[CANCER RESEARCH 41, 4039-4049,
0008-5472/81
/0041-OOOOS02.00
October 1981]
Early Events during Liver Carcinogenesis Involving Two Carcinogen:Protein
Complexes1
Gary R. Blackburn, John P. Andrews, R. Philip Custer, and Sam Sorof2
The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia,
Pennsylvania
ABSTRACT
This report describes the relationships between the levels of
two principal carcinogen:protein
complexes and histological
alterations that occur during carcinogenesis in rat liver by three
carcinogens. An intragastric tracer dose of /v-[9-14C]2-fluorenylacetamide interacts in normal rat liver to form the principal
cytosolic 14C-carcinogen:protein complex (2S; M.W. 14,700).
Continued ingestion of any of three hepatocarcinogens causes
the marked loss of the 2S complex and the concurrent gain of
a large-molecular-weight
carcinogen:protein
complex (7.5S;
M.W. ~150,000) 48 hr following a dose of A/-[9-14C]-2-fluorenylacetamide. The reciprocal changes in amounts of the two
complexes have the following properties. The alterations occur
early during hepatocarcinogenesis.
In contrast to other re
ported early events, chemical carcinogen is directly involved.
The two labeled fluorenyl proteins are the principal 14C-carcinogen:protein complexes in rat liver cytosol. Formation of the
two complexes apparently involves activation of the carcinogen
A/-2-fluorenylacetamide.
The reciprocal effects are brought on
by the ingestion of any of three hepatocarcinogens,
the aro
matic amide A/-2-fluorenylacetamide,
the aminoazo dye 3'methyl-4-dimethylaminoazobenzene,
and the amino acid ana
log ethionine. In contrast, neither their noncarcinogenic chem
ical analogs, fluorene and aminoazobenzene, nor the inducers
of the microsomal monooxygenases,
3-methylcholanthrene
and phénobarbital, nor control diets (without carcinogens)
cause these effects. The loss of the 2S complex was previously
demonstrated by this laboratory to be associated with the loss
of the 2S protein itself (14,700-daltons polypeptide) to which
the carcinogen is bound. The rate of inversion in concentrations
of the two complexes is apparently related to the susceptibility
of the strain and sex of rat to the carcinogen; the greater the
susceptibility the more rapid is the rate. The time of the maximal
inversion coincides approximately with the appearance of pu
tative premalignant lesions. Cessation of A/-2-fluorenylacetamide ingestion results in the symmetrical reversal of the recip
rocal changes in the levels of the two complexes. Liver regen
eration following partial hepatectomy brings about a moderate
inversion of the levels of the two complexes, suggestive of the
possibility that the cell proliferation associated with hepatocar
cinogenesis may be causally related to the effects. Consistent
with this possibility, the 2S protein has a molecular weight like
those of known polypeptide growth regulators. The effects on
the 2S and 7.5S carcinogenrprotein complexes are carcinogen
' Supported in part by USPHS Grants CA-05945, CA-21522, CA-06927, and
RR-05539 from the National Cancer Institute and by an appropriation from the
Commonwealth of Pennsylvania. Presented in part at the 72nd Meeting of the
American Association for Cancer Research, Washington, D. C., April 1981 (1).
2 To whom requests for reprints should be addressed, at The Institute for
Cancer Research, Fox Chase Cancer Center. 7701 Burholme Avenue, Philadel
phia, Pa. 19111.
Received May 8, 1981 ; accepted July 9, 1981.
OCTOBER
1981
19111
group specific, inasmuch as the ingestion of any of three other
types of liver carcinogens, namely, diethylnitrosamine,
aflatoxin Bi, and thioacetamide, does not induce the reciprocal
changes in the levels of the two complexes. The existence of
such carcinogen group specificity suggests that chemical
classes of carcinogens may be grouped on the basis of their
common actions and effects on their target macromolecules.
The generalizations to be derived from a classification of car
cinogen group specificities seem likely to advance understand
ing of underlying events in chemical oncogenesis.
INTRODUCTION
The phenotypic changes that have been associated with the
early onset of chemical carcinogenesis have generally borne
no apparent direct relationship to the oncogenic process (re
viewed in Refs. 9 and 10). We recently described an early
event during liver carcinogenesis that is thus far unique in that
it directly involves the actions of 3 types of hepatocarcinogens.
A tracer dose of the chemical carcinogen [9-14C]FAA3 interacts
principally with a specific polypeptide with a molecular size of
14,700 dallons (2S) in normal liver. Short-term ingestion of
various carcinogens causes marked reductions in the amounts
of both the resultant cytosolic carcinogenipolypeptide
complex
and the polypeptide that binds the carcinogen. Three kinds of
liver carcinogens act in this way: the aromatic amide, FAA; the
aminoazo dye, 3'-Me-DAB; and the amino acid analog, ethio
nine (2). This laboratory also previously described another
event that occurs during liver carcinogenesis by FAA. Ingestion
of FAA also brings on a marked increase in the specificity of
carcinogen-protein
interaction, resulting in the presence of a
relatively basic principal species of liver cytosolic carcinogen:
protein complex with a molecular size of about 150,000 daltons
(7.5S) (25, 26). We report here that early during liver carcino
genesis by any of these 3 carcinogens the lowering in the level
of the 14,700-dalton (2S) carcinogen:protein
complex is cou
pled with a concurrent increase in the content of the 7.5S
complex. Further, this inversion in the concentrations of the 2
complexes approximately correlates in time with the appear
ance of putative premalignant hepatic lesions brought about by
these 3 liver carcinogens.
MATERIALS
AND METHODS
Rats Fed Carcinogens. Male and female rats (initially 100 to 130 g)
of the Sprague-Dawley Spd strain (ARS/Sprague-Dawley,
Madison,
Wis.) and of the Fischer 344 CDF strain (Charles River Laboratories,
Inc., Wilmington, Mass.) were fed ad libitum a grain diet (19) without
3 The abbreviations used are: FAA, N-2-fluorenylacetamide
(2-acetylaminofluorene); 3'-Me-DAB, 3'-methyl-4-dimethylaminoazobenzene;
AFBi, aflatoxin Bi;
DEN, diethylnitrosamine; 4-AAB, 4-aminoazobenzene; i.g., intragastrically; MCA,
3-methylcholanthrene;
PB, sodium phénobarbital.
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G. R. Blackburn et al.
(control) or with the liver carcinogen, FAA (Fluka Chemical Co., Buchs,
Switzerland; Aldrich Chemical Co., Milwaukee, Wis.), at a level of
0.03% for up to 30 weeks.
