 37
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1977 American Association for Cancer Research.
AFL Metabolism
ture or, when applicable, from studies done in this labora
tory. Human samples were obtained from brain-dead bilat
eral kidney donors within 5 mm of the time that life support
measures were terminated. All animal samples were col
lected at the time of sacrifice. The livers were homogenized
with a Potter-Elvehjem homogenizer in either 2.5 volumes
(AFL metabolism experiments) or 6 volumes (AFB, and AFL
kinetic experiments) of ice-cold 0.25 M sucrose containing 3
mM MgCI2 and 50 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid buffer, pH 7.4. This suspension was
centrifuged at 12,000 x umaxfor 30 mm. For some experi
ccx@!
AFL
OH
ments the resulting supernatant (S12) was further centri
fuged at 225,000 x umaxfor 60 mm. The supernatant (5225)
was decanted,and thesediment(P225)was resuspendedin
12
ml
AFP,
AFM,
Chart 1. Metabolism of AFL and its known derivatives by various liver
fractions. MFO, microsomal mixed-function oxidases.
the
homogenizing
mixture.
Both
fractions
(5225
in Erlenmeyer
flasks
containing
the 512 derived
from
1 .4 g
of whole liver, 6 mM G-6-P, and 1 mM NADP in a total volume
of 5 ml of the homogenizing buffer (pH 7.4). In all experi
ments a control flask was included in which the mixture had
been boiled for 20 mm prior to incubation. After a 5-mm
preincubation with shaking at 37°,the indicated amount of
[14C]AFLwas added to each flask. After 30 mm of incubation
the reaction was terminated by mixing with 5 ml of chloro
form. The chloroform layer was removed and combined
with 3 subsequent chloroform extracts of the mixture frac
tion. The chloroform-soluble fraction (which contained 60
to 65% of the starting material) was dried under nitrogen,
redissolved in 0.1 ml of 3% (v/v) acetonitrile in benzene, and
spotted on silica gel TLC plates.
Kinetic Experiments. Incubation mixtures were prepared
in Erlenmeyer flasks containing either the 5225 or P225
derived from 0.3 g of whole liver, 6 mp.i G-6-P, and 1 mM
NADP in a tOtal volume of 2 ml of the homogenizing buffer
(pH 7.4). The incubation flasks were capped and stirred at 4°
for 15 mm under a gentle stream of carbon monoxide. The
CO
AF120
of
and P225) were utilized ri these studies.
Metabolism of AFL. Incubation mixtures were prepared
atmosphere
was
maintained
in
the
flasks
throughout
the
experiments to inhibit metabolism of AFB, and AFL by mi
crosomal mixed-function oxidases. After a 5-mm preincu
bation with shaking at 37°,the indicated amount of [14C]AFL
was introduced through the rubber cap with a syringe. The
flasks were incubated for the indicated time before the
reaction was stopped by shaking with an equal volume of
chloroform. The mixtures were then extracted (yields were
70 to 80% of the starting material), dried, and spotted on
TLC plates as described above.
Identification and Quantitation of Metabolites. The TLC
plates were developed with either chloroform:acetone (9:1,
v/v) or anhydrous diethyl ether. Metabolites were tentatively
identified by cochromatography with known standards in
Table 1
Humans and animals
Species
Sex
used as source of liver samples
Age
Race/strain
AFB, LD50(mg/kg)
Human (5)°Female46 yrCaucasianFemale30
yrCaucasianMale36
yrCaucasianFemale20
yrBlackFemale18
yrCaucasianMonkey
(3)Female
(2)AdultRhesusDog Male
(4)Female
(3)AdultMongrelRabbit
Male
(3)Female4-5
ZealandHamster
SyrianGuinea
(3)Female2
(1)
2.2
(1)
ca.1.0
mos.new
mos.Golden
mos.HartleyTrout
pig (2)Male9-11
(40)Mixed14
gairdneriMouse
mos.Salmo
daysICR
(45)Male30-50
Swiss
(15)b
(12)
ca.0.5
(21)
10.2
(21)
1.4
0.81
60
(4)
(3)
(Edwards, unpub.
