Subcellular Distribution of Some Metallic Cations
in the Early Stages of Liver Carcinogenesis
F. BRESCIANI
ANDF. AURICCHIO
(Institute of General Pathology, University of Naples, Naples, Italy)
SUMMARY
In the early stages of liver Carcinogenesis induced by 4-dimethylaminoazobenzene
the subcellular pattern of calcium, magnesium, sodium, and potassium is sharply
altered. Divalent cations decrease in the nuclear and mitochondrial fractions and in
crease in the residual fraction. In the case of mitochondria,
however, cations decrease
proportionally to the amount of proteins and nucleoproteins in the fraction, and, there
fore, the specific content in calcium and magnesium of mitochondria does not change.
In the nuclear fraction, on the contrary, the quantities of proteins and nucleoproteins
vary only slightly, but the level of divalent cations is strongly reduced and the specific
content of calcium and magnesium in nuclei is sharply altered. Monovalent cations do
not undergo profound alteration in their subcellular distribution but, at a lower degree,
in general follow the pattern of divalent cations.
The mineral content of primary and transplantable hepatomas of the rat shows differences from
that of normal liver (2, 14, 19, 20).
Nothing is known, however, about the subcellu
lar distribution
of minerals in the hepatoma and
preneoplastic
liver except some rough measure
ments of the total content in heavy metals carried
out by Arnold and Sasse (2) using a histochemical
method (sulfide-silver technic).
This paper deals with investigations of the con
tent of some divalent (calcium and magnesium)
and monovalent
(sodium and potassium) cations
in the liver cell fractions, separated by differential
centrifugation, in the early stages of Carcinogenesis.
Liver Carcinogenesis induced by 4-dimethyl
aminoazobenzene
(DAB) was chosen because ex
tensive information about the intracellular
com
position of rat liver after ingestion of azo dyes is
already available, owing to the studies of Price,
the Millers, Potter, and others (21-26).
The present study was undertaken
especially
on the basis of the following considerations :
1. Metals form a variety of complexes with
proteins, but, essentially, three categories can be
recognized (6) : (a) a specific metal takes an in
tegral part in the structure of the protein (metalloproteins) ; (6) the metal ion is bound reversibly but
Received for publication May 21, 1962.
is essential to the stability of configuration of the
molecule or to the association of molecular subunits; (c) the metal ion is readily reversibly bound
and directly affects physical properties of the pro
tein as net charge, aggregation,
solubility, and,
indirectly, configuration and biological functions.
At the present it must be assumed that in the case
of monovalent cations like sodium and potassium
these ions play a role in the ionic atmosphere of
proteins, especially as counter-ions to negatively
charged macromolecules, although it seems that in
part sodium and potassium are also bound in a
more stable way (10, 32). A loss of cations may
therefore affect structure and biological functions
of proteins and nucleoproteins. On the other hand,
primary damages to macromolecules could result
in a release of bound cations, and therefore changes
in the cellular and subcellular concentration
of
ions would be a sign of molecular alterations.
2. Minerals are not uniformly distributed in the
subcellular fractions (10, 16, 28, 32), and selective
modifications
in the intracellular
pattern could
occur with slight changes (or none at all) in the
mineral content of the cell as a whole.
MATERIALS
AND METHODS
BD III (Druckrey [5]) and Wistar male rats
bred in our laboratory
were used. Eighty rats,
1284
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BRESCIANI AND AURICCHIO—Metallic Cations in Carcinogenesis
weighing 150-180 gm. each, were fed a semisynthetic diet (4) containing 2 mg riboflavin/kg.
Forty rats were fed the basal diet, and the remain
ing were fed the basal diet containing 0.5 gm
DAB/kg. Two identical sets of experiments were
carried out 8 months apart. In the first set BD III
rats were used and in the second Wistar rats. On
the average, every rat received a total dose of 170
mg. of DAB at the rate of about 6 mg/day. Four
weeks after the rats were placed on the experi
mental diet, they were killed. The livers were per
fused with 0.25 Msucrose at 4°C. and then forced
through a stainless steel tissue press with holes 1
mm. in diameter. The liver pulp (three to four
livers were pooled) was rapidly weighed and a por
tion removed. The remaining liver pulp was homog
enized in 0.25 M sucrose at 4°C. and fractionated
into nuclei, mitochondria, and supernatant (microsomes + cell fluid) according to Schneider and
Hogeboom (27). Occasionally the nuclei were iso
lated according to the method of Hogeboom et al.
(7), which is recommended for the preparation of
nuclei with small degree of contamination and cytologically closely resembling those seen in the
living cell. No significant difference was found in
the mineral content (calcium was not measured)
of nuclei prepared according to the two different
methods, and therefore the results were combined.