Male Sprague-Dawley Spd rats, initially weighing 100 to 140 g, were
fed up to 6 weeks ad libitum a semisynthetic diet (No. 3 in Ref. 18)
lacking or containing 0.058% of the hepatocarcinogen,
3'-Me-DAB
(Eastman Kodak Co., Rochester, N. Y.).
Female Fischer CDF rats, at the start weighing 100 to 140 g, were
given a semisynthetic diet (28) without or with the liver carcinogen,
0.3% DL-ethionine (Sigma Chemical Co., St. Louis, Mo.), for up to 8
weeks.
Male Fischer CDF rats, weighing 95 to 100 g at the start, were fed
the diet of Wogan and Newberne (33) without and with 1 mg AFB, per
kg diet up to 32 weeks. The diet was prepared and fed as previously
described (17). Dr. Kumar Mainigi, Cornell University, Ithaca, N. Y.,
kindly provided us with initial-trial female CDF rats that had been fed
the same diet without or with 2.5 mg AFB! per kg for 6 weeks.
Male Sprague-Dawley Spd rats, weighing 150 to 200 g, were fed a
commercial stock diet (Wayne Lab Blox; Allied Mills, Inc., Chicago, III.)
and tap water containing 50 mg of the hepatocarcinogen DEN (Eastman
Kodak Co., Rochester, N. Y.) per liter. The daily prepared solution was
available in blackened glass bottles at the rate of 40 ml/rat/day
for 5
days each week, followed by plain tap water during the remaining 2
days, for up to 16 weeks. The protocol was kindly provided by Dr.
William Lijinsky, Frederick Cancer Research Center, Frederick, Md.
Male CDF rats, initially weighing 150 to 200 g, were fed for 25
weeks the synthetic diet of Svoboda and Higginson (29) lacking or
containing 0.032% thioacetamide.
In examination of the reversibility of the effects of the ingestion of
carcinogen, the FAA diet was fed as above to male CDF rats for 2.5
weeks, to female CDF rats for 10 weeks, and to male and female Spd
rats for 30 weeks, followed in each case by the control diet for 4
weeks.
Rats on all diets except those receiving DEN were given acidified
tap water (pH 2.6), in order to control the growth of Pseudomonas
aeruginosa.
Liver Nodules Caused by FAA. Male Sprague-Dawley CFE rats
(Charles River Laboratories, Inc.), weighing 80 g, were subjected to a
protocol of 4 cycles of interrupted feeding, each consisting of 3 weeks
of Ingestion of 0.06% FAA in a synthetic diet (Bio-Serv Corp., Frenchtown, N. J.), followed by its control diet for 1 week (30). Laparotomy
thereafter revealed the presence of several macroscopic nodules in
each liver. The rats bearing the nodules were kindly provided by Dr.
Frederick F. Becker, M. D. Anderson Hospital and Tumor Institute,
Houston, Texas. Following administration of labeled FAA, sacrifice,
and liver perfusions (below), the macroscopic liver nodules and sur
rounding less involved liver were carefully excised and processed as
described below.
Rats Fed Noncarcinogens. In order to evaluate the effects of noncarcinogens that are chemically related to the carcinogens under
study, male CDF rats were fed 0.022% fluorene (equimolar to FAA) in
the same diet as with FAA for 3 weeks (19). Likewise, male Spd rats
were fed 0.045% 4-AAB (equimolar to 3'-Me-DAB) in the same diet as
that with 3'-Me-DAB for 4 weeks (18).
Regenerating Livers. Male Spd rats, maintained on a commercial
stock diet (Wayne Lab Blox), were partially hepatectomized (13), given
the standard tracer amount of [9-'4C]FAA (below) 12 hr later, and
sacrificed 16 hr after the dosing.
Administration of [9-14C]FAA.
Starting on the final day of the
feeding of FAA, fluorene, or their control diet, the rats were given the
control diet for 18 hr, in order to reduce presumed endogenous stores
of FAA and its metabolites. Each rat was then given 10 /iCi of [9-'"C]
FAA per 0.1 ml ethanol per 100 g body weight i.g. (46 to 52 mCi/
mmol; New England Nuclear, Boston, Mass.). Occasional analyses of
the [9-'4C]FAA by thin-layer chromatography in chloroformimethanol
(97:3) on activated silica gel containing fluorescent indicator (Eastman
Kodak Co.) confirmed its 99% radiochemical purity. The rats were then
4040
maintained on the control diet until sacrifice at 48 hr later. The animals
that were fed the other carcinogens or 4-AAB were similarly treated,
except that they were given their respective diets after administration
of the labeled FAA, instead of control diet for 48 hr.
Administration of MCA and PB. Commercial MCA (Eastman Kodak
Co.) was additionally purified by recrystallization from a 10% solution
in hot benzene in near darkness. The product (178-179°) was pure
according
to thin-layer
chromatography
(above) in n-hexane.
A male
CDF rat, weighing 200 g, was fed the stock diet (Wayne Lab Blox) and
acidified tap water ad libitum. On 3 successive days, single doses of
8.0 mg of the MCA in 0.3 ml of corn oil (Mazóla; Best Foods, Englewood
Cliffs, N. J.) were administered i.p. to the rat, followed on the fourth
day by an i.g. dose of 30 fiCi of [9-MC]FAA in 0.3 ml of ethanol. The rat
was fed the same diet until sacrifice 48 hr later.
In another experiment, a male CDF rat, weighing 185 g, was fed the
above stock diet and given PB (crystalline grade; Sigma) at 1.0 mg/ml
in the drinking distilled water ad libitum for 6 days. On the seventh day,
30 /iCi of [9-14C]FAA in 0.3 ml ethanol were given ¡.g.The rat was
maintained on the same diet and water containing PB until sacrifice 48
hr later.
Preparation of Liver Cytosols. Following sacrifice of the rats, all
operations were conducted at 1-4° (24). The livers were immediately
perfused portally with 0.25 M sucrose solution, sampled for histological
analysis, minced, and homogenized in 0.25 M sucrose (1:1, w/v) in a
Potter-Elvehjem homogenizer (A. H. Thomas Co., Philadelphia, Pa.
Size BB glass:Teflon). The homogenates were centrifuged at 100,000
x g for 2 hr, yielding clear, amber liver cytosols containing 25 to 50
mg protein per ml (Lowry assay). The cytosols were adjusted with NaCI
to 0.1 M; brought to pH 7.4 with 0.01 M Tris-chloride buffer, pH 7.5;
and stored at -60° until used.