5.9
(Edwards, unpub
lished)
lished)
Rat (25)
APRIL 1977
Male
30-50 days
a Numbers
in parentheses,
number
b Numbers
in
references.
parentheses,
of animals
F344 (Fisher)
used for the species.
1017
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1977 American Association for Cancer Research.
A. S. Salhab and G. S. Edwards
the 2 solvent systems and by their fluorescence characteris
tics under long-wave (365 nm) UV illumination when viewed
through a filter pack that provides excellent color resolution
among the various aflatoxin metabolites (Kodak Gelatin
Filters CC 20Y, CC 40R, 85B, and 2A; Eastman Kodak,
Rochester, N. V.). The metabolites were removed from the
plates by scraping and quantitated by liquid scintillation
counting. Recovery of radioactive material was routinely
more than 90% of what was spotted on the plates. AFH1and
AFP,
were
usually
not
collected
separately,
as
they
ci
‘U
0
D
-a
chro
matograph very close together. Background counts present
in a zone of the boiled control sample corresponding to
each metabolite were subtracted prior to calculation of the
results. These counts never amounted to more than 0.5% of
the starting material, except for a 1.2% contaminant of the
[14C]AFB,,
which
ran
close
to
the
parent
component
0
Cl)
. C,)
C.,
I
C.)
0
(pre
sumably [14C]aflatoxin B2).
RESULTS
AFL Metabolism by Liver 512 Fractions. A preliminary
experiment was performed to compare the kinetics of AFL
metabolism by liver postmitochondrial supernatant (512)
preparations from AFB,-sensitive versus AFB,-resistant spe
cies (rats and mice, respectively). When incubated with an
NADPH-generating system, both preparations were capable
of metabolizing AFL very rapidly to AFB, and at least 5 other
oxidative metabolites (Chart 2).
Rat preparations made more AFB, from the AFL than
mouse S12 both initially and at longer incubation times. The
mouse preparations, on the other hand, converted more
AFL
to
the
other
oxidative
metabolites.
The
extent
of
AFL
metabolism by preparations from both species had reached
a plateau by 30 mm of incubation, so this time interval was
chosen for our subsequent studies.
AFL
was
metabolized
to
at
least
6
different
metabolites
by
liver 512 fractions from 5 species (Table 2). Several differ
ences between these species were evident. Monkey and
mouse preparations converted at least one-half the starting
AFL
to
AFB,,
while
human
and
rat
preparations
65%
of
the
and
metabolite
AFB2a,
radiolabel
migrated
compounds
known
to
in
be
the
products
region
of
of
AFBI
metabolism. We also found a metabolite that migrated be
tween AFMI and AFB2ain chloroform:acetone (9:1 , v/v) and
that was made in large amounts only when AFL (but not
AFBI)
was
the
precursor.
It
is
likely
that
this
is
a
previously
unknown direct metabolite of AFL, possibly identical to the
“atypical
AFM,―reported by Schoenhard et al. (17).
Such preparations possess the ability to convert AFB,
back to AFL (see Chart 5), so the extent of AFL metabolism
may have been (in part) underestimated by experiments
such as these, which did not preclude this possibility. Sub
sequent experiments were therefore performed under con
ditions that did not permit the microsomal mixed-function
oxidases to function (thus preventing depletion of AFL or
AFB,
1018
by
oxidation)
and
that
assured
the
unidirectional
20
40
INCUBATION
TIME
60
IMIN)
Chart 2. Metabolism of AFL by liver preparations of rats and mice. Fifteen
ml of postmitochondrial supernatant (512) from 4.2 g of liver were incubated
with 1 mM NADP, 6 mM G-6-P, and 100 @g
of [‘4C]AFL
(0.21 @Ci/@moIe).
Two
ml aliquotsofthe
reaction
were removedat2,5,10,15,20,30,and 60 mm of
incubation and were extracted 4 times with chloroform. The chloroform
extracts were dried, spotted on TLC plates, and developed in chloro
form:acetone (9:1 , v/v). AFL (C, rats; 0, mice), AFB (U, rats; 0, mice), and
the pooled oxidative metabolites (AFQI, AFH, AFP, AFM, and AFB@I)(A,
rats; L@,mice) were collectively quantitated by liquid scintillation counting.
