Effect of X-irradiation on liver regeneration

EXPERIMENTAL

AND

MOLECULAR

Effect

of

PATHOLOGY

5, 389-395

X-Irradiation

(1966)

on

Liver

Regeneration’

ENRICO CLERICI, PIERO CAMMARANO,~ AND PAOLO MOCARELLI Institute of General Pathology, University of Milan, Milan, lfaly Received

January

11, 1965

INTRODUCTION Regenerating liver provides the best system for the study of many biochemical aspects of the fast growing mammalian tissue because of its synchronously dividing cell population, and, therefore, it has been extensively employed in radiation studies. It has long been recognized that X-irradiation carried out at different time intervals during the presynthetic period after partial hepatectomy affects several steps of nucleic acid synthesis and may selectively inhibit DNA and RNA production; at later times X-irradiation is practically ineffective (for review, see Clerici, 1961). On this basis, it was of interest to study the influence of X-irradiation on protein synthesis, in order to underline possible changes subsequent to nucleic acid formation. This paper is a report of a series of observations on the in vivo incorporation of m-Leucine-l-l% into the total protein of cell fractions in liver of partially hepatectomized and sham-operated rats given a whole-body X-irradiation within the presynthetic period, and sacrificed at different intervals after X-irradiation. Animals

MATERIALS and experimental design

AND

METHODS

Forty albino rats, Wistar strain, of either sex, bred in the Institute and weighing about 100 gm were used throughout these experiments. After a 12-hr fast, all the animals were either sham-operated or partially hepatectomized, according to the method of Higgins and Anderson (1931). The rats were divided into 2 series, named A and B, each consisting of 20 rats, ten sham-operated and ten partially hepatectomized. Animals included in series A received whole-body X-irradiation 4 hr after operation and were subdivided into 4 subgroups as follows: al and a, each consist of 5 hepatectomized rats, ag and al each consist of 5 shamoperated rats. Series B, acting as unirradiated controls, also consists of 4 subgroups: b, and bZ each consist of 5 hepatectomized rats, b8 and b, each consist of 5 shamoperated rats. All groups of rats were injected intraperitoneally with a single dose of 5 PC (= 1.3 u&f) per 100 gm body weight of nL-Leucine-1-14C 4 hr before sacrifice. Groups al, a3, bl, b:, were killed 8 hr after operation and groups a?, a,, b-, bl were killed 16 hr after operation. All animals were fasted after operation; water was given ad libitum. 1 This paper was supported (C.N.E.N.), Rome, Italy. 3 Present address: C.N.E.N.,

by

a grant

Laboratorio

from

the

Di 389

Comitato

Radiobiologia

Kazionale

per

1’Energia

Animale,

Casaccia,

Rome,

Nucleare Italy.

390

ENRICO

CLERICI,

PIER0

CAMMARANO,

AND

PAOLO

MOCARELLI

nn-Leucine-l-14C (specific activity: 3.8 mc/mM) was purchased from the Amersham Radiochemical Center (Amersham, Bucks, England). Irradiation

procedure

The animals to be irradiated with 1000 R of whole-body X-irradiation were housed, eight at a time, in a Perspex covered rectangular box divided into four compartments; each compartment was of a convenient size to contain two animals in a fixed position. The radiation factors were: 250 kvp, 12 ma, 0.8 mm Cu and 1.0 mm Al filtration (HVL, 1.0 mm 01). The TSD was 40 cm, and the exposure dose rate in air at the position corresponding to the dorsal surface of the animal back was 30 R/min. The average dose rate at the periphery of the container did not appreciably differ from that at the center of the field. Liver fractionation

