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V O L . 24 □ N U M E R O
Acta d e n t. Venezolana 24: 53-57, 1973 effects, such as induction of aplastic
anemia and occassionally leukae­
mia through bone marrow depres­
Partial Inhibition of Microsomal sion in human subjects, chromosomal
alterations in cultures of white blood
cells interference with cell differen­
Protein Synthesis in Rat Liver tiation in planarians
immune responses
suppression of
inhibition of

by Chloramphenicol carcinogenesis in rat liver by amino

azo-dies alleviation of Parkin­
son’s disease etc. It is possible that
O scar V a l b u e n a some of these effects are caused by a
E sth er P eña respiratory impairment subsequent to
N é s t o r F. G o n zález- C a dav id its action on mitochondrial biogenesis,
Universidad C entral de Venezuela or even to direct inhibition of the ac­
Facultad de Ciencias - Escuela de Biología tivity of respiratory enzymes in
A partado 10098 sensitive cells such as those of the bone
Caracas, Venezuela * marrow. However an alternative hypo­
thesis has been proposed, namely a
S U M M A R Y
consequence of a direct partial in hibi­
When incubating rat liver slices with increasing- concentrations of chlo­ tion by CAP of translation on 80-S
ramphenicol, a 25% inhibition of microsomal protein synthesis was observed, ribosomes associated with newly for­
whereas the effect was negligible in the case of five day old rats. However, med m R N A Weisberger claimed
protein synthesis by isolated mitochondria was inhibited similarly in both that CAP can act in this way, showing
cases, and the same occurred with mitochondrial endogenous synthesis in situ,
in the liver slices. that interferes with the binding of exo­
genous m R N A (synthetic polynucleo­
Although the antibiotic does not affect directly the microsomal activity,
when this fraction was obtained from slices of adult liver previously incu­ tides and natural template) to reticulo­
bated with chloramphenicol, a 20% inhibition of protein synthesis was ob­ cyte ribosomes
served, particularly in the medium and high molecular weight fractions (acry- In the present paper we demonstrate
lamide gel electrophoresis). The partial inhibition was observed in vivo in the
membrane bound polysomes. These effects might reflect the reduction of the that CAP when acting on the intact
synthesis of mitochondrial proteins outside the organelle, although a more liver cell, either in vivo or in vitro,
direct interference with the function of cytoplasmic ribosomes can not be reduces the protein synthesizing ability
discarded. of extramitochondrial ribosomes, sug­
gesting a coordination with the in hib i­
IN H IB IC IO N P A R C IA L D E LA S IN T E S IS D E P R O T E IN A M IC RO SO M A L tion of mitochondrial protein synthesis.
E N H IG A D O D E RAT A PO R C L O R A N F E N IC O L

R E S U M E N
M A T E R IA L S AND M E T H O D S
Incubando cortes de hígado de rata adulta con concentraciones crecientes
de cloranfenicol, se observó una inhibición de un 25% en la síntesis proteica Chemicals. The sodium succinate
microsomal, mientras que con ratas de 5 días de edad casi no hubo efecto. salt of chloramphenicol used for inject­
Sin embargo, la síntesis de proteínas por mitocondrias aisladas fue inhibida
similarmente en ambos casos, y lo mismo ocurrió con la capacidad de síntesis ing the rats and the free antibiotic
endógena de las mitocondrias in situ en los cortes. employed in the experiments in vitro
Aunque la fracción microsomal no es afectada por el antibiótico in vitro, were gifts from Parke Davis, Caracas.
cuando se la obtuvo de cortes de hígado adulto previamente incubados con L-[U-^*C] Leucine (331 m C i/m o l),
cloranfenicol, mostró un 20% de inhibición en la síntesis de proteínas, parti­ L-[4,5-=®H] leucine (brought to 2000
cularmente las de mediano y bajo peso molecular (electroforesis en poliacrila- m C i/m m o l), L-[U-“ C] amino acid
m ida). La inhibición parcial se observó in vivo en los polirribosomas asocia­
dos a membrana. Se supone que estos efectos reflejan la reducción de la sín­ mixture (5.4 m C i/m A tom carbon),
tesis de proteínas mitocondriales fuera de la organela, aunque no puede des­ D-threo [methylene-^*C] chloramphe­
cartarse una interferencia más directa con el funcionamiento de los ribosomas nicol (10.2 m C i/m m ol) were purchased
citoplasmáticos. from Amersham/Searle Corporation.

