[CANCER RESEARCH 47, 5861-5867, November 15, 1987]
Comparison of Human and Rat Hepatocyte Metabolism and Mutagenic Activation
of 2-Acety laminofluorene
Kenneth Rudo, William C. Meyers, Walter Dauterman, and Robert Langenbach1
Cellular and Genetic Toxicology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 2 7709 [K. R., K. L.J; Department
of Surgery, Duke University Medical Center, Durham, North Carolina 27710 [W. C. MJ; Toxicology Program, North Carolina State University, Raleigh,
North Carolina 27695 (W. D.J
ABSTRACT
A method has been developed to assess the metabolism and mutagenic
activation of carcinogens using human and rodent hepatocytes in vitro. A
slicing technique which was especially useful for nonperfusable biopsy
and resected surgical human liver tissue was used to prepare the hepa
tocytes. Metabolites of the model carcinogen 2-acetylaminofluorene
(AAF) produced by human and rat hepatocytes were similar and consisted
primarily of 2-aminofluorene with ring hydroxylated products at the 1-,
3-, 5/9-, 7-, and 8-positions produced in addition to /V-hydroxy-AAF.
Sulphate and glucuronide conjugates of ring-hydroxylated metabolites
and 2-aminofluorene were detected. Metabolism and cell-mediated Sal
monella mutagenicity illustrated interindividual variation with human
hepatocytes. Levels of metabolism and mntagenesis were generally higher
with human hepatocytes compared to rat hepatocyte results. The in
creased levels of metabolism and mutagenesis of AAF by human hepa
tocytes compared to rat hepatocytes probably indicates a different sen
sitivity to hepatocarcinogenic effects of AAF on humans as compared to
rats. Understanding differences and similarities between human and
rodent carcinogen activation capabilities should be useful in the extrap
olation of rodent carcinogenesis data to humans.
INTRODUCTION
In order to understand the carcinogenesis process in addition
to testing for agents which cause its occurrence, the factors
involved in the development of this disease must be delineated.
Animal models have been widely utilized for mechanistic stud
ies and as bioassay systems for potential human carcinogens.
However, species differences between humans and animals may
lead to problems in the quantitative extrapolation of the effect
of specific environmental chemicals in the development of
cancer in humans. Differences between human and rat enzy
matic pathways can affect the rates of formation as well as the
levels of reactive metabolites of environmental chemicals. Me
tabolism to reactive intermediates represents one of the signif
icant events in the carcinogenic process which can affect the
accuracy of animal to human extrapolation (1-9). Therefore, a
knowledge of xenobiotic metabolic differences between humans
and test animals should contribute to the reliability of the
extrapolation process. However, in cases in which human tis
sues have been utilized in metabolism studies, a high degree of
individual variation is evident (10-15) and this must also be
considered in the extrapolation process.
Certain aromatic amines, including the model chemical of
this class, AAF2, have been identified as hepatocarcinogens in
Received 3/11/87; revised 6/19/87, 8/10/87; accepted 8/18/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at Cellular and Genetic
Toxicology Branch, National Institute of Environmental Health Sciences, Re
search Triangle Park, NC 27709.
2The abbreviations used are: AAF, 2-acetylaminofluorene; AF, 2-aminoflu
orene; 1-OH-AAF, l-hydroxy-2-acetylaminofluorene; 3-OH-AAF, 3-hydroxy-2-
acetyl-aminofluorene; 5/9-OH-AAF, a combination of 5- and 9-hydroxy-2-acet-
ylaminofluorene; 7-OH-AAF, 7-hydroxy-2-acetylaminofluorene; 8-OH-AAF, 8-
hydroxy-2-acetylaminofluorene; N-OH-AAF, AMiydroxy-2-acetylaminofluorene;
11l'I ( , high pressure liquid chromatography; HBSS, Hanks' balanced salt solu
tion.
5861
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a wide variety of animal species (16-19). These studies have
also illustrated species differences in the metabolism and mu
tagenesis of these compounds. The metabolism and mutagene
sis of AAF have been studied with animal microsomal, S9, and
intact hepatocyte preparations (7, 20, 21) and with human
subcellular preparations (22-25). Previous studies comparing
subcellular and intact cell metabolism and mutagenicity of
aromatic amines in animals have illustrated quantitative and to
a lesser extent qualitative differences between the two prepara
tions (26-29). It has been postulated from these experiments
that intact hepatocytes may simulate the overall metabolic
process (activation and detoxification) as it occurs in the liver
better than cell homogenate preparations (26-29).
Therefore, the present study focuses on the differences in
AAF metabolism and in its metabolic activation to mutagenic
products by hepatocytes from humans and the rat. A modified
slicing technique (30) for hepatocyte preparation from rat and
human liver allowed the use of nonperfusable tissue samples
for experiments. The Salmonella typhimurium assay has proven
to be a sensitive measure of aromatic amine mutagenesis (27,
31) and is used in this study to measure cell-mediated mutagen
esis of AAF. Levels of metabolism, specific metabolite forma
tion, and extent of mutagenicity were measured to determine
interindividual differences in humans and the extent of species
variation. The metabolism and mutagenicity data are discussed