and radioactive protein isolation, sampling, and counting

After the selected period of time, the animals were killed by a blow on the back of the head and their livers immediately perfused both by the venous and arterial routes, with 0.25 1M cold sucrose + 0.005 1c1 Versene isotonic solution. Once removed, the livers were quickly homogenized in the same solution (1 part liver to 10 parts solution) with an all-glass Potter-Elvehjem apparatus, and a small aliquot of the whole homogenate precipitated with trichloroacetic acid (TCA) , final concentration 1%. The remaining homogenate was then fractionated according to the main lines of the Schneider technique ( 1948). Proteins from the whole homogenate, nuclear, mitochondrial, microsomal and supernatant fractions were isolated and purified according to the procedure of Rabinovitz et (II. (1954). They were dried overnight at 50°C weighed and homogenized, in all glass Potter containers, in absolute ethanol to obtain 1 mg protein/O.75 ml. This amount was then plated on stainless steel planchettes (surface = 4.8 square centimeters), dried over CaCls and counted by means of a windowless gas-flow G.M. tube. Sufficient counts were recorded as to afford a statistical error below 35%. Duplicate samples gave reproducible counting rates within 5%. An analysis of variance was applied on the whole set of experimental results in order to check the influence of X-irradiation (I), partial Hepatectomy (H), Time from operation (T) and their interactions (T X H; T X I; I X H; I X H X T) on the different cellular fractions. The significant interactions were further analyzed according to standard statistical methods. RESULTS The incorporation pattern of m-Leucine-l-ilC into protein of the liver fractions of the experimental animals is summarized in Table I. ii nalysis

0 j variance

The analysis of variance, whose final steps are appended to the same table, for sake of clarity, showsonly one significant effect, the H one (p < 0.01) at the level of the nuclear fraction; this indicates (see numerical data in Table I) that early

X-IRRADIATION

AND

LIVER

391

REGENERATION

after partial hepatectomy, the aminoacid incorporation into the nuclear protein of the regenerating liver is depressed as compared to sham-operated controls. Four interactions appear to be significant, namely: I X T in the whole homogenate (p < O.OS), H X T in the mitochondrial fraction (p < O.OS), and I X H in the microsomal (p < 0.01) and supernatant (p < 0.01) fractions. They were analyzed as described below. Analysis

oj the interaction

I X T in the whole

homogenate

The T and H effects and their interaction were verified among all the irradiated and unirradiated rats. A significant H effect (p < 0.01) was underlined in the former; therefore (see numerical data in Table l), the partially hepatectomized X-irradiated rats incorporate less than sham-operatedX-irradiated controls: in other words, X-irradiation of partially hepatectomized rats reduces the protein synthesis rate even below resting values. Analysis

of the interaction

H X T in the mitochondrial

fraction

The H and I effects and their interaction were checked among animals sacrificed at 8 and 16 hr after surgery: following this procedure, a complete loss of significance was observed in both experimental groups, obviously due to halving of degreesof freedom. Analysis

of the interaction

I X H in the microsomal

and supernatant

jractions

In both fractions, the H and T effects and their interaction were controlled among X-irradiated and unirradiated groups of animals. With regard to the microsomes,a significant H effect (p < 0.01) was demonstrated in the unirradiated rats; by observing the numerical data in Table I, it is clear that H enhancesthe protein synthesis of this fraction quite early after surgery and that whole body X-irradiation interferes with this activation. Regarding the supernatant, the same H effect was significant (p < 0.01) in the X-irradiated (instead of unirradiated) group of animals. Therefore (see numerical data in Table I) the aminoacid incorporation rate of partially hepatectomized X-irradiated animals is depressedcompared with sham-operated X-irradiated controls. No difference whatsoever is observed among unirradiated animals. The final steps of each of the above analyses of interaction are also appended to Table I. DISCUSSION Our observations on the aminoacid incorporation into liver protein from partially hepatectomized and sham-operatedrats indicate that, in both experimental conditions, the highest specific activity belongs to microsomes,followed by mitochondria, supernatant and nuclei, and that the specific activity of each fraction, except the nuclear one, ranges above that of the whole homogenate. Furthermore, they show that, in the early phasesof liver regeneration the protein synthesis is significantly increased only at the level of the microsomal and, to a lesser degree, mitochondrial fractions, while they are even depressedin the nuclei, as compared to sham-operatedcontrols.

