2. 22
tumors in mice at doses which were acutely
hepatotoxic (Flaks and Flaks, 1983) raises the
question whether paracetamol is carcinogenic
through a genotoxic or an epigenetic mechanism
(Williams, 1980). Paracetamol and N-hydroxy-
paracetamol, which spontaneously forms NAPQI
(Corcoran et al., 1980), have not been shown to be
mutagenic in Salmonella typhimurium (Wirth et al.,
1980). To address the question of the mechanism
for paracetamol hepatocarcinogenicity, we have
performed studies on bacterial mutagenicity, cova-
lent macromolecular binding, DNA damage as
well as DNA repair with paracetamol and NAPQI.
Materials and methods
Chemicals
[3H-G]Paracetamol (spec. act. 3.0 Ci/mmole,
> 98.5% pure), [14C-9]AAF (spec. act. 52 mCi/m-
mole, > 98% pure) and [Me-3H]thymidine (spec.
act. 47 Ci/mmole, > 96% pure) were obtained
from New England Nuclear, Boston, MA (U.S.A.).
NAPQI was synthesized as described (Dahlin and
Nelson, 1982), and kept under argon at -20°C
until use. Immediately before use it was weighed
and dissolved in DMSO. Other chemicals were
obtained from the following sources: Mycostatin
from Squibb, Twickenham (U.K.), fetal calf serum,
calf serum and Dulbecco's modified Eagle medium
from Gibco, Grand Island, NY (U.S.A.);
horse serum from the National Institute of Public
Health, Oslo (Norway); dexamethazone, insulin,
collagenase (type IV), bovine serum albumin (type
V), ~-aminolevulinic acid, Nonidet P-40, hydroxy-
urea, mitomycin C and deoxyribonucleic acid (type
I) from Sigma, St. Louis, MO (U.S.A.); proteinase
K from E. Merck, Darmstadt (F.R.G.); AAF from
Koch-Light Laboratories, Colnbrook (U.K.);
paracetamol and sodium phenobarbital from The
Norwegian Medicinal Depot, Oslo (Norway); hy-
droxyapatite from Bio-Rad Laboratories, Munich
(F.R.G.). N-OH:AAF was a generous gift from
Dr. S.S. Thorgeirsson, National Cancer Institute,
Bethesda, MD (U.S.A.).
Animals and pretreatments
Male B6 mice, weighing 20-27 g, were obtained
from the Norwegian Research Animal Centre, Na-
tional Institute of Public Health, Oslo (Norway).
They were given a pelleted diet (Norwegian Stan-
dard) and water ad libitum. For induction purpo-
ses phenobarbital 75 mg/kg in saline was given
i.p. 72, 48 and 24 h before isolation of liver cells or
in vivo treatments. For determination of in vivo
macromolecular covalent binding mice were given
1.5-1.8 mCi of [3H]paracetamol, 500 mg/kg, in
0.9% saline i.p. The animals were killed after 4 h.
Preparation of liver subfractions
Livers were homogenized in a Teflon-glass ho-
mogenizer in 2 vol. of sterile, ice-cold 1.15% KC1
containing 20 mM Tris-buffer, pH 7.4, and 10%
glycerol. $9 and microsomal fractions were pre-
pared as described (Dybing and Thorgeirsson,
1977). The $9 fraction was stored as such, whereas
the microsomes were stored in the Tris-KCl buffer
containing 30% glycerol at -70°C until use. Pro-
tein concentrations were determined according to
Lowry et al. (1951) using bovine serum albumin as
standard.
Mutagenicity asssays
Mutagenic activity was assayed with Salmonella
typhimurium TA98, TA100 and TA102. In some
experiments the regular plate assay (Ames et al.,
1975) as described (Dybing and Thorgeirsson,
1977), or a quantitative modification thereof
(Soderlund et al., 1979), were used in the absence
or presence of mouse liver subfractions and cofac-
tors. Freshly dissolved NAPQI in DMSO was
added last in the mutagenicity assays, bacteria
were incubated with NAPQI for 30 min before
plating in the quantitative experiments. In other
experiments substances were tested in cocultures
of S. typhimurium and mouse hepatocytes (Holme
et al., 1983a). Hepatocytes plated on 60-mm dishes
were rinsed, 2 h after plating, with Hanks'-Hepes
buffer, pH 7.4, supplemented with 1% bovine
serum albumin, and then added the same buffer
containing test substance and 0.1 ml of an over-
night culture of S. typhimurium. After 2 h of
co-incubation, the bacteria was collected by
centrifugation and plated to determine the number
of revertants. The genotype of colonies on the
revertant plates were checked according to Ames
et al. (1975). The S. typhimurium strains were
generously provided by Dr. B.N. Ames, University
of California, Berkeley, CA (U.S.A.).