in relation to the rodent to human extrapolation process used
in the prediction of carcinogenic risk.
MATERIALS AND METHODS
Chemicals. [WC]AAF (specific activity, 57.0 fiCi/pmol) was pur
chased from Amersham (Arlington Heights, IL). It was found to be
>99% radiochemically pure by HPLC. Nonradiolabeled AAF, AF, 1-
OH-AAF, 3-OH-AAF, 5-OH-AAF, 7-OH-AAF, 8-OH-AAF, 9-OH-
AAF, and N-OH-AAF were purchased from the National Cancer
Institute (Bethesda, MD). Desferal mesylate was obtained from Ciba
Pharmaceutical Co. (Summit, NJ). 0-Glucuronidase (Type B-10,
100,000 units/mg), ethylene glycol bis(/3-aminoethyl i-iher).v,.V -ti-ira-
acetic acid, arylsulfatase (33 units/mg, Type VIII), calcium chloride,
bovine serum albumin, and r>saccharic-l,4-lactone were purchased
from Sigma Chemical Co. (St. Louis, MO). Dulbecco's PBS (without
calcium and magnesium), Eagle's Minimum Essential Medium (with
out L-glutamine), collagi-nasi-, gentamicin, and HBSS (without calcium
and magnesium) were obtained from GIBCO (Grand Island, NY).
HPLC spectral grade ethylacetate, methanol, acetone, diethyl ether,
ethanol, and isopropanol were purchased from Burdick and Jackson
Laboratories, Inc. (Muskegon, MI). Aquasol-2 and Protosol were ob
tained from Dupont/New England Nuclear (Boston, MA). Trichloro-
acetic acid and dimethyl sulfoxide were purchased from J. T. Baker
Chemical Co. (Phillipsburg, NJ). All treatments, extractions, evapora
tions, and HPLC procedures were carried out under yellow light.
Isolation of Human and Rat Hepatocytes. Human liver tissue was
obtained from nine surgical specimens at Duke University Medical
Center (Durham, NC). Table 1 lists the ages, sex, cause of liver tissue
removal, and viability of liver cells from each individual. Tumor tissue
was separated from normal tissue by the pathology department at Duke
University Hospital. Tissue specimens for this study were transported
 HEPATOCYTE METABOLISM
Table I Liver donor patient data
of Cell
Patient1IIIIIIVVVIVIIVIIIIXAge(yr)304330617131343845SexMaleMaleMaleMaleFemaleFemaleFemaleFemaleFemaleDisease/surgical
procedureOrgan viability92.266.981.670.779.179.092.181.080.0
accident)Rectal
donor (motorcycle
metastasis;partial
carcinoma, liver
hepatectomyFibromellar
left lateral
cancer,liverhepatocellular
metastasis; subtotal left re
sectionHemangioma;
partial left hepatec
tomyAdenocarcinoma
livermetastasis; of the colon,
resectionRetroperitoneal
subtotal left
cancer.partial and liver
hepatectomyAdenocarcinoma
right
livermetastasis; of the colon,
domectomyRight
rightlateral
hepatic mass; subtotal
resectionOrgan
donor%
to the lab in either 4"( ' Euro-Collins solution or Williams E supple
mented with sucrose within 30-45 min of surgical removal. Tissue
digestion was initiated 2-3 h after removal except in Individuals / and
IX in which cases unused organ donor tissue was obtained after trans
port from Pittsburgh, PA, and resulted in another 3 h added to the
time between removal and isolation. Rat hepatocytes were isolated from
6- to 10-wk-old male Fischer 344 rats which were sacrificed via CO2
asphyxiation and the livers immediately removed surgically.
Hepatocytes were isolated from rat and human livers by a modifica
tion of a slicing technique previously described (30). The tissue cubes
were sliced to a thickness of about 0.5 mm with a sterile microtome
blade. About 5-20 g of liver slices per flask were rinsed 6-9 times for
10 min per rinse in 75 ml phosphate buffered saline containing 0.2 m\\
ethylene glycol bis(/3-aminoethyl ether)N,N'-tetraacetic acid in a shak
ing water bath (60 rpm) at 37C.The slices were first digested for 20
min and then 90 min followed by a third digestion of 20 min with
collagenase (250 units/ml) in 200 ml HBSS containing 0.12 mmol/ml
<';i( I. plus 2 ml gentamicin. The cells were filtered through a 100
Micron Nitex nylon screen to remove undigested tissue. Cells from the
initial digest were discarded because of low viability and debris, while
cells from the second and third digests were combined and used.
Viability was determined by trypan blue exclusion staining. Hepatocytes
were carefully transferred by pipets with large bore tips. Approximately
5-12 million hepatocytes per gram of liver were obtained from each
preparation for both human and rat. Isolated human hepatocytes pre
pared by the perfusion technique (32) were generously supplied by Dr.
S. Strom (Duke University Medical Center) and compared for viability
and mutagenic activation to hepatocytes generated via the slicing tech
nique. All of the cells were suspended in complete minimum essential
medium for metabolism studies or in HBSS for mutagenesis studies
with Salmonella.
Extraction of Organic-soluble and Water-soluble Metabolites. Liver
cells suspended in media were exposed to [I4C]AAF for 0.25, 0.5, 1.0,
2.0, and 4.0 h for the time-course study and for 4 h in all other
metabolism experiments. During a 4-h incubation, there was approxi
mately a 10% decrease in viability of rat and human hepatocytes as
determined by trypan blue exclusion. Cell densities for measuring AAF
metabolism by human and rat hepatocytes were 5 million/5 ml in a 50-
ml screw top tube. Each study was conducted with three tubes per cell
preparation and the tubes were maintained sealed in an atmosphere of
5% CO2 in air at a pH of 7.0-7.4 at 37*C in a shaking water bath (30
oscillations/minute). In the dose-response studies with rat hepatocytes,