Animals. Five-day old rats (12.5±

The D (- ) threo isomer of chlo­
ramphenicol (C AP) is a well known
inhiijitor of protein synthesis in bac­
teria isolated mitochondria and
chloroplasts When given in vivo to
partially hepatectomized or new-born
rats it can im pair the synthesis of m i­
tochondrial proteins, with a concomi­
tant decrease in the function of the
electron transport chain, probably by
Requests for reprints should be addressed to
N.G.C.
The present work was supported by a g ran t
from the “ Consejo de Desarrollo C ientífico y
H um anístico” , U .C.V .
interfering with the biogenesis of m i­
tochondria ^ The antibiotic does not
act on the isolated microsomal fraction
from rat liver or 80-S ribosomes
from other sources although it does
inhibit the activity of the ribosomes
from mitochondria and chloroplasts
In bacteria it interferes with
binding of the amino acyl end of the
aatRNA to the 50-S subunit, in hibit­
ing transpeptidation, and presumably
blocking directly the activity of pepti-
dyl transferase
CAP exerts in higher organisms a
variety of biological and pathological
1.5 g) and male young adult rats (150
± 1 0 g) from a closed Sprague-Daw-
ley colony bred at the Instituto Vene­
zolano de Investigaciones Científicas,
Caracas, were used throughout. The
adult rats were starved overnight be­
fore sacrifice.
Procedures. They are modifications
of methods previously described
33, ?,7. 40^ gg detailed in each experiment.
The estimation of CAP was performed
by a turbidimetric assay using Sar-
cina lutea, strain ATCC9341, kindly
given by Dr. Esther Tabernero.

53
 RESULTS

The addition of 1.55 mJVI CAP to

the incubations of slices from adult
liver inhibited the synthesis of m ito­
chondrial proteins by 35% (Table 1).
The contribution of mitochondria to
the formation of its own proteins is
very limited and certainly not higher
than 10% Even assuming that the
antibiotic blocks completely the endo­
genous translation in the mitochondria,
it is evident that the rest of the effect
corresponds to a partial inhibition of
the synthesis of mitochondrial proteins
outside the organelle. This is in agree­
ment with the 27% decrease in the
specific radioactivity of the proteins
associated with the microsomal frac­
tion. However, protein synthesis by C O N C E N TRA TIO N OF D - CH L O R A M PH EN ! C O L ( m M )

cytoplasmic ribosomes in liver slices
from five-day old rats was not affected
by CAP.
To investigate whether the difference
in behaviour between the adult and
new-born liver depends on the concen­
tration of the antibiotic, these experi­
ments were repeated using a wide ran­
ge of concentrations. Slices from adult
liver were significantly inhibited in
concentrations higher than 0.2 mM , to
about 70 to 80% of the control value
in the case of microsomal protein syn­
thesis (fig. lA , reproduced from refei-
ence =*'*). In contrast, no inhibition was
observed with new-born liver at con­
centrations below 1.1 mM, and only at
4 m M the effect was comparable to
that exerted on adult liver (fig. I B ) .
I'iff. 1. E ffect of increasing concentrations of D-chloramphenicol on protein synthesis by liver
slices.
Incubations were performed as described in Table 1, except th a t the concentration of C A P was
varied as ind.'cated. A : young adult liver ; B : five-day old liver ; O : m itochondria ; ^ : microsomal
fractio n : • : homogenate.
T a b le 1
Effect of D-chloramphenicol on protein synthesis by liver slices
Liver slices (1 g.) from 3-4 rats were washed in 9 ml. of the medium
described by Peters & Anfinsen supplemented with 20 m M glucose, at 37 °C,
under O 2 + COo (95:5) for 20 m in. The medium was decanted and replaced
by fresh one containing except leucine, natural L-amino acids (0.05 m M
each) and 1.55 m M CAP. After 30 min. incubation, 1.5 juCi of [^^C]
leucine was added and the incubation proceeded for 60 m in. Non-radioactive
L-leucine (5 mg. in 1 ml. of 0.15 M-NaCl) was then added and the samples
were chilled at 0°C . Subcellular fractionation was carried out as previously
described The determination of radioactivity was done on glass filter discs
Values are means from two experiments.