I

IEH

1r.T

Br

11Xx

1ntsraction

1ntersction

Interaction

Interaction

T

T

of Time from operation

Zffect

?I*patectomy

Of Partial

FTrreot

OF VAQIAnCE

of X-Irradiation

AnALTSIS

Effect

(T)

(Ii)

(1)

EoL~OCis mTB

)

df

I/32

l/32

l/32

l/32

I/32

F

< I

< I

6.02

3.38

c I

1.70

< I

HICEOsOMX9

P

> 0.05

> 0.05

< 0.05

> 0.05

> 0.05

a 0.05

> 0.05

mmomn*TJ

hiiK+ CB)KDBIA

l/32 1 I/32

WCLEI

F

nccm1



2.17

2.03

2.59

7.76


SUPERNATANT

P

> 0.05

> 0.05

> 0.05

> 0.05

> 0.05

< 0.01

> 0.05

F

< 1

5.00

< 1





m1Tocsoms1* P

> 0.05

< 0.05

> 0.05

> 0.05

> 0.05

> 0.05

> 0.05

I

F

<1



12.51

3.82



P

> 0.05

> 0.05

> 0.05

< 0.01

> 0.05

> 0.05

> 0.05

ill?=23

92t20 9a+7

i:ICROSOIi?3

l&1+6

iI%5

I4gal3

< I

< I

2.87

9.50

< 1

< I

-c I

F

suPa.n*TAnT

183t28 79=kIO

11414

151+13

IOaaV

138+16

P

> 0.05

> 0.05

> 0.05

< 0.01

> c.05

> 0.05

> 0.05

103LZO

12.%a

15a*20 :58+14

131112

154L31 1g1*27

214Ul

3.x

T

of Time *mm o*er*tion

Effect

Interaction

of Time from operation

) Effeot

(T)

l/16

l/16

(T) ) l/16

)

< 1

< 1

2.04

> 0.05

> 0.05

> 0.05

< 1

2.34

4.01

> 0.05

> 0.05

> 0.05

Of X-Irradiation

Partial

3x

Interaction

x*eet

Effsot Of Time from

of Partial

HI

T

operation

Hepateatony

T

Hepatectomy

ANALYSI3OFINTNRACTION

Interaction

Efieot

of

ANALYSIS OF INPm*cTIoN Effeat

Ix

(T)

(H)

H

(1)

(H)