3. Macromolecular covalent binding
In vitro. Ice-cold reaction vessels contained
(final concentrations): 2.0 mg/ml microsomal pro-
tein, 0.5 mM [3H]paracetamol (1.6 x 103
cpm/nmole for protein binding, 1.7 X 10 4
cpm/nmole for DNA binding) or 0.05 mM 14C-
AAF (1.5 × 103 cpm/nmole for protein binding or
1.3 × 104 cpm/nmole for DNA binding) in 25 ~1
DMSO, a NADPH-generating system with or
without 0.67 mg/ml DNA in a total volumc of 3.0
ml. Incubations were carried out at 37°C in a
shaking water-bath incubator. Reactions for de-
termination of protein binding were stopped after
15 min by adding 1.0 ml of 30% trichloroacetic
acid (TCA). For determination of DNA binding,
the reactions were stopped after 15 rain with 1.0
ml 0.17 M Na-dodecylsulfate and 0.64 ml 4 M
NaC1 (Neal et al., 1979). Covalent protein binding
was assayed as described (Saderlund et al., 1982).
DNA was extracted from microsomal incubations
with 9.9 ml phenol reagent (50 g phenol, 50 ml
chloroform, and 1 ml isoamyl alcohol) according
to Neal et al. (1979), and washed subsequently
with acetone, chloroform/ethanol (1 : 1) and ether.
DNA was dissolved in Tris-KCl buffer, pH 7.4
(0.1 M). After the 100-~1 addition of a proteinase
K solution (1 mg/ml) the DNA was precipitated
in the presencc of 30% TCA (1.0 ml) and 2.5%
bovine serum albumin (0.5 ml). The precipitate
was collected by centrifugation and DNA was
hydrolysed by heating in 5% TCA at 90°C for 20
min. The amounts of radioactivity incorporated
into the DNA were determined by liquid-scintilla-
tion counting and DNA concentrations were mea-
sured according to Burton (1956).
In vivo. In the in vivo experiments, livers and
femoral muscles were minced and homogenized in
4 vol. of 75 mM NaCI, 10 mM EDTA, and 10 mM
Tris pH 7.8 in a teflon Potter-Elvehjem-type ho-
mogenizer. 100/tl of whole homogenate was used
for protein binding. Purification of DNA was per-
formed essentially as described by Sagelsdorff et
al. (1983) with some modifications. The rest of the
homogenate was centrifuged at 9000 rpm for 20
min. The supernatant was decanted and the pellet
was resuspended in Tris-buffcr (4 mi). A 10%
solution (v/v) of the non-ionic detergent Nonidet
P-40 up to a final concentration of 1.0% was
added to the samples. The samples were incubated
23
at 4°C for 15 min after vortexing and the vortex-
ing was repeated. The crude chromatin was pel-
leted at 2000 rpm for 5 min and the pellet was
resuspended in 0.25 M sucrose buffer (50 mM
Tris-HC1, 25 mM KC1, 15 mM MgCI2, pH 8.0).