the concentration of [I4C]AAF was 0.44, 4.4, 44, and 440 nM with
specific activities of 57 /iCi//imol for 0.44 and 4.4 ^M, 6.52 iiC\/tano\
for 44 /IM, and 1.16 /iCi//miol for 440 M. The concentration of AAF
in all other rat and human hepatocyte metabolism experiments was 4.4
MM.The incubations were terminated by freeze-thawing of the cells 3
times with liquid nitrogen and a 37*C water bath. The pH was adjusted
to 6.5 with 0.5 N HC1 and the media extracted 3 times with diethyl
ether. During each extraction, the tubes were vortexed vigorously for 2
min and centrifuged for 20 min at 2200 x g. The organic layers were
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AND MUTAGENIC ACTIVATION OF AAF
combined, evaporated to dryness under a stream of nitrogen, and stored
at 80C.
Samples were reconstituted in methanol for HPLC analyses.
Aliquots of the organic and aqueous layers were counted in a Packard
Tri Labs 2000CA Liquid Scintillation Counter (United Packard Indus
tries, Downers Grove, IL) to determine the distribution of radioactivity.
Water-soluble AAF metabolites were identified after /3-glucuronidase
or aryl sulfatase treatment of the aqueous phase. To ensure the removal
of all organic-soluble metabolites, a final reextraction of the aqueous
phase with diethyl ether was done before the pH was adjusted to 5.0
with 0.5 N acetic acid. One-half of each aqueous sample (2.5 ml) was
treated with /3-glucuronidase (1000 units) while the other half was
treated with aryl sulfatase (250 units) plus 19.2 mg of D-saccharic-1,4-
lactone (30). The samples were mixed thoroughly and were incubated
at 37Cfor 24 h. After incubation, the pH of the aryl sulfatase samples
was readjusted to 7.5 with l N NaOH and all samples were extracted
as described above with the organic layer being removed following
centrifugation. The samples were then analyzed by HPLC.
The amount of AAF bound to cellular macromolecules was deter
mined as follows. Bovine serum albumin (10 mg) was added to the
combined enzymatically hydrolyzed aqueous phase containing cell de
bris after the final organic extraction. Both bovine serum albumin and
cell debris were precipitated by the addition of 1 ml 20% trichloroacetic
acid to 5 ml of aqueous phase. Each tube was mixed for 2 min and
centrifuged for 10 min at 1100 x g. The precipitate was washed twice
with PBS, 4 times with diethyl ether, and 4 times with 95% ethanol at
which time the extractable radioactivity was at background levels. The
pellet was solubilized in 1 ml Protosol overnight in a 37'C water bath
and neutralized with 1 ml 0.5 N HC1. Aquasol-2 (10 ml) cocktail was
added and each sample counted in the liquid scintillation counter.
High Pressure Liquid Chromatography of AAF Metabolites. The
analysis of AAF metabolites and HPLC conditions is a modification of
methods previously described by Smith and Thorgeirsson (21) and
modified by Langenbach et al. (30). Metabolite identification was
accomplished by co-chromatography with authentic standards. Samples
were chromatographed on a Dupont Model 8800 Chromatograph
equipped with a UV (254 nm) absorbance detector (Dupont Instru
ments, Wilmington, DE). A flow rate of 1.5 ml/min using a Zorbax
C8 column, 4.6 mm (inside diameter) x 15 cm (Dupont Instruments,
Wilmington, DE) was used. Solvent A consisted of a mixture of 0.01
M acetic acid and 0.01% desferal mesylate and solvent B was isopro
panol with 0.01% desferal mesylate. The desferal mesylate prevented
chemisorption of W-hydroxy-AAF to the column (21). The gradient
system used and the separation of standard and radiolabeled metabolites
are shown in Fig. 1. The top profile is the HPLC chromatogram of
each standard metabolite. The lower profile represents the radioactivity
collected corresponding to each separate metabolite produced by rat or
human hepatocytes. The AAF metabolites identified were the 1-, 3-, 5/
9-, 7-, and 8-hydroxy AAF, N-OH-AAF, and AF. Attempts to separate
the 5- and 9-hydroxy-AAF as previously reported (21) were unsuccessful
and therefore these metabolites are combined in subsequent tables. The
8-OH-AAF has been previously reported as a metabolite of AAF in the
rat (33), but the present study is the first HPLC detection of this
metabolite. The 8-OH-AAF and 5/9-OH-AAF were clearly separated
as seen in the radiochromatogram. Eluted fractions were collected
directly into scintillation vials with variable collection times: 1 min/
fraction for the first 2 min, 6 sec/fraction from 2 to 6 min to resolve
closely eluting early metabolites, and 30 sec/fraction until the end of
the Chromatographie procedure. The fractions were counted in a Pack
ard Tri Carb 2000 CA Liquid Scintillation Counter (Packard Instru
ment Co., Downer's Grove, IL) in Aquasol-2 (Dupont/New England
Nuclear, Boston, MA). Medium controls with [I4C]AAF but not con
taining cells were run in each experiment. Recovery of radioactivity
from the column was greater than 90%.
S. typhi muri um Assay. The S. typhimurium assay was performed
according to methods reported by Ames et al. (31) as modified by Hix
et al. (34) for using isolated hepatocytes. The following were added to
2 ml of top agar at 45'C: 100 ^1 of a 16-h nutrient broth culture of S.
typhimurium TA98; the test chemical dissolved in dimethyl-sulfoxide
(50 *il) and HBSS containing the isolated hepatocytes (1 ml). The
volumes varied with cell concentration and amount of chemical per
5862
 HEPATOCYTE METABOLISM AND MUTAGENIC ACTIVATION OF AAF