In Vitro Incubation of Liver Cytosol with [9-'4C]FAA. Six ml of a
fresh clear liver cytosol (54 mg protein per ml) of a normal male Spd
rat (251 g) were mixed with 6.9 /iCi of [9-'"C]FAA (46.16 mCi/mmol)
to a final concentration of 18.6 ¡ÕM.
After being stirred under nitrogen
atmosphere for 2 hr at 1-4°, the sample was applied to a column (30
x 2.5 cm) of Sephadex G-25, previously equilibrated
with 0.01 M Tris-
HCI buffer, pH 7.5, containing 0.1 M NaCI, and eluted with this solution.
The molecular sieving removed the bulk of the unbound labeled carcin
ogen, which would overlap with macromolecules in the subsequent gel
filtration. The excluded macromolecules,
containing 0.24% of the
added label, were pooled, concentrated to 44 mg protein per ml by
Ultrafiltration (UM-10; Amicon Corp., Cambridge, Mass.), and resolved
by molecular sieving (below).
Molecular Sieving of Liver Cytosolic Carcinogen:Protein
Com
plexes. Cytosolic proteins were separated into molecular weight
classes by filtration through a column [197 x 2.5 cm (inside diameter)]
of Sephadex G-200 (Pharmacia Fine Chemicals, Inc., Piscataway, N.
J.) in 0.1 M NaCI and 0.01 M Tris-chloride buffer, pH 7.4 (24). Eluant
fractions (about 5.3 ml) were spectrophotometrically
assayed for pro
tein at 235 nm (24) and for labeled carcinogen:protein
complexes by
ßliquid scintillation spectrometry in Aquasol 2 (New England Nuclear).
Preparation for Histological Study. Samples from the livers, 5 mm
or less thick, were removed after sacrifice, fixed in neutral 10% formol,
embedded in paraffin, sectioned at 5 /im, and routinely stained with
hematoxylin and eosin. Selected sections were stained for glycogen
(periodic acid-Schiff reaction), iron (Prussian blue reaction), connective
tissue (Masson's trichrome), and mucin (mucicarmine). Studies were
limited to light microscopy.
Criteria for Histological Diagnosis. The presumptive premalignant
lesions were evaluated according to a modification of the scheme
suggested by the Workshop on Classification of Specific Hepatocellular
Lesions in Rats (Table 1) (27). The lesions were first grouped as being
initial, intermediate, or neoplastic. These groups were then divided into
specific types. A deviation was noted very early after carcinogen
ingestion, prior to the Workshop-described
focal cellular alterations.
The initial lesion, which we term "primary hepatocellular deviation,"
generally
occurred
in the periportal
parenchyma
CANCER
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of a
VOL. 41
Early Events during Liver Carcinogenesis
of the 7.5S 14C-carcinogen:protein
Table 1
Modified classification
of putative premalignant
hepatocellular
lesions in rats
The classification system derives from that of Squire and Levitt (27). Our
modifications are in italics. The presumptive premalignant lesions are listed in
sequential order of increasing neoplastic progression, with the exception of VII.
Initial lesions
I. Primary hepatocellular
Per/porta/
Diffuse
II. Ductular proliferation
Cuboidal cell
Oval cell"
deviation
Intermediate lesions
III. Hepatocellular dysplasia, diffuse
IV. Foci of cellular alteration
Clear cell foci
Eosinophilic or ground-glass foci
Basophilic foci
Mixed-cell foci
Neoplastic lesions
V. Neoplastic nodules
VI. Hepatocellular carcinoma
Well differentiated
Moderately differentiated
Poorly differentiated
With glandular and/or papillary formation
VII. Cholangiofibrosis
VIII. Cholangiocarcinoma
IX. Hepatocholangiocarcinoma
deeper intensity of hepatocytic staining with hematoxylin and eosin
owing to an increase of cytoplasmic basophilic granules, often accom
panied by a 2-cell width of hepatocytic plates (Fig. 2). Glycogen
content of the cells was usually increased over that in the centrilobular
hepatocytes (Fig. 8), a feature of chemical carcinogenesis reported to
result from decrease in glucose-6-phosphatase
and nucleotide polyphosphatase (8). The primary hepatocellular deviation was also found
to be diffuse in its distribution in the lobule (Fig. 5). In the latter, the
hepatocytic plates were frequently multicellular in width, and the sinus
oidal pattern was distorted (Fig. 3).
Proliferation of biliary ductules was a common initial lesion in the
chemical carcinogenesis
in the liver, although the extent varied with
the agent. The least occurred with ethionine, and the most occurred
with 3'-Me-DAB.
A further change from the Workshop classification was the desig
nation of the intermediate lesion termed "diffuse hepatocellular dyspla
sia" (Fig. 9). In this stage, the liver parenchyma was generally affected,
and much of the lobular pattern was obscured. The affected hepato
cytes were large with polyploid nuclei and conspicuous nucleoli, and
without focal or nodular distribution.
Consideration was given to ductular epithelium as a second source
of neoplasia via "oval cell" proliferation (9). Hepatocellular carcinoma
or Cholangiocarcinoma,
alone or in unison, appeared on occasion to
derive from this pathway. The relationship of Cholangiofibrosis
to
oncogenesis was not clear from the material studied. No distinction
was attempted between hyperplastic or regenerative nodules and
neoplastic nodules (27), in that virtually all were induced by carcino
gens and at least some contained putative precursor cells of carcino
mas.
RESULTS
CarcinogerrProtein
Complexes during Liver Carcinogen
esis by FAA. An i.g. tracer dose of [9-'4C]FAA interacts in
normal rat liver to form a principal cytosolic '"C-carcinogen:
protein complex. This complex has a molecular size of 14,700
daltons and belongs to the 2S class of liver cytosolic macromolecules (2). Continued ingestion of the liver carcinogen FAA
brings about the early loss of this complex and concurrent gain
OCTOBER 1981
complex with a molecular
size of about 150,000 daltons. Table 2 and Chart 1 demon
strate the progressive alterations in the relative concentrations
of the 2 liver cytosolic complexes in rats of both sexes of 2
strains of rats.
Male CDF rats responded most rapidly to the actions of FAA,
achieving in 3 weeks both a lowering in the relative concentra
tion of the 2S complex to one-third that at the start and a 3-fold
gain in the 7.5S complex (Table 2). Charts 14 and 2 illustrate
these changes. We recently reported that the lowered level of
the 2S complex is also associated with the loss of the 14,700dalton polypeptide to which the fluorenyl carcinogen is bound
in the 2S complex (2). Most male CDF rats fed the FAA diet
died at Days 21 to 25.