After this, 0.88 M sucrose buffer was added to the
bottom of the tube and the tubes were centrifuged
at 3000 rpm for 20 min. The pellet was washed
once with Tris buffer and centrifugated again at
3000 rpm for 20 min. The pellet was then sus-
pended in 10 ml of lysing medium (8 M urea, 0.24
M sodium phosphate buffer, pH 6.8, 10 mM
EDTA, 1% (w/v) SDS) and homogenized. Pro-
teins were extracted under intensive shaking for 10
min with CIP (chloroform : isoamylalcohol : phe-
nol 25:1:25). The resulting suspension was sep-
arated into layers by centrifugation at 14000 rpm
for 20 min. The aqueous layer was washed again
with CIP. The aqueous solution was extracted
twice with 25 ml of ether to remove trace amounts
of phenol. The aqueous solution was thereafter left
standing overnight at room temperature and was
applied to an HA column. Dry HA (2 g/g liver)
was suspended in 0.014 M sodium phosphate
buffer, pH 6.8 (6 vol.). The slurry was swirled
gently and heated at 85°C for 15 min then was
left untouched for 10 min. The fine particles were
decanted. The remaining slurry was resuspended
in MUP (8 M urea, 0.16 M sodium phosphate
buffer, pH 6.8). The suspension was allowed to
settle. The slurry was then added to glass columns
(1 cm x 25 cm). The column was allowed to equi-
librate and MUP was let run off. Thc aqueous
solution containing nucleic acids was loaded on
the column and the elution was monitored at 260
nm. Proteins and RNA were washed from the
column with MUP at a flow rate of 0.5 ml/min
until the transmission had returned to background
values. Two bed volumes of 14 mM sodium phos-
phate buffer, pH 6.8, was used to purge off the
ui'ea. DNA was eluted with 0.48 M sodium phos-
phate buffer, pH 6.8, and 25-30 ml of the DNA
solution was collected. The samples were dialyzed
at 4°C against 8 1of 0.2 M NaCI (24 h). DNA was
precipitated by adding 2 vol. ethanol and storing
at -20°C for at least 12 h. The DNA was centri-
fuged for 20 min at 1500 rpm, the supernatant
decanted and DNA washed with acetone. The
DNA was hydrolysed in 10% HC104 and the ra-
5. dioactivity determined by scintillation counting.
The amount of DNA was determined by diphenyl-
amine assay (Burton, 1956). 100 ~1 of whole ho-
mogenate was added to 1.0 mi of cold 5% TCA,
vortexed and was let standing for 10 min. Cova-
lent protein binding was assayed as described
(Soderlund et al., 1982). The amount of protein
was measured by the Lowry assay (1951).
DNA damage in hepatorna cells
DNA damage of Reuber H4-II-E rat hepatoma
cells was determined by alkaline elution (Kohn et
al., 1981). The cells were grown in Dulbecco's
minimal essential medium supplemented with 10%
calf serum and 10% fetal calf serum and 60 U/ml
mycostatin. DNA was labelled by growing 2.5 ×
105 cells in 0.05 /~Ci 3H-TdR/ml for 24 h, fol-
lowed by growth in non-radioactive medium for
an additional 24 h. The cells were deposited on a
2.0-/~m pore size polyvinyl chloride filter (Milli-
pore Corp., Bedford, MA, U.S.A.) using mild suc-
tion, and lysed with 5 ml of a 2% sodium lauryl
sulfate solution containing 0.02 M Na2EDTA, 0.1
M glycine, pH 10.0, which was allowed to drip
through the filter by gravity. After lysis, 2 ml of a
0.5 mg/ml solution of proteinase K in the 2%
sodium lauryl sulfate lysing solution was pumped
through the filter at 0.035 ml/min, followed by a
0.02 M EDTA solution adjusted to pH 12.3 with
tetrapropyl ammonium hydroxide pumped at the
same rate for a total of 15 h.
For chemical exposure 100-/~1 stock solutions of
paracetamol and N-OH-AAF in DMSO were ad-
ded per 10-ml fresh medium. NAPQI was dis-
solved immediately before exposure of the cells in
DMSO and added directly to the cell cultures.
After exposure of cells for 1 h, medium with test
chemicals was removed and the cultures rinsed
with cold growth medium. The cells were removed
with a calcium-free Hanks' balanced salt solution
containing 0.1% trypsin and 0.2% EDTA.
3-h fractions were collected and processed for
liquid-scintillation counting, as described by Kohn
et al. (1981). The elution rate constant, k, was
determined as described by Sina et al. (1983).
Cytotoxicity was measured either immediately after
1-h exposure with the test chemicals or after in-
cubating the cultures in fresh medium for another
23 h, by determining trypan blue uptake (Laishes
et al., 1978).