Therefore, all hepatocytes including those from rat liver were

prepared by the slicing technique in subsequent studies.
The dose responses and cell number responses for human
and rat hepatocyte-mediated Salmonella mutagenesis are shown
in Figs. 2 and 3. Each Roman Numeral in the figures represents
a separate human donor (see Table 1) with results from Indi
viduals lll-Vll illustrated because only these cases yielded a
sufficient number of cells necessary to do complete dose and
cell number response experiments. In addition, mutagenesis
O
data for Individuals /, //, and IX were obtained using 10 ng/
TJ AF/plate at a cell concentration of 1 x IO6 cells/plate. Hepa
Z tocytes from these individuals induced 651, 226, and 481 rev-
X
O. DOSE RESPONSE OF HUMAN ANDRAI HEPATOCYTE
I
co MEDIATED MUTAGENESIS (AF-lxlO CELLS/PLATE)
1350

TIME(min.)
Fig. 1. Profiles of HPLC chromatogram ( ) and corresponding radioactiv
ity chromatogram ( ) of AAF metabolites. Separation via HPLC was achieved
under the following condition; Segment 1, 28% isopropanol with 0.01% desferal
mesylate:72% 0.01 M acetic acid with 0.01% desferal mesylate for 20 min;
Segment 2, 100% isopropanol with 0.01% desferal mesylate for 10 min.