In each cell fraction and in the liver pulp proteins
were determined gravimetrically (22) and DNA
and RNA according to Webb and Levy (33-34).
The mineral content was determined in the fol
lowing way. The liver pulp and the subcellular
fractions were dried at 90°C. for 6 hours and ashed
at 500°C. for 12 hours. The ashes were dissolved
in 10 N HC1, dried under an infrared lamp, and
finally taken up in a measured quantity of 0.1 N
HC1. Sodium and potassium were measured by
flame photometry (Beckman DU) with compari
sons of the solution of ashes with solutions of cat
ions which had a known concentration of electro
lytes similar to that of liver tissue. Calcium and
magnesium were determined as follows: The solu
tion of the sample in 0.1 N HC1 was passed through
a column (7 X 70 mm.) of a purified cation ex
change resin (Dowex 50-X8; 200-400 mesh); and,
after a washing with ion exchange water, the cat
ions were eluted with 6 N HC1. The eluate was
neutralized with NH4OH (methyl red as indicator)
and calcium precipitated as calcium oxalate. Mag
nesium in the supernatant and calcium in the pre
cipitate, after having been dissolved in l N HC1,
were determined by complexometric titration ac
cording to Holasek and Flaschka (8, 9). Prelimi
nary trials showed no intrinsic loss of calcium and
magnesium from the resin under the experimental
1285
conditions used for the purification of the ash solu
tions. The purification of the samples by cation
exchange resin was carried out because complexo
metric titration when applied to the nonpurified
ash extracts is unreliable, according to our experi
ence, because of uncertainty in the color-turning
point of the indicator (Eriochrome T Schwarz).
Extremely precise measurements are possible with
the modified procedure described above.
RESULTS
Calcium and magnesium are firmly bound to
the cell particulate fractions. In fact, preliminary
experiments have shown that the concentration of
the divalent cations in the mitochondrial and nu
clear fractions did not change significantly when
mitochondria and nuclei, prepared according to
the method of Schneider and Hogeboom (27), were
washed 2 additional times with 0.25 M sucrose at
4°C. These results are in agreement with previous
work, and we refer to the paper of Thiers and
Vallee (32) for comparison of data from different
sources.
The levels of DNA, RNA, and protein in the
liver and subcellular fractions from normal and
DAB-fed rats are in agreement with those of Price
et al. (23), and therefore no absolute figures are
reported in this paper.
Table 1 presents the whole cell content (liver
pulp) and the subcellular pattern of calcium and
magnesium in livers of normal and DAB-fed rats.
In normal liver the largest quantity of calcium
was found in the mitochondria, and only a small
portion in the residual fraction. Nuclei, however,
showed the highest specific calcium content per
gm. protein and nucleoprotein. Ingestion of DAB
caused a decrease of the calcium content in the
whole cell and altered the subcellular distribution
of the cation. The decrease of calcium in the whole
liver pulp after ingestion of the carcinogen was
accounted for by a decrease of the nuclear and
mitochondrial calcium while the cation increased
in the residual fraction (Table 1).
In normal liver cells magnesium is distributed
differently from calcium. The largest quantity of
magnesium was found in the residual fraction.
Nuclei contained only a small portion of the mag
nesium of the whole cell, although the concentra
tion of this cation in the nuclear fraction differed
little from that of calcium. The distribution of
magnesium in the cell was closely proportional to
that of proteins and RNA. In fact, magnesium
content expressed per gm. protein and RNA does
not vary much in the different cell fractions,
whereas the specific content of calcium per gm.
protein or nucleoprotein was 20-24 times higher
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1286
Cancer Research
in nuclei than in the residual fraction. DAB in
gestion caused only a slight decrease in the mag
nesium content of the whole liver pulp, but the
subcellular distribution of the cation was sharply
altered. The changes were a decrease of the nuclear
and mitochondrial magnesium while the cation in
creased in the residual fraction.
It should be pointed out that there is a basic
difference in the nature of the decrease of cations
in the mitochondrial and nuclear fractions. In the
case of mitochondria, in fact, the specific content
in calcium and magnesium per gm. protein or
nucleoprotein does not change, although the di
valent cations content of the fraction per gm. fresh
tissue is sharply reduced—i.e., the fraction in foto
is reduced.
In the nuclear fraction, on the contrary, there
Vol. 22, December
1962
is a strong decrease of the specific content of cal
cium and magnesium after DAB ingestion, i.e., the
decrease of calcium and magnesium is due to an
actual release of the divalent cations from nuclei.