25
DNA repair in hepatocytes
The two-step technique for isolation of rat
hepatocytes (Seglen, 1975) as described elsewhere
(Holme et al., 1983b), was modified for isolation
of mouse hepatocytes. The mice were anesthetized
with ether and the portal vein was cannulated with
a 20 gauge intravenous needle. The liver was per-
fused in situ with a Ca2 *-free Hanks' bicarbonate
buffer containing 0.25 mM EGTA, for 3-5 min,
then with Hanks' bicarbonate buffer containing 5
mM Ca2 ~ and 0.6% collagenase for 8-10 min. A
perfusion rate of 10 ml/min was maintained for
both perfusates using non-recirculating conditions
by a cut in the inferior caval vein. Before disper-
sion of the hepatocytes the gall bladder was re-
moved. Livers from 4-5 mice were pooled and
preincubated for 20 min at 37 °C in Hanks'-Hepes
buffer containing 1% bovine serum albumin, and
then centrifuged 3 times at 50 × g for 30 sec. In 4
Expts. with pooled hepatocytes from 4-5 mice, the
mean cell yield was 44 + 6 (S.D.) million cells per
mouse. The mean viability of the cells was 91 + 3
(S.D.) per cent as determined by trypan blue ex-
clusion. Hepatocytes were incubated 37°C as sta-
tionary monolayers (7 × 104 cells/cm2) in 35-mm
or 100-mm Falcon tissue-culture dishes for de-
termination of cytotoxicity and DNA repair, re-
spectively. Viability of the hepatocytes was de-
termined by trypan blue exclusion (Laishes et al.,
1978) and by the release of LDH (Auforo et al.,
1978) into the culture medium. Unscheduled
DNA-repair synthesis was measured by liquid-
scintillation counting of 3H-TdR incorporated into
nuclear DNA (Althaus et al., 1982) as described
(Holme et al., 1984). Monolayers of hepatocytes
were incubated with 10 mM hydroxyurea for 1 h
before exposure with test chemicals. Medium con-
taining hydroxyurea and 0.62 ~tCi/ml, 3H-TdR
and test substance in 0.5% DMSO was added to
the hepatocytes 3 h after plating and the experi-
ments were terminated after an incubation time of
18-19 h. Some cultures were exposed to UV light
for 30 sec at a distance of 6 cm using a Model
UVSL-25 Mineralight lamp (Ultra-Violet Prod-
ucts, San Gabriel, CA, U.S.A.) with a multiband
254-366 nm at 220V, 50 Hz and 0.12 A. In each
experiment nuclear DNA was isolated from 3 dis-
hes, as described elsewhere (Holme et al., 1984).
Nuclear DNA content was measured according to
Burton (1956).
6. 26
Results
Mutagenicity in Salmonella typhimurium
Paracetamol was not mutagenic in S. typhimu-
rium TA98, TA100 or TA102, tested in the pres-
ence of mouse-liver subfractions ($9 or micro-
somes) and cofactors, either in the regular plate
assay (data not shown) or in a quantitative assay
where revertants and survivors were plated sep-
arately (Table 1), or in coculteres with mouse
hepatocytes (Table 2). No marked bacterial cyto-
toxicity of paracetamol was evident under the
conditions employed. On the other hand, NAPQI
was markedly cytotoxic for the bacteria, especially
in the absence of an activation system (Table 1).
Of interest to note was that the strain TAI02
appeared to be somewhat more resistant to the
cytotoxic effects of NAPQI than TA98. Under
extreme cytotoxic conditions (less than 1% survival)
some of the colonies on the revertant plates showed
an altered genotype so that a calculation of re-
vertants per 10 6 survivors appeared to increase in
relation to the spontaneous reversion frequency.
However, this was an inconsistent finding after
having performed many experiments, a statistical
significance could therefore not be ascribed to this
phenomenon.
Macromolecular covalent binding
Paracetamol was readily converted to inter-
TABLE 2
MUTAGENICITY TESTING OF PARACETAMOL IN
Salmonella typhimurium TA98 WITH HEPATOCYTE
ACTIVATION
Test substance His' Revertants
per plate
DMSO (control)
Paracetarnol
N-OH-AAF
15+12
0.5 mM 30+ 7
1.0 mM 22+ 3
5.0 mM 20+ 3
10.0 mM 26+ 3
20.0 mM 22+ 5
0.1 mM 908_+25
Test substances were added to co-culture.s of S. typhimurium
TA98 and isolated hepatocytes pooled from 5 untreated mice.
The values are mean + S.D. of 3 incubations.
mediates which bound covalently to mouse liver
microsomal protein in the presence of NADPH
(Table 3). This occurred at a rate which was higher
than that of the hepatocarcinogen AAF. When
DNA was added to microsomal incubations,
paracetamol also bound to this macromolecule,
although at a rate appreciably slower than that of
AAF. In preliminary experiments, intrapcritoneal
injections of 500 mg/kg paracetamol to PB-
pretreated B6 mice caused extensive liver necrosis.