Each point represents the mean of 2 determinations on the pooled livers of 3
animals.
tabolism of AFL to AFB, (or vice versa ), as detailed below.
Localization and Some Requirements of AFL Dehydro
genase Activity. Rat liver 512 was separated into 5225 and
P225
by
differential
centrifugation
in
an
attempt
to
localize
the enzyme(s) responsible for the conversion of AFL to
converted
nearly all the AFL to AFB,, most of which remained as such
without extensive further metabolism. The dog preparations
apparently were also very active in converting AFL to AFBI,
although only 15.9% of the total was found as AFB, after the
incubation period. This can be surmised by the fact that
AFM,
5
me
AFB,.
A
preliminary
experiment
(not
discussed)
showed
that
centrifugation for 1 hr at 105,000 x g did not effect an
adequate separation of AFL dehydrogenase activity be
tween the pellet and supernatant or between this activity
and the opposing AFB, reductase (AFB, —@
AFL) activity.
The 5225 fraction contained much less AFL dehydrogenase
activity than the P225 fraction (Table 3), which accounted
for almost all the activity present in the 512.
Heat denaturation destroyed all AFL dehydrogenase ac
tivity, while omission of NADP from the medium markedly
decreased activity in both 512 and P225 fractions. Storage
of the P225 fraction for up to 1 month at —70°
did not affect
AFL dehydrogenase
activity,
while omission
of G-6-P from
the P225 incubation medium resulted in a 15 to 30% in
crease in activity (results not shown), probably due to an
increased NADP:NADPH ratio. Incubation of the prepara
tions in the presence of carbon monoxide completely in
hibited the production of the other oxidative metabolites
without significantly affecting AFL dehydrogenase activity
(Table 3).
Therefore in subsequent studies in which AFL dehydro
CANCER
RESEARCH
VOL. 37
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1977 American Association for Cancer Research.
AFL Metabolism
2Metabolism
(S12)512
Table
of AFL by postmitochondrial
liver fractions
@Ci/@tmoIe)
fractions from 1.4 g of liver were incubated for 30 mm with 20
@g
of [‘4C]AFL
(0.43
theresidue
in 5 ml of mediumcontaining 1 mMNADPand 6 mMG-6-P.After chloroform extraction
was separated by TLC, and the metabolites were quantitated by liquid scintillation count
fluorescencecharacteristics
ing. Metabolite identification
was based on comigration with authentic standards and
under UV illumination
as detailed in “Materialsand Methods.― Results
are ex
±S.D.
pressedas the mean percentageof total chloroform-soluble material associatedwith each spot
Plateswere spotted
cpm/sample.Human
such that there were approximately 7000
MouseCompound
4)AFL
(n = 5)
Monkey
(n = 3)
Dog
(n = 4)
15.2AFB,
2.6 ±3.1
10.3 ±5.1
11.0 ± 1.3
4.6 ±2.5
19.0 ±
15.9 ±11.0
71.6 ±3.5
47.1 ±
8.4AFQ,
77.0 ±1.1
53.4 ±6.4
3.8 ±1.4
4.3 ±3.2
Trace
1.9 ±0.7
2.5 ±
8.7AFMI
+ AFP,
3.7AFM2a
2.5 ±1.6
5.5 ±3.6
5.8 ±2.4
5.5 ±3.7
Trace
29.8 ±11.9
6.3 ±4.6
3.6 ±0.9
11.3 ±
5.5 ±
4.4 ±3.0
4.7 ±2.5
35.1 ±11.0
3.0 ±3.5
in2 (5225)fractions from 0.3 g of liver were incubatedfor 30 mm
mal
of[14C]AFL
ml of a mediumcontaining 1 mMNADP,6 mMG-6-P,and 10j.@g
@Ci/j.trnoIe). After chloroform
extraction
the
separated by TLC, and the AFB, product (as well as the re
TLCplates
maining AFL)was quantitated by liquid scintillation counting.
cpm/sample.Numbers
were spottedsuch that there wereat least5000
radioactivityassociated
are the percentages of chloroform-soluble
Metabolitemaining
reIncubation
conditions
(AFBI) pro
duced
(%)
fractionCompletemedium
2
75.6Complete
15.8
0.5Complete
medium (heat denatured)―
92.7
25.0Complete
medium —NADPb
76.9ider5225 medium + carbon monox-
68.0
18.1
4.7 ± 2.5
ide. Although the pH optimum for this activity was 6.5 (Ch&t
3), we chose to perform subsequent experiments at pH 7.4,
thereby allowing a direct comparison of the 2 enzymatic
activities under conditions that more closely approximate
the in vivo situation.