Table 2 Viability and Salmonella mutagenesis comparison ofAF and AAF by

hepatocytes prepared by the slicing and perfusion technique
Both techniques were performed on liver from each individual in the table.
TA98 revenants"

Individual I Individual III

Sliced Perfused Sliced Perfused
AFconcentration0*
jig/Plate1
^g/plate10
ig/plate25
(ig/plateAAF
Fig. 2. Effect of AF concentration and human and rat hepatocyte-mediated
'922197182250ND90.147725827927182245815624022373
mutagenesis of Salmonella TA98. Results are from individual human cases and
jig/plate)Viabilit/(%)1863203298NO'.
(25
the average of three rat experiments using 1 x 10* hepatocytes/plate. The
revenant values are the average of 2 plates/concentration. The variation between
TA 98 revenant counts based on 2 plates/point at a cell concentration of 0.5 the 2 averaged revenant values is 15%of the stated value. Zero axis values,
x 10* hepatocytes/plate. dimethyl sulfoxide controls.
'' Dimethyl sulfoxide control for background revenant measurements.
c ND, not done. 1750-1
1350- AAF(25|ig/pJOU
'' Viability determined by trypan blue exclusion procedure. 950-
1300
TOO
plate. The agar mix was vortexed gently and poured on Vogel-Bonner
Medium E plates and incubated inverted at 37C.The mixture of test
chemicals, hepatocytes, and bacteria was preincubated for 30 min at
37C.Each experiment was carried out with duplicate plates per treat
500

ment point. A negative control with dimethyl sulfoxide instead of AF

or AAF was run as well as a rat liver S9 positive control (using 2-
aminoanthracene) to monitor the quality of each assay. Colonies were
counted 48 h later. 300w

RESULTS 200 m

Cell-mediated Salmonella Mutagenesis. Because preliminary

studies indicated that many human liver samples received were
not sufficiently perfusable to yield adequate numbers of viable
hepatocytes, the possibility of using the slicing technique on
these samples was investigated. The data in Table 2 indicate
that hepatocytes obtained by the slicing technique (30) and the
perfusion technique (32) from liver samples from the same
individual were comparable in their viabilities and in their
abilities to activate AAF and/or AF to Salmonella mutagens.
CELLS PER PLATE X 10
Fig. 3. Effect of human and rat hepatocyte number on Salmonella TA98
revenants, a, revenant values for AF at 25 *ig/plate; b, revenant values for AAF
at 25 ig/plate.Results are from individual human cases and the average of three
rat experiments. The revenant values are the average of 2 plates/cell number.
The variation between the 2 averaged revenant values is 15% of the stated value.
Zero axis values, dimethyl sulfoxide controls.

5863

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 HEPATOCYTE METABOLISM AND MUTAGENIC ACTIVATION OF AAF

ertants/plate, respectively. As shown in Fig. 2, levels of Sal- TIME COURSE METABOLISM OF AAF