During isolation in aqueous media, and despite
repeated washing, a quota of the original content
of sodium and potassium is retained by nuclei and
mitochondria (10, 32). The figures in Table 2,
therefore, should be regarded as measurements of
the monovalent cations "adhering" to nuclei and
mitochondria after the three washings with 0.25 M
sucrose. Hence, sodium and potassium content was
expressed as ^eq/gm fresh tissue or per gm. pro
tein, DNA, and RNA; and no attempt was made
to give figures per gm. water in fraction.
Ingestion of DAB caused an identical per cent
decrease ( —17 per cent) of sodium and potassium
TABLE1
DISTRIBUTION
OFCALCIUM
ANDMAGNESIUM
INLIVERFRACTIONS
OFRATSFEDDAB*
DNA
RNAIN
PROTEIN
TISSUENone3.16±0.0971.11±0.0561.70±0.0910.24±0.0103.0510.10±0.3841.14±0.0662.76±0.0486.00±0.2409.90P„r
FRESH
FRACTIONNone5401,880945771,7251,9301,5351,935DAB
FRACTIONNone1,7205615,490576DAB9951494,320228A%-42-73-21-61fiEO/OM
IN
FRACTIONNone24.8470.1845.003.6879.0071.3070.9088.20DAB20.0020.4445.605.9083.5030.6059.20103.00A%-20-71+
IN
FRACTIONLiver
ercent100365489810011275997DAB2.12±0.1110.34±0.0201.29±0.0630.37±0.0142.009.20±0.4120.52±0.0381.66±0.0516
5-660+95+18-49-12+42
(Mean)(SM)Nuclei
pulpt
(Mean)(SM)Mitochondria
1+60+
(Mean)(S«)Residual
(Mean)fraction}
(Su)RecoveryLiver
9-54-40+
(Mean)(SM)Nuclei
pulpf
6-57-17+17MEQ/GM
(Mean)(S«)Mitochondria
(Mean)(SM)Residual
(Mean)fraction}
(Su)Recovery/¿EQ/GM
SM = standard error of mean.
* 4-Diinethylarainoazobenzene
13JiEQ/GM
80±0
3508.90P~_ercent1001C6118951006187498A%-33-69-24+54-
fed 6 rag. per day and per rat for 4 weeks.
t Liver after being forced through a stainless
removing connective tissue.
t Microsomes and nonparticulate
cell fluid.
steel tissue press with holes 1 mm. in diameter,
which process is effective
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in
BRESCIANIANDAURICCHIO—Metallic
Cations in Carcinogenesis
in the liver pulp. The subcellular fractions also
showed reduction in their content of monovalent
cations between a minimum of —12per cent and
a maximum of —31per cent (Table 2). As for the
divalent cations, sodium and potassium in mito
chondria decrease proportionally to the whole
fraction, whereas in nuclei their specific content is
reduced. Sodium and potassium also decreased in
the residual fraction.
DISCUSSION
It is generally assumed that proteins and nucleic
acids bind divalent cations to their negatively
charged sites. Owing to their large number of phos
phate groups, nucleic acids are particularly suit
able for the binding of divalent cations. Recent
evidence, however, supports the view that adenine
1287
and guanine of nucleic acids form five-membered
chelates with divalent cations (35). Many authors
suggest, moreover, that calcium and magnesium
act as a metallic bridge between DNA and protein
(12, 13). On the other hand, evidence exists that
proteins are bound to nucleic acids by interference
of the negatively charged phosphate groups of
nucleic acids with positive groups of proteins. Free
negative charges (phosphate) on nucleoproteins
are, however, still available (3).
The possibility that ingestion of DAB could pro
duce calcium and magnesium loss from nuclei
through an indirect effect, such as loss of nucleic
acids or proteins and consequently of the bound
divalent cations, seems hardly acceptable. On the
contrary, DNA and proteins are slightly increased
in the nuclear fraction after feeding the carcinogen,
TABLE2
DISTRIBUTION
OFPOTASSIUM
ANDSODIUM
INLIVERFRACTION
OFRATSFEDDAB*
RNAIN
PROTEININ
DNAIN
FRACTIONNone11,1506,7001,50018,300DAB13,1006,0801,65019,100+18FRACTIONNone35,5001,995DAB25,3001,410A%-29-29MEQ/QM
FRACTIONNone51024769835DABK+49018972710A%-
TISSUENone65.30+
FRESH
0.9203.95+
2.3403.22+
FRACTIONLiver
4-24+
(Mean)(S«)Nuclei
pulp t
9+11+
(Mean)(SM)Mitochondria
0.1502.70+
0.1802.01±
0.09056
0.13046.78+
(Mean)(SM)Residual
(Mean)fraction
(SM)RecoveryLiver
ÃŽ
4-15iiEQ/GM
4Na+21.70+
.75+
1.68052.01Percent100648797A%-17-19-26-18/1EQ/GM
3.41063.40Percent100648797DAB54.00±
4-16-
(Mean)(SM)Nuclei
pulp t
0.8401.21+
1.0101.08+
0.0501.32±
0.0700.91±
0.06019.16+
0.08015.79+
,8006118,460474-28-223,7002,0507346,1804,3702,0407466,400+181+
(Mean)(SM)Mitochondria
4-1511
(Mean)(SM)Residual
(Mean)fraction!