Comparison of AFB, Reductase and AFL Dehydrogen
ase Activity among Species. The relative rates of conver
sion of AFB, to AFL by liver 5225 fractions from 8 species
are illustrated in Chart 4A. AFB, reductase activity was most
active
with each compound.Substrate(AFL)
(%)51
(n =
0.6AFH,
Table 3
Subcellular localization AFLdehydrogenase
and cofactor requirements of rat liver
activityPostmitochondrial
(51 2), microsomal (P225), and postmicroso
residuewas(0.43
Rat
(n = 5)
in the rabbit
and trout
preparations,
which
could
convert 26% of the starting AFB, to AFL in the 1st 2 mm of
incubation. We have recently shown (16) that several other
species of fish are equally active in this regard.
Because fish body temperatures rarely, if ever, are as high
as 37°,we repeated this experiment with trout liver 5225
fraction incubated at 20°.Under these conditions conver
sion of AFBI to AFL was only 26.5% at 30 mm of incubation
fractionComplete
7.8Completemedium
0.8Completemedium
83.6
93.7
(heat denatured)°
12.4Complete
medium —NADPb
82.8
77.0Complete
medium
14.8
92.9
11.3Complete
medium
81.3
—NADPb
68.0ider
medium + carbon monoxcontaining
liver
CU
was
made
as
were
usual
except
100%
carbon
LU
>
z
z
0
C.,
0
24.5
preparation
4
>
boiled
that
no
0
C.,
.@
U.
4
4
LL2
for
20
U.
o
0
30 mm before addition of the [‘4CJAFL.
medium
I0
‘U
I.
0
0.6Complete
medium (heat denatured)―
b Incubation
0
0
fractionComplete
flasks
4
81.9
7.3ideeP225medium + carbon monox-
a Incubation
0
IL
6
CL
NADP
was added.
C Incubations
were
performed
in
a
monoxide
at
mosphere.
genase activity was to be measured, the incubations were
done with liver P225 (insuring minimal conversion of the
product AFBI back to AFL by AFB, reductase) in the pres
ence of carbon monoxide. Under these conditions, the pH
optimum for AFL dehydrogenase activity was between 7.4
and 8.0 (Chart 3). Similarly, AFB, reductase activity was
measured in the 5225 fraction (which contains little AFL
dehydrogenase activity) in the presence of carbon monox
0
0
4
6.0
8
10
pH
Chart 3. pH optimum of cytosol AFBI reductase (AFBI -+ AFL) (A) and
microsome-associated AFL dehydrogenase (AFL -+ AFB,) (•)
from rat liver.
Microsomal (P225) or cytosol (5225) fractions from 0.3 g of liver were sus
pended in a total of 2 ml of medium containing 1 mM NADP and 6 mM G-6-P.
Five @.tg
of [‘4C]AFL
or [“C]AFB,
(0.43 @Ci/@moIe)
were added to the P225
and5225preparations,respectively.Afterthe mixtureswereincubatedin the
presence of carbon monoxide for 30 mm, the metabolites were quantitated
asdetailedin the legendfor Chart2. Eachpoint is the meanof 2 determina
tions.
APRIL 1977
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1977 American Association for Cancer Research.
1019
A. S. Salhab and G. S. Edwards
active. Trout P225 fraction also had comparatively low ac
tivity, which was reduced even further (to 13.8% conversion
of AFL to AFBI in 30 mm) if the incubation was conducted at
A
IL
20°. In fact,
4
0
LU
I.
‘U
>
2
0
C.)