When the mice were given radiolabelled para-
cetamol (1.5-1.8 mCi per mouse), 1201 + 150
pmoles [3H]paracetamol bound covalently per mg
liver protein when measured 4 h after treatment
(Table 3). In this experiment, only 23 pmoles
[3H]paracetamol bound covalently per mg muscle
protein. Hepatic DNA, purified by hydroxyapatite
chromatography, was found to contain 15 + 9
pmoles bound paracetamol per mg DNA. Liver
DNA extracted and purified from untreated mice
gave a value of 4 pmoles/mg DNA. The level of
1.0
I-
I-
z
o
a
|
F-
z
Q
z
_o
I-
U
0.8
0.7
0.6
0.5
o14
0.3
0.2
CONTROL
pHAA 10"2M
NAPOI 5 10-5M
NAPOl 10-4M
N-OH-AAF 10-4M
I
~NAPQI 2,5 10-4M
0.1 I I i I I
3 6 9 12 15
ELUTION TIME (HOURS)
Fig. 1. DNA damage in Reuber hepatoma cells after exposure
to paracetamol, NAPQI and N-OH-AAF. The cells were in-
cubated for 60 rain in presence of various concentrations of the
compounds. The data shown are from one representative ex-
periment.
7. 27
TABLE 3
COVALENT BINDING OF [3H]PAP,ACETAMOL AND [14C]2-ACETYLAMINOFLUORENE TO MOUSE-LIVER MACRO-
MOLECULES IN VITRO AND IN VIVO
Test substance Covalent binding in vitro Covalent binding in vivo
Protein DNA Protein DNA
(pmoles/mg (pmoles/mg (pmoles/mg (pmoles/mg
protein/min) l)NA/min) protein) DNA)
[3H]Paracetamol 100.4+4.1 2.6+_0.8 1 201 + 150 15+ 9
I14C]AAF 76.0 + 4.8 7.6 + 1.0 N.D. N.I).
N.D., not determined.
In vitro studies were performed by incubating 0.5 mM of radiolabelled substrates with 2 mg/ml mouse-liver microsomes, a
NADPH-generating system with or without 0.67 mg/ml DNA. For determination of in vivo macromolecular covalent binding
phenobarbital-pretreated mice were given 500 mg/kg [3l--l]paracetamol 4 h before the animals were killed.
Values are means+ S.D. of 4 incubations or 4 animals, respectively.
paracetamol binding to DNA in treated mice was
calculated to give a CBI (Lutz, 1979) of 1.2.
DNA damage in hepatoma cells
Alkaline elution of cellular DNA is a sensitive
assay for the determination of DNA damage (Sina
et al., 1983). When paracetamol was added to
Reuber rat hepatoma cells grown in culture, no
increase in the elution rate of DNA was observed
(Fig. 1, Table 4). On the other hand, NAPQI
caused extensive DNA damage at concentrations
of 0.05-0.25 mM, resulting in the formation of
single-strand breaks. This was not due to a general
cytotoxic effect of NAPQI under these experimen-
tal conditions as determined by cellular trypan
blue uptake, since NAPQI did not cause cytotoxic-
ity measured after an incubation period of 1 h
(Table 4). However, NAPQI caused a concentra-
tion dependent cytotoxic effect when assayed 24 h
after exposure. The proximate carcinogen N-OH-
AAF also caused considerable DNA damage in
the Reuber cells as measured by alkaline elution
(Fig. 1, Table 4).
DNA repair in hepatocytes
Damage to cellular DNA can also be assessed
by the determination of an increase in unsched-
uled DNA synthesis in isolated hepatocytes grown
"FABLE 4
EFFEC~I" OF PARACETAMOL, NAPQI AND N-OH-AAF EXPOSURE ON ALKALINE ELUTION OF DNA AND CYTO-
TOXICITY IN REUBFR IIEPATOMA CELLS
Test substance Elution rate constant Cytotoxicity
(% of control)
1 h 24h
DMSO (control) 0.010 + 0.002 0 + 0 0 + 0
Paracetamol 10 mM 0.012 + 0.004 0 + 0 0 + 0
NAPQI 0.05 mM 0.041 +0.019 0+0 36+ ll
0.10 mM 0.075 + 0.036 2 + 2 81 + 3
0.25 mM 0.173+0.068 5+1 100+ 0
N-OH-AAF 0.10 mM 0.155+0.029 0~-0 0+ 0
Reuber cells, prelabelled with 3H-TdR, were exposed to test substances for 1 h. The cultures were then rinsed and prepared for the
alkaline elution assay or cytotoxicity was determined immediately or after incubation for further 23 h.