monella revenants increased with AF concentration for both 40OO

human and rat hepatocytes. Each human case exhibited a higher
mutagenic response at 10 and 25 ng/plate than was observed
with rat hepatocytes, although at the 1 fig/plate dose the differ
ences were smaller. In Fig. 3, a and >,
the cell number responses
for AF and AAF, respectively, indicate that at all cell concen
trations, the mutagenic response was higher with human he
patocytes than with rat hepatocytes. In Figs. 2 and 3, the
variation in the ability to activate the aromatic amine by the
different human samples is apparent. However, rat hepatocytes
prepared from different animals exhibited a relatively consistent
level of mutagenic activity. For example, in studies at 10 /ug/
plate AF at a cell concentration of 1 x 106 cells/plate, the range
for eight human samples was about 6-fold (198-1225 rever-
tants/plate) with the variation by rat hepatocytes being less
than 1.5 fold.
Metabolism of AAF. To determine concentration and times
for AAF metabolism studies, dose-response studies with rat
hepatocytes and a time course experiment with human and rat
hepatocytes were conducted. In Table 3, the overall percentage
of metabolism and percentage of metabolism to water-soluble
products by rat hepatocytes were measured at four AAF con
centrations. With increasing AAF levels the percentage of dose
metabolized decreased. In addition, the ratio of percentage of
water-soluble:percentage of organic-soluble metabolism de
creased with increasing AAF concentration.
Fig. 4 illustrates the time-dependent rat and human hepato-
cyte metabolism of AAF separated into different metabolite
fractions. Rat and human hepatocytes showed an increase in
the amount of organic-soluble metabolite formation with time;
however, hepatocytes from Humans (HL) IV and V demon
strate peaks in ether extractable metabolites at 1 and 2 h,
respectively (Fig. 4a). Levels of human hepatocyte organic-
soluble metabolite formation from these three cases were higher
at all time points than in the rat. Both rat and human hepato
cytes also showed a time-dependent increase for water-soluble
metabolites and for covalently bound products (Fig. 4, b and c).
Despite lower metabolism, rat hepatocytes had equal or higher
levels of covalent binding of AAF than the three human cases.
Total AF production increased with time although human
hepatocytes deacetylated AAF to a greater extent than rat
hepatocytes (Fig. 4d). CX-OH-AAF (summation of all carbon
hydroxylated metabolites) formation exhibited variability
among human hepatocytes with peaks at l h for Individual IV,
2 h for Individual V, and an increase over the 4-h time period
for Individual f7and by rat hepatocytes. N-OH-AAF formation
varied in the three different human hepatocyte preparations,
was lower in the rat hepatocytes than in Individual V and VI,
but was similar to the hepatocytes from Individual IV. Based
on the results from the dose-response (Table 3) and time-course
experiments (Fig. 4), human and rat hepatocyte metabolism
Table 3 Dose response metabolism of AAF by rat hepatocytes
All experiments were 4-h incubations containing 5x10* hepatocytes in 5 ml
medium/flask with 3 flasks/dose.
Concentration0M)0.44 of total AAF of water-soluble
metabolized"26.7 metabolism20.2
8.1
4.4 19.6
44 9.9 3.2
440% 3.3% 0.50
The percentage of total AAF metabolized and percentage of water-soluble
metabolism were based on the initial amount of [MC]AAF added. Total percentage
of AAF metabolized includes the percentage of organic-soluble and the percentage
of water-soluble metabolism.
5864
6000 3000
4000 20OO
-1000
3000
2000 !
IOOO
600
ZOO
120 240 15 30 60 120 24O
TIME(minutes)
Fig. 4. Effect of time on the metabolism of AAF by human and rat hepatocytes.
Results are from individual human hepatocyte cases (HL) IV, V. and VI and from
the average of three rat experiments. Ether-extractable and water-soluble metab
olite values represent the sum of individual metabolites for each group. C,-OH-
AAF values represent the sum of all carbon-ring hydroxylated metabolites. AF,
N-OH-AAF, and d-OH-AAF values represent the sum of ether-extractable
products and glucuronide plus sulfate-conjugated products. All experiments were
conducted for 4 h at a [14C]-AAFconcentration of 4 nM using 5x10' hepatocytes
in 5 ml medium with 3 flasks/experimental point.
Table 4 Metabolism by human and rat hepatocytes
All experiments were done at 4 n\i | "( | V\ I for 4 h in flasks containing 5 x
1d" hepatocytes in 5 ml medium.
Human"
II III IV VI VII VIII Rat"
% of total AAF 49.4 36.4 45.9 19.8 42.5 38.4 40.1 31.8 19.6 3.3
metabolized*
% of water-soluble 45.4 23.2 28.0 7.3 16.5 19.6 8.2 19.8 8.1 0.77
metabolism
" Each human hepatocyte value represents 1 experiment and rat hepatocyte
values represent the average of 3 experiments.
h The percentage of AAF metabolized and percentage of water-soluble metab
olism were based on the initial amount of [I4C]AAF added. The total AAF
metabolized represents the percentage of organic-soluble and water-soluble me
tabolism.
with 4 UMAAF was measured at 4 h. In Table 4, total metab
olism by hepatocytes from each individual human case and the
average of 3 experiments for the rat are shown. Hepatocytes
from Individual / had the highest overall metabolism (49.4%)
of AAF and highest metabolism to water-soluble products
(45.4%) with Individual IV exhibiting the lowest metabolism
(19.8% and 7.3%, respectively). The range of conjugative me
tabolism by human hepatocytes was greater (7.3-45.4%) than
the range of overall metabolism (19.8-49.4%). Except for In
dividual IV, human hepatocyte metabolism of AAF to organic
and water-soluble products was greater than rat hepatocyte
metabolism.
The ranges of specific metabolite levels formed by hepato
cytes from the eight human cases and the rat are shown in
Table 5. The major organic-soluble metabolites formed by both
human and rat hepatocytes were AF, 7-OH-AAF, and the