(SM)Recovery/1EQ/GM
2+
4
0.84017.78100658899-17-11-31-18170763428216464332390.99021.69100668810018.00+
SM = standard error of mean.
* 4-Dimethylaminoazobenzene
fed 6 mg. per day and per rat for 4 weeks.
t Liver after being forced through a stainless
removing connective tissue.
t Microsomes and nonparticulate
cell fluid.
steel tissue press with holes 1 mm. in diameter,
which process is effective
in
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1288
Cancer Research
and the observed decrease of RNA of approximate
ly 10 per cent could account for only a small per
centage of the total cation loss.
It is, therefore, necessary to conclude that the
nucleic acids and the proteins of liver nuclei of rats
fed the carcinogen actually bind a smaller quan
tity of divalent cations than under normal condi
tions. At the present status of our knowledge a
definite mechanism for the cation release cannot be
given although several reasonable hypotheses can
be presented:
1. The carcinogen molecules could replace the
cations on the negatively charged sites of the nu
clear macromolecules. DAB is in fact a strong posi
tively charged molecule.
2. Metabolic products of DAB could seques
trate the cations by formation of metal chelates.
3. Actual reaction of the metabolic products of
DAB with nucleic acids and proteins could occur.
Miller, Miller, and Hartmann
(18) have found
that one of the most important
metabolites
of
2-acetylaminofluorene
is N-hydroxy-2-acetylaminofluorene. They postulated that either this com
pound or the deacetylated
derivative could react
directly with nucleic acids or proteins. It can be
foreseen that a similar mechanism would act also
in the case of DAB, as well as for many other car
cinogenic amines, amides, and azo dyes (18). As a
consequence of the alterations in the nucleic acids
and protein structure, a net loss of cations could
result.
The decrease of calcium and magnesium in the
mitochondrial
fraction is roughly proportional to
the decrease of protein and RNA of the fraction.
Therefore, a quantitative
reduction of the cell
mitochondria accounts for the cation decrease. The
decrease of liver cell mitochondria in rats fed azo
dyes has already been demonstrated
(1).
The lack of a specific release of cations from
mitochondria in contrast with nuclei could be due
to different environmental
conditions (pH, redox
potential, etc.) in these cell particles or because
the molecules of the carcinogen are metabolized
differently by mitochondria.
Monovalent
cations do not undergo profound
alterations in their subcellular distribution but, at
a lower degree, in general follow the pattern of
divalent cations. In the residual fraction, in con
trast to divalent cations, monovalent cations are
decreased.
Nuclei and mitochondria
isolated in aqueous
media retain only a part of their original content
of monovalent cations (10, 29, 30). It is currently
assumed that the portion of "adhering"
sodium
and potassium plays a part in the ionic atmosphere
of negatively charged sites of macromolecules. An
Vol. 22, December
1962
alteration of nuclear macromolecules which causes
release of divalent cations, therefore, is expected to
produce some loss of monovalent cations as well.
In the case of mitochondria a reduction of the
whole fraction accounts for the cation decrease.
The decrease of monovalent cations in the residual
fraction, finally, seems consistent with a partial
alteration of the cell capacity to retain these ions.
As stated in the Introduction,
calcium and mag
nesium are intimately associated with protein and
nucleic acids, and their release cannot be without
consequences for the physical and chemical prop
erties of the macromolecules and, hence, for their
biological functions. In fact, loss of calcium and
magnesium causes splitting of nucleoproteins
(12,
13) and mutations (15, 17, 31); calcium and mag
nesium protect bacteriophage
from inactivation
(11), and recent evidence suggests that magnesium
has a role in the mechanism of nucleoprotein re
production (35).
ACKNOWLEDGMENTS
The authors wish to express their thanks to Mrs. Tyra
Korling-Bresciani for the English translation and to Dr. Salva
tore Califano, Istituto Chimico, University of Naples, for
helpful discussion.
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Subcellular Distribution of Some Metallic Cations in the Early
Stages of Liver Carcinogenesis
F. Bresciani and F. Auricchio
Cancer Res 1962;22:1284-1289.
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