Means+ S.D. of 4-8 determinations.
9. 29
in monolayer culture (Althaus et al., 1982). Experi-
ments with paracetamol showed that the drug
increased DNA-repair synthesis in control mouse
hepatocytes at concentrations of 5.0 mM and
higher (Table 5). At these concentrations cellular
toxicity, as determined by an increased leakage of
LDH (Table 5) or by trypan blue exclusion (not
shown), also became evident. No significant in-
crease in unscheduled DNA synthesis was found
with concentrations of NAPQI up to 0.25 mM.
When using hepatocytes from mice pretreated with
PB, a lowering of the concentration threshold for
both increased DNA-repair synthesis as well as
cytotoxicity was noted. A brief exposure of the
cells to UV-light was used as a positive control for
DNA damage in these experiments.
Discussion
The present investigation was carried out in an
attempt to address some of the questions per-
taining to the mechanism(s) for paracetamol
hepatocarcinogenicity (Flaks and Flaks, 1983) in
mice. Whether or not paracetamol could be shown
to display genotoxic effects should be of impor-
tance when assessing the risk to humans of para-
cetamol intake. These studies were in part made
possible through establishment of a procedure
(Dahlin and Nelson, 1982) for the synthesis of the
proposed ultimate reactive metabolite of para-
cetamol, NAPQI.
For many carcinogens, mutagenicity in Salmo-
nella typhimurium is a very good predictor of
carcinogenic activity (McCann et al., 1975). How-
ever, for some chemical classes there is not a good
correlation between mutagenicity and carcinogen-
icity (Rinkus and Legator, 1979), in part this can
be due to a nongenotoxic mechanism of carcino-
genic action. Paracetamol did not cause mutations
in S. typhirnurium, neither in the presence of mouse
liver subfractions or with mouse hepatocytes as
activating system. Interestingly paracetamol was
not mutagenic for the new strain TA102 which is
sensitive to active oxygen mutagens (Levin et al.,
1982). Testing of the major ultimate reactive
metabolite of paracetamol, NAPQI (Dahlin et al.,
1984), also did not shown mutagenic activity in the
presence of metabolic activation systems. In the
absence of activation, concentrations of NAPQI
above 25 /xM were extremely cytotoxic. Thus, the
liver subfractions protect the bacteria from NAPQI
cytotoxicity, which in part may be explained by a
reduction of NAPQI back to paracetamol
(Corcoran et al., 1980). Under severe cytotoxic
conditions, a moderate increase in the reversion
rate with NAPQI was found. However, this was an
inconsistent finding. NAPQI, which is very short-
lived and reactive (Dahlin et al., 1984), presuma-
bly interacts preferentially with vital bacterial
macromolecules so that the organism dies even if
its DNA also should have premutational lesions
from NAPQI exposure. Similar to the present find-
ings were those reported earlier for N-hydroxy-
paracetamol (Wirth et al., 1980), which sponta-
neously breaks down to NAPQI (Corcoran et al.,
1980).
In addition to its avid binding to protein, it
could be shown that paracetamol bound cova-
lently to exogenously added DNA in a microsomal
incubation system. The DNA-binding rate was
slower than that of the hepatocarcinogen AAF,
whereas the reverse was true with respect to pro-
tein binding. This presumably reflects differences
in the chemical reactivity and stability of the re-
spective electrophiles of the two carcinogens. In
addition to the well-known fact that paracetamol
binds covalently to liver proteins in vivo (Jollow ct
al., 1973), it could also be demonstrated that
paracetamol binds covalently to hepatic DNA.
However, the level of binding was two orders of
magnitude lower than that reported for AAF, as
normalized by determination of the CBI of Lutz
(1979). In this study it was found that paracetamol
had a CB! of 1.2, whereas the moderately strong
hepatocarcinogen AAF has a CBI of 560 in rats
(Lutz, 1979). Further, it is important to note that
the covalent binding of paracetamol was demon-
strated at a hepatotoxic dose. If this covalent
binding only occurred in cells which would later
die, such a DNA interaction would not lead to
mutation, an event which most probably is in-
volved in initiation of carcinogenesis.