The histological features of the livers of the male CDF rats
were examined after FAA ingestion, in search of correlations
with the observed changes in the carcinogen:protein
com
plexes. In 6 of 13 rats fed FAA for 1 week, there were minor
phenotypic changes consisting of periportal primary hepato
cellular deviation in 5 livers and early ductular proliferation in
3 livers (Fig 2). The other 7 livers were like those of the 3
controls (Fig. 1). After 2 weeks, there were primary hepatocel
lular deviations that were periportally distributed in one liver
and diffuse in 6 livers. Ductular proliferation of greater degree
appeared in 7 livers, in which 3 were of the ovalocytic type
(Fig. 3). Three of 10 livers remained histologically normal. At 3
weeks, 14 FAA-fed rats invariably displayed marked primary
hepatocellular deviations in which 2 were periportal and 12
were diffuse. Some livers had considerable hepatocellular dys
plasia, and most had plates of multicellular width and loss of
sinusoidal pattern. Ductular proliferation was present in 13
livers; 8 were of ovalocytic type, of which 2 had extensive
penetration of the lobules (Fig. 4). Foci of cellular alteration
were conspicuous in 2 livers, a frank neoplastic nodule was
found in one, and another gave evidence of early Cholangiofi
brosis. Thus, by 3 weeks, all livers of the male CDF rats fed
FAA had putative premalignant initial lesions, and the 8 control
livers were essentially normal. It is noteworthy that this duration
of time coincided with that of the maximal inversion in the
relative contents of the 2S and 7.5 carcinogeniprotein
com
plexes (above). Among the longer survivors, 1 had clear cell
and basophilic foci of cellular alterations at 5 weeks, another
had ductular and oval cell proliferation completely disrupting
lobules, and 2 were richly studded with neoplastic nodules
heavily laden with glycogen. In the 2 rats fed FAA for 10 weeks,
the entire liver parenchyma was replaced by cells rich in
glycogen in ill-defined nodules without lobular pattern. The 2
that lived for 36 weeks had developed multinodular well-differ
entiated hepatocarcinomas.
In female CDF rats fed FAA, most of the loss in relative
content of the 2S complex and the gain in that of the 7.5S
complex in the liver cytosols occurred during the first 3 weeks
of FAA feeding (Table 2; Chart 1B). With further ingestion of
this hepatocarcinogen, there was a decrease in the amounts of
both adducts up to 30 weeks.
The histological changes in the livers of the female CDF rats
fed FAA occurred more slowly than in the corresponding males.
At 3 weeks, all livers of the females showed the initial lesion,
periportal primary hepatocellular deviation. It is noteworthy
that, at this same duration of FAA feeding, the females had
undergone most of the inversion in the contents of the 2S and
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G. R. Blackburn et al.
Table 2
Relative content ofcarcinogen:protein
TreatmentFAAFluoreneControl
complexes in rat liver cytosols
(%)2S24(16-35)21127
of
analyses331142"1111121111112e21112e11241222222226e4"3224322422344234121"C-carcin
(wk)01230123103001235101330012361030333030302.5
fed
(8-17)282639(31-41)14(13,
(4-8)34(27,
41)22171512926(25,
16)242934342322(17,27)3432373620212825
27)191917121181425(20,31)2420222313824(21,
26)24242533462612(10,
(25,
27)28
14)11
(24-30)3425
(10-13)1615(14,
28)29
(22,
30)33
(28,
16)18(13,24)14(11,
4control1
FAA +
39)26
(28,
16)15(10,
4control30
0 FAA +
29)16(13,
(23,
20)16(12.
4control30
FAA +
18)34
19)21
4control6.5
FAA +
38)28
(31 ,
24)19
(18,
pro-tocol-FAA)Liver
(reversal
control1
4control4
29)34
(28,
34)410
(34,
11)16(14,
(9.
17)820(18,
(FAA)Surrounding
nodules
cycles4
(FAA)Reversai
protocol(FAA)Control
liver(FAA)3'-Me-DAB4-AABControl
(3'-Me-DAB)EthionineControl
(ethionine)DENControl
cycles01246414e01.6481.6488160No.
13)15(10-19)20(16,
(8,
21)24
23)15(14,
15)9
(9-11)9
(8-10)20
(20-28)22(18,
27)26(21,
31)40
(38-43)31
(27-34)13(11,
20)28
(20,
15)18(15,20)13(10-16)22(21,24)16(14,
30)29
(25,
(24-33)24
25)30
(23,
33)11
(28,
(7-18)3
(0.5-4)3
(3-4)36
17)49
(48-50)59
(48-72)49(38-61)13(12,
38)45(44-46)40
(34,
14)15(11-18)13(11-16)2318(17,
(36-47)1924
25)257.5S12
(23.
18)27
(DEN)StrainCDFSpdCDFCDFSpdCDFSpdCDFCFECFESpdSpdSpdCDFCDFSpdSpdSexMFMFMMFMFMFMFMFMMMMMFFMMTime
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VOL. 41
Early Events during Liver Carcinogenesis
Table 2—Continued
Strain
Treatment
Sex
Time fed (wk)
No. of
analyses3
4C-carcinogen
(%)
2S
7.5S
AFB,Control
30)20(19-23)37
(25.
17)15(14-16)12(11,
38)3339
(37,
12)1411
30)15(12-21)45
(29,
11)18(12-22)8
(11,
46)34
(44,
9)14 (8,
35)34
(32,
15)21
(14,
(AFB,)ThioacetamideControl
(thioaceta-mide)MCAPBPartial
23)16132612
(19,
38)342618271316(16,
(30,
days6
days28
hr28
hepatectomySham-operatedCDFCDFCDFCDFCDFCDFSpdSpdMFMFMMMMMM1016326101632625253
hr123212322211321928
Each analysis was of one rat, except where indicated.
One analysis was of the pooled cytosols of 2 rats. The other analysis was of one rat.
Single analysis was from the pooled cytosols of the indicated number of rats.
Duplicate analyses, each of 2 rats.
1 FAA, CDF ?
DIET, weeks
Chart 1. Reciprocal changes in the relative contents of the liver cytosolic 2S
and 7.5S ['"C]fluorenyl carcinogen:protein complexes during periods of ingestion
of 3 carcinogens by rats of different strains and sexes. Rats were fed the
indicated carcinogen, then given a tracer dose of [9-'"C]FAA, and sacrificed 48
hr later. The relative contents of the carcinogen:protein
complexes were deter
mined after gel filtration of the liver cytosols. Arrows, start of the feeding of diet
without carcinogen (control) and the beginning of the reversals of the observed
reciprocal changes.