3 I T

l/l6

l/l6

l/16

df

l/16

l/16

l/16

df

F 1

0.05

P

< 1

C 1

10.31

> 0.05

> 0.05

< 0.01

P

rats

X-Irrlviiated F

> 0.05

> 0.05

>

< 1

< 1

<

F

P

< 1

c 1

a 0.05

> 0.05

P > 0.05

F

rats

> 0.05

> 0.05

zs 0.05

2.15

mlrradlated

-z 1

< 1

3.55

394

ENRICO

CLERICI,

PIER0

CAMMARANO,

AND

PAOLO

MOCARELLI

Such results are in good agreement with those of other investigations on the labeled aminoacid incorporation into the total protein and into the protein of some subcellular structures of the regenerating liver (Hultin and Von der Decken, 1957; Von der Decken and Hultin, 1958; Hammarsten et al., 1956); they also confirm our preliminary observations on the same matter (Clerici et al., 1965). It is worth recalling that soon after surgery, the liver stump left in situ undergoes regressive changes (cell vacuolation, fatty infiltration, necrosis; glycogen and protein loss) similar but even more extensive than those observed after a prolonged fasting. During this phase, the outstanding aspect of regeneration, i.e., the synthesis of new proteins, is strongly hampered. As a main feature of the X-irradiation procedures, it was found that, during the early stages of liver regeneration, whole body X-irradiation inhibits both the incorporation of labeled leucine into the soluble protein fractions, or, more properly, prevents the increase of metabolic activity which would be expected about 16 hr after partial hepatectomy, and contemporaneously keeps the microsomes from increasing their protein synthesis. It should be recalled that radioactivity found in the soluble protein fraction after administration of labeled aminoacids in viva is likely to represent newly formed proteins which have passed from the cellular site of protein synthesis into the soluble phase, and that direct demonstration of transfer of radioactivity from microsomesto radioactive serum albumin has been reported in a cell-free rat incorporation system (Von der Decken and Campbell, 1962). On this basis, it may be assumedthat X-irradiation during the early stages of liver regeneration prevents the synthesis of protein moleculeswhich are either necessary to overcome the parenchymal loss or which pass into the blood stream after synthesis by liver cells. It has also been reported that (1) in regenerating liver, synthesis of the nuclear RNA occurs during the very early stages of liver regeneration preceding the onset of both DNA and protein synthesis; (2) a whole-body X-irradiation carried out within 6 hr after partial hepatectomy brings about a marked inhibition of the synthesis of nuclear RNA, whereas no such inhibition is observable in animals X-irradiated at later times after surgery (Welling and Cohen, 1960). These findings and our results may be consistent with the view that the decreased synthesis of cytoplasmic proteins found in animals X-irradiated within 6 hr after surgery is due to the disturbance in the synthesis or turnover of nuclear RNA’s (Hiatt, 1962) acting as templates in the production of the new protein molecules necessaryto support liver regeneration. An impairment of RNA polymerase (Weiss, 1960) synthesiscausing in turn decreasedsynthesisof nuclear RNA templates, might be tentatively postulated as a leading event in the radiation injury. Further studies to clear this point are in progress. SUMMARY The in viva incorporation of m-Leucine-l-r% into the total protein and the protein of cell fractions in liver of partially hepatectomized rats, given a whole-body X-irradiation 4 hr after surgery (i.e., within the so-called presynthetic period of liver regeneration) was investigated. The main feature of our results indicates that the whole-body X-irradiation inhibits the incorporation of labeled aminoacid into the soluble, nuclear, and microsomal fractions of regenerating liver as compared with unirradiated partially hepatectomized control rats.

X-IRRADIATION

AND

LIVER

REGENERATION

395

On the basis of recent information on the mechanism of protein synthesis, such data are tentatively explained as resulting from disturbance in the synthesis or turnover of nuclear RNA’s acting as templates in the production of the new protein molecules necessary to support liver regeneration. REFERENCES E. (1961). La rigenerazione de1 fegato. Recenti Progr. Med. 31, 429-472. E., CAMMARANO, P., and MOCARELLI, P. (1965). Protein synthesis in the early stages of liver regeneration. Experientia. 21, 143-144. HAMMARSTEN, E., AQVIST, S., ANDERSON, E. P., ELIASSON, N. A., and THOREL, B. (1956). Turnover of polynucleotides and proteins in regenerating rat liver studied with glycine-Nl”. Acta Chem Stand. 10, 1568-1575. HIATT, H. H. (1962). A rapidly labeled RNA in rat liver nuclei. J. Mol. Biol. 5, 217-229. HICCJNS, G. M., and ANDERSON, R. M. (1931). Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Avch. Pathol. 12, 186-202. HULTIN, T., and VON DER DECKEN, A. (1957). The incorporation in vitro of labeled amino acids into the proteins of regenerating rat liver. Exptl. Cell Res. 13, 83-87. RABINOVITZ, H., OLSON, M. E., and GREENBERG, D. M. (1954). Independent antagonism of aminoacid incorporation into protein. J. Biol. Chem. 210, 837-849. SCHNEIDER, W. C. (1948). Intracellular distribution of enzymes. III. The oxidation of octanoic acid by rat liver fractions. J. BioZ. Chem. 176, 259-266. VON DER DECKEN, A., and CAMPBELL, P. N. (1962). Aminoacid transfer from aminoacyl-ribonucleic acid to serum albumin by the microsome fraction from rat liver. Biochem. /. 82, 448-454. VON DER DECKEN, A., and HULTIN, T. (1958). The activity of microsomes from regenerating rat liver in aminoacid incorporating systems. Ezptl. CeZZ Rex. 14, 88-96. WEISS, S. B. (1960). Enzymatic incorporation of ribonucleoside triphosphates into the interpolynucleotide linkage of ribonucleic acid. Proc. N&Z. Acad. Sci. U.S. 46, 1020-1030. WELLING, W., and COHEN, J. A. (1960). Disturbance of RNA turnover in the cell nucleus by X-irradiation in the early phase of rat-liver regeneration. Biochem. Biophys. Acta 42, 181-182. CLERICI, CWRICI,