For many compounds, detection of DNA
single-strand breaks by alkaline elution (Kohn et
al., 1981) correlates well with mutagenic and
carcinogenic activity (Sina et al., 1983). In the
present study, rat hepatoma cells with a low capac-
ity for cytochrome P-450 metabolism (Owens and
10. 30
Nebert, 1975) was used. It was thus not surprising
that paracetamol did not cause DNA damage in
this system. However, the ultimate reactive
metabolite NAPQI was sufficiently stable and
lipophilic to traverse cellular membranes and cause
DNA single-strand breaks at a time where NAPQI
showed no cytotoxic effect. Thus, these single-
strand breaks were probably not caused by an
immediate, general cytotoxicity. However, after an
additional incubation time of the cells for 23 h
NAPQI cytotoxicity became manifest, in agree-
ment with findings using isolated rat hepatocytes
(Holme et al., 1983b).
Induction of unscheduled DNA synthesis, a
measure of DNA repair, has also been shown to be
a good indicator for genotoxic carcinogens (Alt-
haus et al., 1982). When mouse-liver hepatocytes
were incubated in the presence of paracetamol at
concentrations which caused cytotoxicity, an in-
crease in DNA-repair synthesis was demonstrated.
This finding was presumably related to metabolic
activation of paracetamol, since cells from mice
pretreated with PB showed a lower concentration
threshold for DNA-repair synthesis induction than
did cells from untreated mice. In contrast to the
experiments with the hepatoma cells, addition of
NAPQ! to the hepatocyte culture medium did not
elicit a response indicative of DNA damage in this
system. This difference in cytotoxicity vs. geno-
toxicity of NAPQI in the two cell systems may be
related to differences in the cell membranes, cellu-
lar shape and size, in cellular reduction capacity
and/or in number of cellular nucleophilic sites
between the mouse hepatocytes and the rat
hepatoma cells.
In summary, paracetamol or its ultimate reac-
tive metabolite has been found to show genotoxic
effects in 3 out of 4 test systems, only bacterial
mutagenicity did not give a significant positive
response. However, the covalent binding of para-
cetamol to DNA and the increased DNA alkaline
elution and DNA-repair synthesis only show that
paracetamol may cause DNA damage. This is not
a surprising finding taken together with the fact
that paracetamol is known to be metabolized to an
electrophilic intermediate which binds covalently
to nuclcophilic sites. Whether such an initial DNA
damage will be properly repaired or the cells will
die before a mutation becomes fixed is an open
question. The finding that the genotoxic effects of
paracetamol were observed at levels which caused
delayed cytotoxic effects, indicates that cytotoxic-
ity of paracetamol may predominate compared to
genotoxicity. Thus, the necrogenic effects of para-
cetamol, which would be expected to cause a strong
proliferative stimulus to an initiated liver cell, are
probably of major importance for paracetamol-in-
duced hepatocarcinogenicity in mice (Flaks and
Flaks, 1983). In this connection, it is important to
note the apparent dose-thresholds for both acute
liver damage (Mitchell et al., 1973a) and liver-
tumor formation (Flaks and Flaks, 1983) corre-
lates with the non-linearity of the relationship
between paracetamol dose and covalent protein
binding (Mitchell et al., 1973b; Mudge et al.,
1978). However, the present findings taken to-
gether with the report that paracetamol may cause
chromosome aberrations in Chinese hamster cells
in vitro (Ishidate and Yoshikawa, 1980) indicate
that it is important to further examine paraceta-
mol for possible mutagenic effects.
Acknowledgements
This study was supported by The Royal
Norwegian Council for Scientific and Industrial
Research and by grant GM-25418 from The Na-
tional Institutes of Health (U.S.A.).
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![22
tumors in mice at doses which were acutely
hepatotoxic (Flaks and Flaks, 1983) raises the
question whether paracetamol is carcinogenic
through a genotoxic or an epigenetic mechanism
(Williams, 1980). Paracetamol and N-hydroxy-
paracetamol, which spontaneously forms NAPQI
(Corcoran et al., 1980), have not been shown to be
mutagenic in Salmonella typhimurium (Wirth et al.,
1980). To address the question of the mechanism
for paracetamol hepatocarcinogenicity, we have
performed studies on bacterial mutagenicity, cova-
lent macromolecular binding, DNA damage as
well as DNA repair with paracetamol and NAPQI.