It is possible that the inhibition ob­

Y O U N G A D U L T EAT S F IV E - D A Y O L D RAT S
served in protein synthesis by cytoplas­
mic ribosomes is due to the interrup­ CONTROL CAP CONTROL CAP
tion of the elaboration of mitochon­
A B S O LU T E C H A N G E AS A BSO LU T E C H A N G E AS
drial proteins outside the organelle VALUE REFERRED VALUE REFEERED
which would follow immediately the TO C O N T R O L TO C O N T E O L
block of endogenous mitochondrial
dpm /m g. % dpm /m g. %
protein synthesis by the antibiotic. In protein protein
that case it could occur that the m ito­
chondria of new-born liver are less
sensitive to the antibiotic, and as a H O M O G EN A T E 3260 — 25 7065 — 21
consequence microsomal protein syn­ M IT O C H O N D R IA 1870 — 35 3945 — 21
thesis remains resistant at this age. M IC R O S O M E S 8850 — 27 13060 — 3
How-ever, when isolated mitochondria
were incubated in vitro with [^‘‘C]
leucine, no significant difference was barrier to CAP in the new^-born liver, formed with this system. To dismiss
detected in the degree of inhibition of is that when slices were incubated in the possibility that the inhibition ob­
protein synthesis between new-born the presence of cycloheximide to sup­ served could simply derive from an
and adult liver (Table 2 ). The entran­ press the background derived from ATP depletion due to a respiratory
ce of CAP into the liver slices cor­ microsomal protein synthesis, the endo­ deficiency through the inactivation of
responds to an active transport and genous mitochondrial activity was in ­ an electron transport system such as
the antibiotic is concentrated 3-5 fold hibited in the same degree as wdth the N A D H dehydrogenase protein syn­
in the new-born tissue under the con­ isolated mitochondria (Table 2 ). thesis was tested using ribosomes iso­
ditions used for the incubations, as lated from liver slices incubated with
shown with [^^C] CAP and by estimat­ Considering that the effect on pro­ CAP. The microsomal fraction obtain­
ing the antibiotic with a microbiologi­ tein synthesis by cytoplasmic riboso­ ed from the CAP— treated slices was
cal method. But undoubtedly the best mes was apparent only on adult liver, 19% less efficient, on the average, than
proof for the absence of a permeability the rest of the experiments were per­ the control (Table 3 ).

54
 Fig. 2. Polyacryiamidc gel electrophoresis of
proteins synthesized in v itro by the microsomal
traction obtained from liver slices incubated
w ith D-chloramphenicol.

The microsomal fraction from incubated liver

was obtained and treated as described in Table 3,
except that the total volume of incubation for
this fraction was 1 ml, and the concentration
of p H ] leucine was 25 /iC i/m l. Samples were
precipitated w ith trichloroacetic acid and treated
as in (39) by dissolving in 1% sodium dodecyl
sulphate (S D S ), 1% mercaptoethanol, 0.1 M
sodium phosphate buffer pH 7.4, heating at
90 °C for 6 m in ., and dialysing against the same
buffer diluted 1/10. Polyacrylamide gel electro­
phoresis was carried out in 0.9 X 8 cm. gels
(400 /¿g protein) at 4 m A /g e l for 16 h. Gels
were 10% acrylamide, 0.1% SDS, 0.1 M phos­
phate, 0.26% ethylendiacrylate, 10% glicerol,
0.075% am m onium persulphate, 0.06% TBM ED
and the electrophoresis buffer was 0.1% SDS,
0.1 M sodium phosphate p H 7.4. C ounting and
staining of duplicate gels was done as previously
described (40). O----- O control; « ----- •
C A P treated sample.