0
CL
4
IL
among
the
8 species
tested
there
seemed
to
be
2
fairly distinct groups: those with a high (>3) ratio of AFB,
reductase activity to AFL dehydrogenase activity (rabbit and
trout) and those with a high ratio (>3) of AFL dehydrogen
ase activity to AFB, reductase activity (monkey, rat, man,
hamster, mouse, and guinea pig) (Chart 5). The former
group consists of 2 species that are extremely sensitive to
the effects of AFB,, while the latter group is generally less
sensitive to this toxin (although human sensitivity is un
known).
0
20
0
DISCUSSION
0
20
INCUBATION
TIME
30
IMIN)
The 1st report of conversion of AFL to AFB, was made by
Patterson (14) with duck liver homogenates. The present
study expands upon his work by examining the in vitro
metabolism of AFL in a number of species (including man)
and by partially characterizing the enzymes that catalyze the
interconversion between AFB, and AFL.
AFL was rapidly
metabolized
to AFB, by postmitochon
4
drial (512) fractions of all 5 species tested (Table 2). Rat and
human preparations converted more than 70% of the AFL to
0
AFB,, while somewhat
0
tions from mouse or rhesus monkey livers. These prepara
tions also oxidized AFL to compounds that comigrate with
0
IL
aLU
a-
less AFB, was present in the prepara
AFH,, AFQ,, AFP,, AFM,, and AFB2a. Our experiments
‘U
>
2
0
did
not distinguish between AFL and its major metabolite,
AFB,, as the prime source of these other metabolites. Thus
it is not clear whether AFH, was produced directly from AFL
or whether it was formed via the AFB, and AFQ, intermedi
C.,
IL
4
@
AFB, -.-—+
@
0
10
INCUBATION
20
TIME
P225
AFL
—p
AFB,
I
30
1
= RANGE
j
IMIN)
Chart 4. A, conversionof AFB,to AFLby livercytosol(5225)fractionsin
the presenceof carbon monoxide.5225 fractions from 0.3 g of liver were
incubated for various times in a total volume of 2 ml containing 1 mM NADP, 6
mM G-6-P, and 5 @Lg
of [‘@C]AFB
(0.43 @Ci/@moIe)
in a carbon monoxide
atmosphere. The incubation media were extracted with chloroform, and the
metabolite (AFL) was quantitated by liquid scintillation counting after separa
tion by TLC. Source of liver: 0, human (n = 3); •,hamster (n = 3); 0,
monkey(n = 2); U, rat (n = 3); & mouse(n = 3); A, guineapig (n = 2); x,
trout (n = 3);G, rabbit (n = 3). Each point is the mean of a single determina
tion made on the specified number of liver preparations. B, conversion of
z
0
U,
‘U
40
>
z
0
C.)
AFLto AFB,by liver microsomal(P225)fractions in the presenceof carbon
monoxide.P225fractions from 0.3 g of the livers shown in Chart 44 were
suspended in 2 ml and incubated as described for Chart 4A, except that
[“C]AFL
(instead of [‘4CJAFB)
was used as the substrate. Symbols are the
same as for Chart 44.
,Ss,
10
versus 51.0% in paired controls incubated at 37°.S225
preparations from humans and 5 other species had less
than 15% of the activity of the rabbit and 37°trout samples
(Chart 4A), with the guinea pig being least active (only 0.5%
conversion of AFBI to AFL in 30 mm).
The reverse activity, measured as P225-associated AFL
dehydrogenase, showed a very different pattern of species
distribution (Chart 4B). Human and hamster preparations
were most active in this regard, while rabbits were least
1020
@]@:
Lr@
.t
.0
.g@-2
@S
S
S
a.,
C,SC'
IS
Chart5. Speciescomparisonof in vitro ability to convert[‘4CIAFL
to AFB,
(stippled bars) or [‘4C]AFBI
to AFL (solid bars). Liver fractions (5225 and
P225) were incubated for 30 mm as described in the legends for Chart 4, A
andB. Eachbar is the meanof a singledeterminationon at least2 prepara
tions. G. Pig, guineapig.
CANCER
RESEARCH
VOL. 37
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1977 American Association for Cancer Research.
AFL Metabolism
ates. Similarly, the other oxidative metabolites could have
arisen, at least in part, from a 2-step conversion of AFL
(e.g., AFL —+
AFH, —+
AFQ,), although no such pathway has
been reported.