Materials and methods
Chemicals
[3H-G]Paracetamol (spec. act. 3.0 Ci/mmole,
> 98.5% pure), [14C-9]AAF (spec. act. 52 mCi/m-
mole, > 98% pure) and [Me-3H]thymidine (spec.
act. 47 Ci/mmole, > 96% pure) were obtained
from New England Nuclear, Boston, MA (U.S.A.).
NAPQI was synthesized as described (Dahlin and
Nelson, 1982), and kept under argon at -20°C
until use. Immediately before use it was weighed
and dissolved in DMSO. Other chemicals were
obtained from the following sources: Mycostatin
from Squibb, Twickenham (U.K.), fetal calf serum,
calf serum and Dulbecco's modified Eagle medium
from Gibco, Grand Island, NY (U.S.A.);
horse serum from the National Institute of Public
Health, Oslo (Norway); dexamethazone, insulin,
collagenase (type IV), bovine serum albumin (type
V), ~-aminolevulinic acid, Nonidet P-40, hydroxy-
urea, mitomycin C and deoxyribonucleic acid (type
I) from Sigma, St. Louis, MO (U.S.A.); proteinase
K from E. Merck, Darmstadt (F.R.G.); AAF from
Koch-Light Laboratories, Colnbrook (U.K.);
paracetamol and sodium phenobarbital from The
Norwegian Medicinal Depot, Oslo (Norway); hy-
droxyapatite from Bio-Rad Laboratories, Munich
(F.R.G.). N-OH:AAF was a generous gift from
Dr. S.S. Thorgeirsson, National Cancer Institute,
Bethesda, MD (U.S.A.).
Animals and pretreatments
Male B6 mice, weighing 20-27 g, were obtained
from the Norwegian Research Animal Centre, Na-
tional Institute of Public Health, Oslo (Norway).
They were given a pelleted diet (Norwegian Stan-
dard) and water ad libitum. For induction purpo-
ses phenobarbital 75 mg/kg in saline was given
i.p. 72, 48 and 24 h before isolation of liver cells or
in vivo treatments. For determination of in vivo
macromolecular covalent binding mice were given
1.5-1.8 mCi of [3H]paracetamol, 500 mg/kg, in
0.9% saline i.p. The animals were killed after 4 h.
Preparation of liver subfractions
Livers were homogenized in a Teflon-glass ho-
mogenizer in 2 vol. of sterile, ice-cold 1.15% KC1
containing 20 mM Tris-buffer, pH 7.4, and 10%
glycerol. $9 and microsomal fractions were pre-
pared as described (Dybing and Thorgeirsson,
1977). The $9 fraction was stored as such, whereas
the microsomes were stored in the Tris-KCl buffer
containing 30% glycerol at -70°C until use. Pro-
tein concentrations were determined according to
Lowry et al. (1951) using bovine serum albumin as
standard.
Mutagenicity asssays
Mutagenic activity was assayed with Salmonella
typhimurium TA98, TA100 and TA102. In some
experiments the regular plate assay (Ames et al.,
1975) as described (Dybing and Thorgeirsson,
1977), or a quantitative modification thereof
(Soderlund et al., 1979), were used in the absence
or presence of mouse liver subfractions and cofac-
tors. Freshly dissolved NAPQI in DMSO was
added last in the mutagenicity assays, bacteria
were incubated with NAPQI for 30 min before
plating in the quantitative experiments. In other
experiments substances were tested in cocultures
of S. typhimurium and mouse hepatocytes (Holme
et al., 1983a). Hepatocytes plated on 60-mm dishes
were rinsed, 2 h after plating, with Hanks'-Hepes
buffer, pH 7.4, supplemented with 1% bovine
serum albumin, and then added the same buffer
containing test substance and 0.1 ml of an over-
night culture of S. typhimurium. After 2 h of
co-incubation, the bacteria was collected by
centrifugation and plated to determine the number
of revertants. The genotype of colonies on the
revertant plates were checked according to Ames
et al. (1975). The S. typhimurium strains were
generously provided by Dr. B.N. Ames, University
of California, Berkeley, CA (U.S.A.).](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)