In order to rule out a possible con­

tamination of the microsomal fraction
with mitochondrial fragments or bac­
teria, which could be inhibited by CAP
persisting in the fraction, another sim i­
lar experiment was carried out, but
this time adding the antibiotic also in
vitro (100 jU.g/ml) to the system ob­
Tab le 2 tained from incubated liver slices. No
inhibition at all was observed in the
Inh ib itio n of mitochondrial protein synthesis by D-chloramphenicol control, and the microsomes from the
slices previously incubated with CAP
In experiment A mitochondria were isolated from 5 g. of rat liver by the remained inhibited similarly after CAP
procedure of Roodyn et al. and incubated at 30 °C for the periods indicated, was added again to the subcellular
in a medium containing: sucrose (100 m M ), K C l (40 m M ) , EDT A (1,3 m M ), system.
nicotinamide (20 m M ), K succinate (10 m M ), Ka H P O 4 (16 m M ), A M P (4 When the proteins synthesized in
m M ), N A D (0.5 m M ), M g SO4 (8 m M ), amino acid mixture (50 jU,g/ml), vitro by the microsomal fraction ob­
mitochondrial protein (5 m g /m l), [^“C] leucine (1 ftC i/m l), and when in d i­ tained from liver slices incubated with
cated CA P (1 m M ), all at pH 7.2 and in a total volume of 0.6 ml. Samples of CAP were fractionated by polyacryla­
0.1 ml. were taken for radioactivity determinations and sterility tests. All mide gel electrophoresis, it was observ­
operations were performed under aseptic conditions After 48 h. in Bacto ed that the inhibition was more evi­
nutrient broth at 37°C, bacteria were less than 500 colonies/mg protein. dent in the region of medium and low
In experiment B., liver slices w^ere incubated as described in Table 1, except molecular weight proteins (Fig. 2 ).
that cycloheximide was present (500 /xg/ml) in the fresh medium of incuba­
Since in the case of regenerating
tion, with or without CAP (1.55 m M ) . Each flask contained 3 [xCi of [^‘‘C]
liver, González-Cadavid & Ramirez
leucine. The control sample was that containing cycloheximide alone.
observed that CAP given in vivo in ­
duced a partial and short inhibition of
the protein synthesizing ability of the
Y O U N G A D U L T RATS F IV E - D A Y O L D RAT S isolated microsomes, we decided to see
CONTROL CA P CONTROL CAP whether this effect could be obtained
with normal liver. In our case the re­
ABSOLU TE C H A N G E AS A BSO LU T E C H A N G E AS
sults were not consistent and therefore
VALUE REFERRED VALUE REFERRED
TO C O N T R O L TO C O N T R O L we investigated the behaviour of free
and membrane bound polysomes ex­
dpm /m g. % dpm /m g. % pecting to find a differential effect. A
M IT O C H O N D R IA protein protein
single 100 mg injection of CAP was
given to rats and 60 m in after, poly-
A ) ISO LA T E D somal fractions were prepared and
30 min. 435 — 82 1280 — 76 incubated under conditions of protein
60 min. 830 — 84 2010 — 80 synthesis. Table 4 shows an average of
21% inhibition in the case of the mem­
B) IN SITU
brane bound polysomes, as opposed to
(Liver slices) 446 83
a 19% stimulation in the free polyso­
60 min.
mes and no significant effect in the