In contrast to the preparations mentioned above, dog 512
fractions converted 65% of the AFL to at least 3 compounds
that migrated in the same region as AFM, and AFB2a.One of
these metabolites appears to be a previously unreported
aflatoxin that is made in relatively large quantities (>25%
yield) when AFL (but not AFB,) is the precursor. Dog prepa
rations are incapable of making AFQ,, AFH,, and AFPI from
AFL (or AFB,; G. S. Edwards, B. Khan, C. M. Parker, and
A. S. Salhab,
have
in common
unpublished
with
results),
preparations
a characteristic
from
rainbow
they
trout.
The rates of AFL metabolism by 512 fractions from rats
and mice were compared (Chart 2). Mouse 512 was slightly
faster than the 512 from rats in reaching peak amounts of
AFB,
production,
although
the
rat
preparations
converted
more AFL to AFB, than mouse 512 at all time intervals. This
may be because the S12 from mice was more active than
that from rats at converting the AFB, to the other metabo
lites (AFQ,, AFH,, AFP,, AFM,, and AFB2a),thereby deplet
ing the AFBI pool faster.
The dehydrogenase enzyme(s) responsible for converting
AFL to AFB, was apparently associated with the microsomal
fraction of rat liver (P225) and required a hydrogen acceptor
(NADP; other hydrogen acceptors were not tested). This
enzyme(s) was not inhibited by carbon monoxide, implying
that it was not dependent on a heme-containing system, nor
was it inhibited by the absence of G-6-P or G-6-P dehydro
genase. It was most active in P225 preparations from hu
mans and hamsters and was least active in P225 from rab
bits(andtroutincubatedat20°).
An enzyme(s), AFB, reductase(s) with the opposite activ
ity was found in the cytosol fraction (5255) and apparently
required an NADPH-generating system but was not in
hibited in the presence of carbon monoxide. It was most
. active
in
5225
fractions
from
rabbit
and
trout
and
was
virtually absent in the guinea pig. This latter finding was
confirmed in separate experiments (G. S. Edwards, C. M.
Parker, B. Khan, and T. D. Rintel, unpublished data) that
demonstrated no AFL production by guinea pig S12 frac
tions even in the absence of CO. Thus a pattern seems to be
evident in these results: liver preparations from species
that are very sensitive to AFB, (LD50< 1.0 mg/kg) had a high
ratio of AFB, reductase activity to AFL dehydrogenase ac
tivity, while preparations from less sensitive species showed
the opposite pattern. This pattern of AFB,-AFL intercon
version in human liver preparations places man among
those species that are not extremely sensitive to AFBI
(along with monkey, guinea pig, hamster, rat, and mouse).
In absolute terms aflatoxin is still very toxic and carcino
genic to most of these species, and in any case the pattern
APRIL
is too crude to be considered predictive for species of un
known AFB, sensitivity.
ACKNOWLEDGMENTS
We are indebted to Dr. Len Friedman for his interest and support, to Dr.
Glenn Geelhoed for supplying the human samples, to Dr. Fred Abramson for
mass spectrometry, to the Maryland State Trout Hatchery for trout, and
especially to B. Khan for excellent technical assistance.
REFERENCES
1. Adamson, R. H., Correa, P., and Dalgard, D. W. Brief Communication:
Occurrence of a Primary Liver Carcinoma in a Rhesus Monkey Fed
Aflatoxin B,. J. NatI. Cancer Inst., 50: 549—553,
1973.
2. Alpert, M. E., and Davidson, C. 5. Mycotoxins: A Possible Cause of
Primary Carcinoma of the Liver. Am. J. Med., 46: 325—329,
1969.
3. Bauer, D. H., Lee, D. J., and Sinnhuber, R. 0. Acute Toxicity of Aflatox
ins B and G in Rainbow Trout (Salmo gairdneri). Toxicol. AppI. Phar
macol., 15: 415—419,
1969.
4. Butler, W. H. Acute Toxicity of Aflatoxin B in Guinea Pigs. J. Pathol.
Bacteriol., 91: 277-280, 1966.
5. Campbell, T. C., and Hayes, J. R. The Role of Aflatoxin Metabolism in Its
Toxic Lesion. Toxicol. AppI. Pharmacol., 35: 199-222. 1976.