7.5S carcinogerrprotein
complexes (above). At 5, 6, and 7
weeks, the primary hepatocellular deviation was diffuse; at 10
weeks, it appeared in 8 of 10 rats in association with ovalocytic
ductular proliferation in 5 rats, marked hepatocytic dysplasia
in one rat, focal cellular alteration in 3 rats, and neoplastic
nocules in 8 rats. One liver at 30 weeks and 3 livers at 34
weeks contained moderately differentiated hepatocellular car
OCTOBER 1981
cinoma. The susceptibility of both sexes of CDF rats to the
hepatocarcinogenic
actions of FAA has been reported previ
ously (31, 32).
Feeding FAA to Spd rats caused similar but slower changes
in the amounts of the 2S and 7.5S complexes. Most of the
effects occurred during the initial 5 weeks in the males and
during the first 10 weeks in the females (Table 2; Chart 1, C
and D). The results were irregular, possibly a consequence of
the single assays. However, at these 2 durations of FAA inges
tion, histological lesions had just begun to appear (below). The
slower rate of the reciprocal changes in the Spd rats than in
the CDF rats is consistent with the greater susceptibility of the
CDF rats to the hepatocarcinogenic
actions of FAA (16).
The first histological lesions in the livers of the Spd males
were seen at 5 weeks feeding of FAA as the intermediate
lesion; they were large foci of clear cell alteration. By 10
weeks, ductal proliferation, diffuse hepatocytic dysplasia, and
neoplastic nodulation had developed. Thereafter, up to 30
weeks, hepatocellular carcinoma was found in 5 rats, in one
case coupled with cholangiocarcinoma.
Among the Spd female
rats, less than 10 weeks of FAA ingestion caused no evident
histological alteration. However, at 10 weeks, 3 livers showed
periportal primary hepatocellular deviation, 4 livers contained
neoplastic nodules, and 4 livers had no detectable lesion. One
rat each at 13 and 20 weeks had similar nodules, and 4 rats at
25 weeks and beyond had well to moderately differentiated
hepatocytic carcinoma. Thus, the times of the maximum inver
sion of the levels of the 2S and 7.5S carcinogeniprotein
com
plexes, namely, at 5 weeks in the males and 10 weeks in the
females, were generally the times of the initial histological
lesions.
In contrast to the marked inversions in the amounts of the
2S and 7.5S carcinogen:protein
complexes brought about by
the hepatocarcinogen FAA, feeding the related noncarcinogen,
fluorene, at an equimolar concentration for 3 weeks had vir
tually no effect in male CDF rats (Table 2). Also, the corre4043
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G. R. Blackburn et al.
300
400
500
600
700
800
900
VOLUME, ml
Chart 2. Loss of the 2S and increase of the 7.5S [MC]fluorenyl carcinogen:protein
complexes during liver carcinogenesis
by FAA. Male Fischer 344 rats were fed
a grain diet with FAA (top) or without carcinogen [control (bottom)] for 3 weeks. The liver cytosolic macromolecules of individual rats were resolved into molecular size
classes by extensive gel filtration through columns (dimensions indicated) of Sephadex G-200. The concentration of bound [MC]fluorenyl derivative is greater in the
control profile than in that from the carcinogen-fed rat (see ordinate). Shaded areas, 2S and 7.5S carcinogen: protein complexes. Details are provided in the text.
These profiles were reported in another form (2).
sponding control diet at 3 weeks in CDF males and at 30 weeks
in CDF females induced little, if any, significant difference in
the levels of the complexes from those present at 0 week
(Table 2; Chart 2). Lastly, the control livers were histologically
normal.
Reversal of FAA-induced Alterations in Carcinogen:Protein
Complexes. Cessation of FAA ingestion brought on the rever
sal of the inversions in the levels of the 2S and 7.5S fluorenyl
carcinogen:protein complexes. A regimen of control diet for 4
weeks was sufficient to undo the effects of the prior ingestion
of FAA for 2.5 weeks in male CDF rats, 10 weeks in female
CDF rats, and 30 weeks in male and female Spd rats (Table 2).
The reciprocal nature of the reversals is illustrated in Chart 1,
A to D. However, these reversals were not accompanied by
complete regression of the putative premalignant histological
lesions in the livers of examined rats. Four CDF male rats fed
FAA diet for 2.5 weeks followed by control diet for 4 weeks
retained the initial phenotypic changes observed prior to the
withdrawal of carcinogen, i.e., primary hepatocytic deviation
and early bile duct proliferation. In contrast, the CDF females
in which 8 of 10 had neoplastic nodules at 10 weeks in the
previous series (above) now showed none, but the lesions
present matched those of the CDF males. Spd males fed FAA
for 30 weeks followed by control diet for 4 weeks again had
neoplastic nodules and carcinomas, as noted with the uninter
rupted feeding (above), while the Spd females showed merely
4044
the primary hepatocytic deviation. The conclusion, therefore,
seems to be justified that the inversions in the levels of the 2S
and 7.5S complexes, at least as elicited by FAA, require the
continued ingestion of carcinogen. However, at least some of
the accompanying histological alterations in the livers do not
require continued exposure, in that they remain after 4 weeks
withdrawal of the carcinogen.
Carcinogen:Protein Complexes in Neoplastic Liver Nod
ules Caused by FAA. Four cycles of interrupted FAA ingestion
yielded rat livers bearing macroscopic nodules and less deviant
surrounding livers. The nodules in general contained only low
levels of all cytosolic carcinogen:protein complexes of different
molecular sizes, compared to complexes in the rats continually
fed the carcinogens for the shorter durations listed in Table 2.
This lowering of the levels of all cytosolic complexes was also
progressively evident with feeding of FAA for extended unin
terrupted periods, especially at 30 weeks. The liver nodules
have a reduced capacity to activate the procarcinogen (11), a
process that appears to be necessary for the formation of the
various complexes (see below). The nodules thus appear to
represent a state that is beyond those of livers following the
short-term ingestion of carcinogen. In accord, the nodules had
only low levels of both the 2S and 7.5S complexes (Table 2).
On the other hand, the surrounding livers possessed low levels
of 2S complex and moderately high concentrations of the 7.5S
complex. The findings are in agreement with the histological
CANCER
RESEARCH
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Early Events during Liver Carcinogenesis
determinations that the macroscopically
selected neoplastia
liver nodules contained relatively large regions of early hepatocarcinomas. The surrounding liver tissue presented every
variant of lesion from diffuse hepatocellular alteration and
dysplasia through neoplastia nodulation to hepatocellular car
cinoma; ductular activity in some specimens was striking and
suggestive of cholangiocarcinoma.