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 HEPATOCYTE METABOLISM AND MUTAGENIC ACTIVATION OF AAF

unknown metabolite(s). The primary glucuronide- and sulfate- studies because of their advantages when compared to isolated
conjugated product formed by human and rat hepatocytes was subcellular preparations (20, 27, 34, 35). Intact hepatocytes
the 7-OH-AAF although sulfate and glucuronide conjugates catalyze Phase I and Phase II reactions at rates and in the
were formed with 8-OH-AAF, 5/9-OH-AAF, 1/3-OH-AAF, N- sequence that they occur in vivo (28), as well as preserving their
OH-AAF, and AF. Levels of organic-soluble and water-soluble metabolizing capabilities for a longer period of time than sub-
N-OH-AAF formation were low compared to other metabolites cellular fractions (36). Differences in levels of mutagenic activ
formed by both human and rat hepatocytes. AF formation was ity are also apparent between subcellular fractions and intact
highest in Individuals V, VI, and VU and lowest in Individuals hepatocytes (27, 34). Levels of AF and AAF mutagenic activity
/ and VIII (data not shown). It is interesting that while total previously measured (20, 23, 30, 35, 37-39 and data not shown)
metabolism or water-soluble metabolism with human hepato are higher in S9 preparations than intact hepatocytes from
cytes varied 2.5- and 6-fold (Table 4), respectively, the range of humans and rats possibly due to a lack of detoxifying pathways
individual metabolites such as AF (organic-soluble) varied 35- that are present in intact hepatocytes but not in subcellular
fold while glucuronide and sulfate conjugates of 7-OH-AAF preparations. Previous investigation of AAF metabolism by
varied 30- and 36-fold, respectively, in eight human cases (Table human liver microsomes (23, 40) did not detect AF, although
5). deacetylase activity has been measured in microsomal fractions
from guinea pigs, dogs, hamsters, mice, and rats (41) and with
DISCUSSION intact hepatocytes in the present study. Other studies have
illustrated extensive species differences in the metabolism and
In the present study, human hepatocytes were compared to mutagenesis of many environmental compounds (7-10, 30, 38,
rat hepatocytes for their ability to metabolize AAF and AF and 39, 42-45) and these results also may be useful in the extrapo
to gain insight into interindividual variations in the human lation of animal data to represent hazard levels in humans (45).
population for AAF metabolism. The slicing technique used to In the present study, rat and human hepatocytes metabolized
prepare hepatocytes in the present study is valuable because it AAF in a qualitatively similar fashion. The major metabolite
utilizes samples from biopsy or resected tissue that are too produced by rat and human hepatocytes was AF. Evidence of
small or are otherwise not perfusable. In six of nine cases in the importance of AF as an AAF metabolite is provided by our
this study, the tissues obtained were not perfusable. The slicing findings that levels of AF mutagenesis were substantially higher
technique therefore allows studies to be done on a more frequent than with AAF and that AF was the major metabolite formed
basis with human hepatocytes and yields hepatocytes with a while N-OH-AAF, which is a mutagenically active metabolite
viability and mutagenic activity comparable to hepatocytes pre isolated primarily in microsomal preparations (21, 23, 40, 46)
pared via the perfusion technique (Table 2). was a minor metabolite in both human and rat hepatocytes. It
Intact hepatocytes are useful in metabolism and genotoxicity should be noted, however, that in hepatic microsomal prepa
rations N-OH-AAF was more mutagenically active than AF
Table 5 Metabolism of AAF to ether-extractable products and glucuronide and (47). Because AF may be further metabolized (48, 49) and N-
sulfate-conjugated metabolites by human and rat hepatocytes OH-AAF can be metabolically changed primarily via deacety-
All experiments were done using 4 nM [MC]AAF for 4 h. Hepatocytes were
incubated with 5 x IO6cells in 5 ml medium. lation to a reactive form (18, 38, 50), the relative contribution
(pumi.1 of these two compounds to the mutagenic process cannot be
(pmol/assay)"43-89328-42618-53736-87727-98117-58077-35823-16918-18077-47810-547-7186-50825-17517-17551-44322-28419-312490-7824261-2552205-22
assay)*551-70482-10887-192126-506148-315196-32839-14147-13774-7838-7914-5314-42144-38621-2418-2819-6120-2917-762025-36
clearly established.
MetaboliteUnknownEther-extractableGlucuronideSulfate7-OH-AAFEther-extractableGlucuronideSulfate8-OH-AAFEther-extractableGlucuronideSulfate5/9-OH-AAFEther-extractableGlucuron
The C-hydroxylation of AAF by human and rat hepatocytes
represents a step in the detoxification process. In addition to
the normal C-hydroxylated products the tentative identification
of 8-OH-AAF was reported in rat hepatocytes in this study and
previously (30) and in human hepatocytes for the first time.
The 8-OH-AAF had also been detected in rat urine (33). The
unknown peak in the HPLC chromatogram and 7-OH-AAF
were the major organic-soluble C-hydroxylated metabolites
with the 5/9-OH-AAF, 3-OH-AAF, 1-OH-AAF, and 8-OH-
AAF appearing in roughly equivalent levels in hepatocytes from
human individuals. The 7-OH-AAF was the primary glucuro
nide and sulfate conjugate formed by human and rat hepato
cytes. It has been previously reported that C-hydroxylated glu-
curonides are the only detectable water soluble metabolites
formed in rat hepatocytes (7). In the present study both conju
gates were detected using longer enzymatic times than in pre
vious sulfatase and 0-glucuronidase hydrolysis experiments
(51). AF-conjugated products were also identified (Table 5)
based on the extractable radioactivity after sulfatase and ,-
glucuronidase treatment, but the structural nature of the con
jugates could not be positively identified.
Quantitative differences in rat and human hepatocyte metab
olism were evident when comparing levels of overall organic-
soluble and water-soluble metabolite formation with human
hepatocyte metabolism higher than that by rat hepatocytes.
" Ranges of human hepatocyte metabolites from 8 individuals. This was also true for water-soluble metabolite formation in 7
* Ranges of rat hepatocyte metabolites from 3 animals. of 8 human cases in which the specific metabolite levels, espe-
5865