6. Detroy, R. W., and Hesseltine, C. W. Aflatoxicol: Structure of a New
Transformation Product of Aflatoxin B,. Can. J. Biochem., 48: 830—832,
1971.
7. Edwards, G. S., Rintel, T. D., and Parker, C. M. Aflatoxicol as a Possible
Predictor for Species Sensitivity to Aflatoxin B, (AFB,). Proc. Am. Assoc.
Cancer Res., 16: 133, 1975.
8. Garner, R. C. Chemical Evidence for the Formation of a Reactive Afla
toxin B, Metabolite by Hamster Liver Microsomes. Federation European
Biochem. Soc. Letters, 36: 261-264, 1973.
9. Garner, R. C., Miller, E. C., and Miller, J. A. Liver Microsomal Activation
of Aflatoxin B, to a Reaction Derivative Toxic to Salmonella typhimurium
TA 1530. Cancer Res., 32: 2058-2066, 1972.
10. Gopalan, C., Tulpule, P. G., and Krishnamurthi, D. Inductionof Hepatic
Carcinoma with Aflatoxin in the Rhesus Monkey. Food Cosmet. Toxicol.,
10: 519-521, 1972.
11. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. Detection of
Carcinogens and Mutagens in the Salmonella Microsome Test: Assay of
300 Chemicals. Proc. NatI. Acad. Sci. U. S., 72: 5135-5139, 1975.
12. Newberne, P. M., Russo, R., and Wogan, G. N. Acute Toxicity of Afla
toxin B, in the Dog. Pathol. Vet. (Basel), 3: 331-340, 1966.
13. Patterson, D. S. P. Hydroxylation and Toxicity ofAflatoxins. Biochem. J.,
125:19P-20P,1970.
14. Patterson, D. S. P. Metabolism as a Factor in Determining the Toxic
Action of the Aflatoxins in Different Animal Species. Food Cosmet.
Toxicol., 11: 287-294, 1973.
15. Rao, K. 5., and Gehring, P. J. Acute Toxicity of Aflatoxin B, in Monkeys.
Toxicol. AppI. Pharmacol., 19: 169-175, 1971.
16. Salhab, A. S., and Edwards, G. S. Production of Aflatoxicol from Afla
toxin B, by Postmitochondrial Liver Fractions. J. Toxicol. Environ.
Health, 2: 583—587,
1977.
17. Schoenhard, G. L., Lee, D. J., Howell, 5. E., Pawlowski, N. E., Libbey, L.
M.,
and
Sinnhuber,
R. 0.
Aflatoxin
B, Metabolism
to Aflatoxicol
and
Derivatives Lethal to Bacillus subtilis GSY 1057 by Rainbow Trout (Salmo
gairdneri) Liver. Cancer Res., 36: 2040-2045, 1976.
18. Schoental, R. Hepatotoxic Activity of Retrosine, Senkirkine and Hydrox
ysenkirkine in New Born Rats and the Role of Epoxides in Carcmnogene
sis by Pyrolizidine Alkaloids and Aflatoxins. Nature, 227: 401-402, 1970.
19. Shank, R. C., Bhamarapravati, N., Gordon, J. E., and Wogan, G. N.
Dietary Aflatoxins and Human Liver Cancer, IV. Incidence of Primary
Liver Cancer in Two Municipal Populations of Thailand. Food Cosmet.
Toxicol., 10: 171-179, 1972.
20. Stich, H. F., and Laishes, B. A. The Response of Xeroderma Pigmento
sum Cells and Controls to the Activated Mycotoxins, Aflatoxins and
Sterigmatocystin. Intern. J. Cancer, 16: 266-274, 1975.
21 . Wogan,
G. N. Chemical
Nature
and Biological
Effects
of the Aflatoxins.
Bacteriol. Rev., 30: 460-470, 1966.
1977
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1977 American Association for Cancer Research.
1021
Comparative in Vitro Metabolism of Aflatoxicol by Liver
Preparations from Animals and Humans
Abdulazim S. Salhab and Gordon S. Edwards
Cancer Res 1977;37:1016-1021.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/37/4/1016
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1977 American Association for Cancer Research.