55
 cleoxychoíate treated polysomes (C- T able 3
ribosom es). The stimulation observed
Protein synthesis in vitro by the microsomal fraction isolated from slices of
with the free ribosomes was constant,
adult liver incubated with D-chloramphenicol
but the effect on the membrane bound
polysomes was somewhat variable and
Liver slices were incubated as described in Table 1, except that no radio­
even negligible in one experiment.
activity was added. The reaction was stopped with ice-crushed isolation medium
However, the bound/free ratio was (MgCL, 5 m M ; KCl, 25 m M ; Tris-Cl, pH 7.8 at 20°C, 50 m M ; sucrose, 250
consistently inhibited. mM) and the slices were washed twice in the same medium at 0°C. The
microsomal and the pH 5 fractions were prepared by the procedure of von
DISCUSSION der Decken & Campbell The latter fraction was obtained from non-incubated
liver. Incubations were performed for 30 min. at 37°C in a medium contain­
We have shown that CAP can act on ing; sucrose (90 m M ), Mg += (6 m M ), K+ (25 m M ), Tris-Cl pH 7.8 at 20°C
the intact hepatocyte in situ, either in (21 m M ), P E P (10 m M ), GTP (0,25 m M ), ATP (2 m M ), piruvate kinase
(50 /xg/m L), microsomal R N A (0.15-0.25 m g /m l.), pH 5 fraction protein
vivo or in incubations of liver slices,
(1 m g /m l.), and radioactive amino acids as stated, all in a total volume of
interfering slightly with protein syn­
0.5 ml. Values are average of three incubations.
thesis by 80-S ribosomes, through a
mechanism not derived from a respira­ CONTROL CAP
tory impairment and affecting prefe­
ABSOLUTE V A LU E CHAN GES AS R EF ER RE D
rentially the membrane bound polyso­
TO C O N T R O L
mes. The fact that the antibiotic in h ib ­
its the mitochondrial ribosomes where­ dpm /m g. %
protein
as does not exert any direct effect on
microsomal protein synthesis, suggests
[ “ C] A M IN O A C ID S
that the small but constant decrease in
activity observed in the latter could a) 0.25 jU.Ci/ml.
be the consequence of the inhibition Experiment 1 1010 — 13
Experiment 2 1380 — 21
of mitochondrial protein synthesis
through a tight coordination between b) 5 |aCi/ml.
both systems. Membrane bound polyso­ Experiment 3 62300 — 8
Experiment 4 61950 — 25
mes might be more affected because
they seem to be more involved in the [ ‘H] L E U C IN E
elaboration of proteins to be trans- 25 (U,Ci/ml.
fered to mitochondria than the free Experiment 5 28110 — 25
ribosomes AVERAGE — 19
An alternative explanation, that can
not be dismissed by the present expe­
riments, is that CAP when acting on
the whole cell interferes directly with
the function of the 80-S ribosomal T able 4
system, either by affecting factors of
chain elongation, or by inducing small Effect of D-chloramphenicol in vivo on protein synthesis in vitro by
changes in ribosomal conformation, C-ribosomes and membrane-bound and free polysomes
such as has been shown for the E. coli
Rats were injected intraperitoneally with 1 ml. of 0.15 M-NaCl containina
system
or not 100 m g /m l. of CAP. The rats were killed 60 min. later and free and
The small stimulation of free poly­ bound polvsomes prepared according to the technique of Ragnotti et al. but
somes by CAP in vivo is probably using 2.0 M-sucrose as bottom layer. Incubations were performed for 30 min.
related to an as yet unexplained pheno­ at 37°C in a medium similar to the one used in Table 3, except that polysomal
menon; the increase in protein syn­ R N A was present at 1 to 2 m g /m l. The concentration of [^^C] amino acids
thesis in normal rat liver observed was 0.125 jU,Ci/ml. Values are average of four separate experiments.
after long treatments with CA P in
vivo \ following a period of in h ib i­ CONTROL CA P
tion and which has also been ob­
ABSOLUTE V A L U E CHAN GES AS R EF ER RE D
tained in vitro with microsomes iso­ TO C O N T R O L
lated from these treated animals
Apparently the effect of the antibiotic
dpm /m g. %
RNA
in vivo is biphasic after each adminis­
tration, first inhibition and later stim­
ulation, in a way resembling the oscil­ Membrane bound polysomes 9890 — 21
lations of cytochrome synthesis observ­ Free polysomes 13920 + 19
ed in continuous culture of Saccharo­ C-ribosomes 13210 — 4
myces carlsbergensis and Candida Bound
utilis following the addition and re­ — 32
Free “
moval of CAP

56
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57