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 HEPATOCYTE METABOLISM AND MUTAGENIC
cially AF, 7-OH-AAF, and 5/9-OH-AAF, were appreciably
higher in humans than rats. However, there was a higher
conversion of total water-soluble metabolites by rat hepatocytes
to glucuronide and sulfate conjugates (>85%) than by human
hepatocytes (40-67%), although humans exhibited a higher
overall water-soluble metabolite level. Although it was not
investigated in the present study, a possible reason for this
discrepancy is that more glutathione and amino acid conjugates
of AAF metabolites are produced by human hepatocytes than
are produced by rat hepatocytes. The higher level of such
conjugates produced by human hepatocytes may indicate a
species difference between humans and rats in the sensitivity
and susceptibility to the hepatocarcinogenicity of aromatic
amines. Rat, guinea pig, and hamster differences in hepatocar-
cinogenic susceptibility have been reported (18, 19) and Holme
and co-workers (7) have illustrated differences in the ability of
these species to detoxify AAF.
Another factor to be considered when extrapolating animal
data to humans is the wide degree of interindividual variation
in humans. The extent of interindividual variation in humans
is well documented with the emphasis divided on factors related
to environment or heredity (9, 52, 53). Genetic factors, nutri
tion, drug intake, cigarette smoking, and exposure to pollutants
are but a few of the factors that modify the way human enzy
matic systems and in particular the cytochrome P-450 system
metabolize foreign and endogenous substances (52, 54-56). The
metabolic polymorphisms and substrate specificities of certain
P-450 isozymes in the hydroxylation of compounds such as
debrisoquine and spancine (4, 5, 9, 37), populations divided
into slow and fast acetylators (38, 57), and findings that have
identified multiple P-450s all attest to a widespread variation
in humans. Such variation may not be seen in inbred laboratory
animals or animals maintained under identical environmental
conditions.
The variability in individual AAF metabolites by human
hepatocytes probably results from the polymorphism of P-450
isozymes. It has been shown that in human liver microsomes
two phenotypes, slow and fast metabolizers, exist in the metab
olism of AAF (22). However, the extent of the variability of
human hepatocyte-mediated metabolism and mutagenesis of
AAF was markedly lower than the variation reported in pre
vious studies of human metabolism of other compounds by
various tissues (11-15, 58). This lower variability in the present
study may be due to the consistent use of fresh suspensions of
hepatocytes from surgical tissue instead of cultured cells from
autopsy samples or from expiant cultures which were utilized
in many of these previous studies (10, 11, 59). The human
variability in deacetylase activity is interesting as it reflects
individual polymorphism and the possibility of phenotypic ac
tivity comparable to slow versus fast acetylator populations (38,
57). The high levels of AF produced by human and rat hepa
tocytes suggest evidence for the role of deacetylation in the
formation of genotoxic AAF products in rats and humans. A
possible relationship between metabolism to AF and AAF
mutagenesis was seen in human hepatocytes but was not evident
with rat hepatocytes (data not shown). This possible difference,
combined with the higher levels of AAF mutagenesis and me
tabolism by human hepatocytes, may alter the direct extrapo
lation of rat carcinogenesis data as far as an effect from aromatic
amines in humans would be concerned.
The current study shows the practicality and utility of inves
tigating the metabolism and activation of carcinogens with
human tissues. Such data when used in combination with rodent
bioassay data and other results from frequently used short-term
5866
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ACTIVATION OF AAF
assays can be useful for risk assessment of environmental chem
icals to humans.
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Comparison of Human and Rat Hepatocyte Metabolism and
Mutagenic Activation of 2-Acetylaminofluorene
Kenneth Rudo, William C. Meyers, Walter Dauterman, et al.

Cancer Res 1987;47:5861-5867.

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