This assortment of altera
tions accounted for the observed intermediate level of inver
sions in the concentrations of the 2 complexes.
Lack of Carcinogen Binding to 2S and 7.5S Proteins in
Vitro. The need for the metabolism of carcinogen in the pro
duction of the 2S and 7.5S 14C-carcinogen:protein complexes
was next examined. Cold incubation of [9-""C]FAA with fresh,
clear liver cytosol in vitro failed to produce any detectable
amount of these complexes. Virtually all of the labeled bound
carcinogen was instead associated with the class of the 4S
macromolecules. The indication is that the formation of the 2S
and 7.5S complexes requires that the carcinogen be metabolically driven, i.e., presumably activated and directed to the
specific target proteins.
Carcinogerv.Protein
Complexes during Liver Carcinogen
esis by 3'-Me-DAB. Feeding the hepatocarcinogen, 3'-MeDAB, to male Spd rats caused early marked reciprocal changes
in the levels of the 2S and 7.5S [14C]fluorenyl carcinogen:
protein complexes. As shown in Table 2 and Chart 1E, there
was a symmetrical inversion in the levels of the 2 complexes,
so that by the fourth week of azocarcinogen ingestion the
concentration of the 2S complex had approximately halved and
that of the 7.5S complex had approximately doubled. We
recently reported a molecular size profile of the liver cytosolic
fluorenyl carcinogen:protein
complexes in a male Spd rat fed
the 3'-Me-DAB diet for 4 weeks (Chart 2 in Ref. 2). Evidently,
there was a decreased level of 2S complex and an elevated
concentration of the 7.5S complex. Furthermore, the loss of
the 2S complex was associated with the simultaneous loss of
the 14,700-dalton polypeptide to which the fluorenyl carcino
gen is bound in the 2S complex (2). This aminoazo dye has
previously been found to be hepatocarcinogenic in both sexes
of Spd rats (3, 23, 31).
Histological examination revealed that, after the first week of
ingestion of 3'-Me-DAB, 6 of 9 livers showed the initial lesion,
periportal and diffuse primary hepatocellular deviation, along
with early ductular sprouting in 4 livers (Fig. 5). By 2 weeks, 7
of 8 livers showed diffuse hepatocellular deviation, consider
able dysplasia, and in one, a neoplastic nodule. Ductular pro
liferation of the ovalocytic type was becoming active (Fig. 6).
After 4 weeks of feeding, 5 livers showed striking oval cell
proliferation and neoplastic nodulation (Fig. 7), and 4 livers
contained foci of cholangiofibrosis.
By the sixth week, 4 of 5
rats had developed well-differentiated
hepatocellular carci
noma, while the other had neoplastic nodulation. Thus, the
times of appearance of the initial and intermediate histological
alterations (primary hepatocellular deviation, dysplasia, and
ductular proliferation) overlapped with the inversion in the
contents of the 2S and 7.5S fluorenyl carcinogen:protein com
plexes observed at 4 weeks. The time of maximum oval cell
proliferation and neoplastic nodulation (4 weeks) coincided
with the time of maximal changes in the levels of the 2 complexes.
In contrast to the hepatocarcinogen, 3'-Me-DAB, the related
noncarcinogenic aminoazo dye, 4-AAB, caused no inversion in
the levels of the 2S and 7.5S complexes (Table 2). Livers from
OCTOBER 1981
the rats fed 4-AAB displayed no lesions. In addition, ingestion
of the control diet for 4 weeks had little if any effect on the
contents of the 2 complexes (Table 2), a point also evident in
our recently reported molecular size profile of the liver cytosolic
fluorenyl carcinogen:protein
complexes in a control male Spd
rat (Ref. 2, Chart 2). Finally, the control livers were histologically normal.
Carcinogen:Protein Complexes during Liver Carcinogen
esis by Ethionine. As with FAA and 3'-Me-DAB, ingestion of
the hepatocarcinogenic
amino acid analog, ethionine, by fe
male CDF rats brought on early and marked inversions in the
concentrations of the 2S and 7.5S [MC]fluorenyl carcinogen:
protein complexes. Most of the changes had occurred at 1.6
weeks. However, these changes were maximal at the fourth
week (Chart 1F), when the concentration of the 2S complex in
liver cytosol had dropped to about one-tenth of that present at
the start of the regimen and the level of the 7.5S complex had
risen approximately 4-fold (Table 2). The level of the 2S com
plex fell to 3% of the total labeled complexes in cytosol, while
that of the 7.5S complex rose to 59%. Of all the carcinogens
studied in the different strains and sexes of rats, the effects of
ethionine in the female CDF rats were the most extreme. This
is also supported by our recently reported molecular size
profile of the liver cytosolic fluorenyl carcinogenrprotein
com
plexes in such a female CDF rat fed ethionine for 4 weeks (Ref.
2, Chart 3). Only a barely detectable amount of 2S complex
was present, and the 7.5S complex was virtually the only
[14C]fluorenyl carcinogen:protein
species in the liver cytosol.
The loss of the 2S complex was associated with the concurrent
loss of the 14,700-dalton polypeptides to which the [14C]fluorenyl carcinogen is bound in the 2S complex (2). Further,
most of the changes in the levels of the 2 complexes occurred
by the 1.6th week in the present study, at approximately the
time of the first apparent premalignant lesions in the livers of
such rats (below). The hepatocarcinogenicity
of ethionine in
female CDF rats is well known (6, 7, 31).
Histological examination of the livers of the female CDF rats
fed ethionine revealed the early presence of cellular alterations.
Two rats fed the carcinogen for 2 days showed periportal
primary hepatocellular deviation. After 1.6 weeks, 5 of 7 livers
had diffuse extension of this initial deviation and also marked
dysplasia. In the other 2 livers, the deviation was still periportal
with glycogen retention (Fig. 8). At 4 weeks, the lobules were
diffusely involved in each of 4 rats (Fig. 9). At 8 weeks, there
was ductular proliferation in all 7 livers (Fig. 10) and early
neoplastic nodules in 2 of these. Thus, at 1.6 weeks, when
most of the inversion in levels of 2S and 7.5S had occurred,
most of the livers displayed diffuse primary hepatocellular
deviation and marked dysplasia.
In contrast to the ethionine-fed rats, matched rats fed the
control diet exhibited no significant change in the levels of the
2 complexes (Table 2). The point is also attested to by the
recently demonstrated molecular size distribution of the liver
cytosolic complexes of rats fed the control diet for 4 weeks
(Ref. 2, Chart 3). The control livers were histologically normal.
Carcinogen:Protein
Complexes during Hepatocarcinogenesis by DEN, AFB,, and Thioacetamide. In marked con
trast to the 3 hepatocarcinogens,
FAA, 3'-Me-DAB, and ethio
nine, the 3 liver carcinogens, DEN, AFB,, and thioacetamide,
did not bring about the inversions in the concentrations of the
2S and 7.5S ['"C]fluorenyl carcinogen:protein complexes. The
4045
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G. R. Blackburn et al.
levels of these 2 adducts were essentially similar to their
concentrations
in corresponding
control liver cytosols in
matched experiments (Table 2). This lack of change was ob
served over the periods of 16 weeks of DEN administration, 32
weeks of AFB, feeding, and 25 weeks of thioacetamide inges
tion. These treatments were in each case sufficient to induce
putative premalignant hepatic lesions. The indication is that the
induction of the reciprocal changes in the levels of the 2S and
7.5S carcinogen:protein
complexes in liver cytosols is char
acteristic of a group of carcinogens and not of others.
The histological effects of ingestion of these 3 carcinogens
were examined in a few animals. At 8 weeks of administration
of DEN to male Spd rats, 2 of 2 livers displayed periportal
primary hepatocellular deviation. At 16 weeks, 2 of 2 livers had
the same lesion, and one also had neoplastic nodules. After 16
weeks of AFBt feeding, 4 of 4 male CDF rats showed periportal
primary hepatocellular deviation. At 32 weeks, 4 of 4 livers of
such rats had neoplastic nodules. At 4 weeks of thioacetamide
ingestion by male CDF rats, 2 of 3 rats had minimal centrilobular
necrosis of hepatocytes. At 25 weeks, there was centrilobular
dysplasia and extensive cholangiofibrosis in 2 of 2 livers. Thus,
the indication appears to be that the production of putative
premalignant liver lesions by DEN, AFBi, and thioacetamide is
not accompanied by a lowering in content of 2S complex and
an elevation in level of 7.5S complex.
CarcinogenrProtein
Complexes after Administration
of
MCA and PB. Repeated administrations of MCA and PB to
male CDF rats at doses sufficient to elevate the levels of
microsomal drug-metabolizing enzymes (5) did not evoke the
reciprocal changes in the concentrations of the 2S and 7.5S
complexes (Table 2). It therefore appears that the depression
in the level of the 2S complex and the elevation in the 7.5S
complex are not associated with the induction of those systems.
Carcinogen:Protein
Complexes in Regenerating Liver. In
an effort to ascertain whether the reciprocal changes in the 2S
and 7.5S complexes result from an increased rate of normal
growth of liver cells, rats were partially hepatectomized, and
their ability to form the [14C]fluorenyl protein complexes was
determined. At 28 hr following partial hepatectomy, the regen
erating livers exhibited moderate reciprocal changes in the
levels of the 2 complexes, when compared to livers of shamoperated controls (Table 2). See also Ref. 2. The moderate
nature of the modulations suggests the possibility that an
increased rate of growth may in part be causally involved in
the reciprocal changes, possibly in conjunction with additional
factors as well.
DISCUSSION
The loss of the 2S carcinogen:protein complex and the gain
in the 7.5S carcinogen:protein complex in liver cytosol during
hepatocarcinogenesis
are unique in several ways. Chemical
carcinogens are directly involved (formation of carcinogen:
protein complexes), in contrast to most other reported associ
ations with carcinogenesis. This inversion in concentration
involves 2 principal species of carcinogen:protein
adducts in
the cytosol of the organ undergoing oncogenesis. The loss of
the 2S complex was recently demonstrated to be accompanied
by the concurrent loss of the protein (14,700-dalton polypeptide) to which the carcinogen is bound (2). Three hepatocarcinogens of diverse types act in this manner, namely, the aromatic
4046
amide FAA, the aminoazo dye 3'-Me-DAB,
and the amino acid
analog ethionine. The reciprocal alterations in the levels of the
2 complexes occur early during the hepatocarcinogenesis
by
the 3 kinds of carcinogens. In contrast, their noncarcinogenic
chemical analogs, fluorene, and 4-AAB, induce no such
change. The rate at which the effects occur is apparently
related to the susceptibility of the strain and sex of rat to the
carcinogen; the more the susceptibility, the greater is the rate.
The maximal inversion in the levels of the 2 complexes approx
imately coincides in time with the appearance of initial and
intermediate premalignant lesions in the livers of the rats fed
these carcinogens. Finally, the inversion is reversed by the
cessation of carcinogen administration, although some of the
histopathological aberrations are not.
The levels of the 2S and 7.5S carcinogen: protein complexes
appear to be linked. Carcinogen brings about the loss of the
2S complex and the concurrent gain of the 7.5S complex, and
then the withdrawal of carcinogen causes the symmetrical
reversal of this inversion. The 2 complexes may reside in the
same liver cells. It seems unlikely that changes in the level of
one complex in one cell would bring on a linked response of
another complex in another cell. Alternatively, the inversion
may in part be a reflection of an alteration in linked cell
populations brought on by carcinogen. The relationship be
tween the 2 complexes may not be a direct molecular one,
such as that represented by a protein precursor and its product.
This indication is supported by our preliminary finding that
purified 2S complex and purified 7.5S complex do not immunologically cross-react." The tentative judgment is therefore
that the linkage between the 2S and the 7.5S complexes may
be mediated by related cellular responses to the interactions
of the 2 target proteins with the 3 kinds of chemical carcinogens.
A process related to hepatocarcinogenesis
appears to be
involved in the inversion of the levels of the 2S and 7.5S
carcinogen:protein
complexes in liver cytosol. Three chemi
cally different types of carcinogens, an aromatic amide, an
aminoazo dye, and an amino acid analog, all bring on this same
effect. In contrast, neither 2 noncarcinogenic chemical analogs
of the carcinogens nor control diets (no carcinogens) do so.
Also ineffective are PB and MCA, 2 potent inducers of the
microsomal monooxygenases that metabolize many carcino
gens. Thus far, the only positive indication concerning the
basis of the effect is the induction of a partial inversion of the
levels of the 2 complexes during liver regeneration following
partial hepatectomy. Additional study is required to determine
whether cell proliferation, such as that attending both liver
regeneration and chemical hepatocarcinogenesis,
is causally
related to the inversion in the content of the 2 complexes. It is
intriguing to speculate that the demonstrated losses of both the
2S carcinogen:protein
complex and its polypeptide moiety
(14,700-dalton polypeptide) during liver carcinogenesis by the
3 carcinogens may pertain to the inactivation and loss of a
growth regulator by carcinogen. Polypeptide growth factors of
the size of the 2S carcinogen:protein
complex are known (4,
12, 22).
The identities of the 2 target proteins from which the 2S and
7.5S carcinogen:protein complexes derive are unknown. Ketterer eÃ-al. (14, 15) have reported on the existence in liver
cytosol of a 14,000-dalton "A protein" that forms a minor
' G. R. Blackburn and S. Sorof. unpublished observations.
CANCER
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VOL. 41
Early Events during Liver Carcinogenesis
azoprotein following azocarcinogen ingestion (14,15). Ketterer
has claimed the A protein to be the same as the fatty acidbinding protein of Mishkin ef al. (20) and Ockner ef al. (21).
We are reluctant at present to relate either protein to the 2S
protein [14,700-dalton polypeptide (2)], in part because of the
existence of at least 6 isoelectric protein species of this molec
ular size in rat liver cytosol.4
The formation of the 2S and 7.5S carcinogenrprotein
com
plexes appears to involve the activation of the carcinogen FAA.
Cold incubation of [9-14C]FAA with fresh liver cytosol in vitro
failed to produce these complexes. Further, the levels of both
complexes decreased with extended ingestion of FAA, con
sistent with the progressive lowering in ability to activate this
carcinogen (11 ). By this same criterion as well as by histopathology, the liver nodules of rats fed FAA discontinuously were
in a relatively late stage of chemical carcinogenesis, inasmuch
as they contained low levels of both complexes. The histologically less aberrant liver surrounding the macroscopic nodules
had more of both adducts and displayed low levels of 2S
complex and high levels of the 7.5S complex. Lastly, that
carcinogen is covalently bound to their target proteins is indi
cated by its nonextractability from the purified complexes using
a series of organic solvents of different polarities."
The effects of chemical carcinogens on the levels of the 2S
and 7.5S carcinogen:protein
complexes are carcinogen group
specific. Three kinds of liver carcinogens of one group, i.e.,
FAA, 3'-Me-DAB and ethionine, cause marked inversions in
the content of the 2 complexes, while 3 other types of liver
carcinogens of another group, i.e., DEN, AFBi, and thioacetamide, do not. There is no known basis for this difference in
response. The existence of this carcinogen group specificity
suggests that the different chemical classes of carcinogens
can be grouped on the basis of their common actions and
effects on their target macromolecules. The great chemical
diversity of the known chemical carcinogens makes it likely
that no universal common entry into the process of chemical
carcinogenesis may exist. Different direct-acting and activated
carcinogens, depending on their particular chemical affinities,
may react with certain macromolecules more so than with
others, thereby initiating and/or promoting oncogenesis at
different biochemical levels in the cell. The generalizations to
be derived from the carcinogen group-specific common actions
on cellular macromolecules seem likely to advance understand
ing of underlying events in chemical oncogenesis.
ACKNOWLEDGMENTS
We are grateful to Dr. Frederick F. Becker, M. D. Anderson Hospital and
Tumor Institute, for rats bearing liver nodules; Dr. Zbynek Brada. Papanicolaou
Cancer Institute, for trial ethionine-fed rats; Dr. Kumar Mainigi, Cornell University,
for trial rats fed AFB,; and Dr. William Lijinsky, Frederick Cancer Research
Center, for the protocol of administration of DEN to rats. We acknowledge the
dedicated assistance of Susan Schnabel, J. Mark Danley, Ruth Bender, and
Grace Kroetz.
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Fig. 1. Perfused liver of a female CDF rat fed the control grain diet for 11 days. Lobules are undisturbed, and sinusoids are separated by hepatocytic plates of
single-cell width.
Fig. 2. A, liver of a male CDF rat fed FAA diet for 1 week. Periportal primary hepatocellular deviation, an initial sign of putative premalignant change, is indicated
by polychromasia of cells and blurred plates surrounding portal triads. In fl, the plates are multicellular in width, and the cytoplasm is granular. There is beginning
proliferation of biliary ductules.
Fig. 3. A, liver of a male CDF rat fed FAA diet for 2 weeks. The primary hepatocellular deviation has spread throughout the lobules. Plates are no longer
discernible. Ductal and ductular proliferation is extensive. B, a predominance of "oval cells."
Fig. 4. A, liver of a male CDF rat fed FAA diet for 3 weeks. The ductular proliferation has completely disrupted the hepatocellular parenchyma, persistent areas
being present in the lower portion and extending up through the center. B. a junction between oval cells and hepatocytes.
Fig. 5. A, liver of a male Spd rat fed 3'-Me-DAB diet for 1 week. Diffuse primary hepatocellular deviation has effaced the lobular pattern, and bile ductal
proliferation is conspicuous. B, cellular deviation.
Fig. 6. Liver of a male Spd rat fed 3'-Me-DAB for 2 weeks. Ductular proliferation
a small island.
Fig. 7. Liver of a male Spd rat fed 3'-Me-DAB for 4 weeks. Background
has involved the parenchyma extensively, islands of hepatocytes
persisting. B,
parenchyma is occupied largely by ductules and oval cells supporting neoplastic nodules,
the margin of one such being shown in B.
Fig. 8. A, liver of a female CDF rat fed ethionine for 1.6 weeks. Periportal primary hepatocellular deviation is evident. Cells and plates are indistinct. Hepatocytes
also display considerable dysplasia, best seen in B and also present in the lobular periphery. There is virtually no ductular reaction.
Fig. 9. A, liver of a female CDF rat fed ethionine diet tor 4 weeks. The parenchyma shows diffuse hepatocellular dysplasia. B, variations in cellular size, shape, and
staining quality and presence of large nucleoli. No ductular proliferation is evident. Mottling is due to patchy glycogen retention.
Fig. 10. A. liver of a female CDF rat fed ethionine for 8 weeks Hepatocellular dysplasia and marked glycogen retention are conspicuous, the latter best seen in
B. A modest gradient of ductular proliferation is now evident.
4048
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Early Events during Liver Carcinogenesis Involving Two
Carcinogen:Protein Complexes
Gary R. Blackburn, John P. Andrews, R. Philip Custer, et al.
Cancer Res 1981;41:4039-4049.
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