Influence of d -galactosamine upon the nad-metabolism in rat liver

INFLUENCE OF D-GALACTOSAMINE UPON NAD-METABOLISM IN RAT LIVER*

Robert

Koch-lnstitut.

H. KRikER. R. GRATZ and H. GRAHN Nordufer 20. D-1000 Berlin 65, F.R.G.

THE

[Tel. (030) 450 32301

Abstracts After application of wgulxtosamine a hepatitis develops in the rat liver. This can be prevcntcd by different agents, including tryptophan. Yet it has not been possible to give definitive conclusions about the mechanism of galactosamine hepatitis. In this paper we report about the influence of galactosamine on the NAD metabolism.

I. wgalactosamine inhibits the NAD synthesis initiated by nicotinamidc in normal and adrenalectomizcd animals. 2. The NAD sInthesis from tryptophan is prevented m normal animals. in adrennlectomized ones however there is an increase of NAD in the presence of 1,.g:llactos~unine. 3. I)-galactosamine reduces the activity of the ADPR transfcrase. 4. Inhibitors of the ADPR transferase prevent the palactosamine hepatitis. 5. From the results presented we conclude that the ADPR transferase plays an important role in the development of the galnctosamine hepatitis.

INTRODCICTIOiX

Kcppler it trl. (196X) observed development of heputitis in the liver of rats following injection of D-galactosamine. This ell’ect can be prevented by different agents: uridine. erotic acid (Farber c’t rll.. 1973; Keppler et a/.. 1974) NH,‘-ions (Pausch c’f trl., 1977). anorganic phosphate in culture of hepatocytes (Sterman er trl., 197X). x,-macrofetoprotein (van Cool ef t/l.. 1979). r.x-Dithiodicapronsliure (Wegmann et (I[.. 1979). tryptophan (KrGger c’f trl.. 1979. 1980. 1981). For the time being it has not yet been possible to draw definitive conclusions as to ;i uniform mechanism of I,-galactosamine induced hepatitis. We dealt with the NAD metabolism under intluencc of galactosamine because tryptophan especiall! in combination with L-methionine inhibits the I,-galactosamine induced hepatitis. In this paper WC report on the results of the experiments. MATERIALS

L)c,t~,r,,lirlirlit,II 0f %,1D + rV.4DIIZ This v,as performed according to Nissclbnum & Green (lY69). The sum of NAD + NADH, (itmol, g liver) is stated Mith the a\eragc value and standard deviation. Ul,tu~,ltr,lcltio,1 o/ ADPR

trrm~rrtrsr

Nuclei Here isolated according to Blohel & Potter ( 1966). The method of Kidaell & Burdette ( 1974) was used. The experiments were performed in the presence of DNAse. Uercwriwrro/~ The method

0f’GOT trc,riritj described

by Bergmeyer

(lY74) was used.

RESC LTS

AND METHODS

We obtained from Roth (Karlsruhe): wgalactosamineH(‘I: Merck (Dnrmstodt): nicotinamtde. I>L-tryptophan. prednisolone. caffeine: Scrva (Heidelberg): hromdesouyuridlne. guanosine. adenosine. erotic acid: Schharz-Mann (Orangchurg. U.S.A.): t.-methionine: Sigma (Miinchen): I-methylnicotinamide. henzamidc: (Boehringer. Mannham): DNAsc I: Amersham-Buchler (Braunschweig): [‘??]NAD. WC thank E. Lily (Kalamazoo) for providing 5-methqlnicotinamide and Dr J. Klosa (Berlin) for nicotinamide-orotat and nicotinamide-coeffeinat.

Rats of Wistar strain Bundesgesundheitsamtes.

ectomized animals received 0.15 M NaCI over a period of 5 days and were used then for the experiments. All sub stances were given intraperltoneally.

(Zentrale Versuchstieranlage des Berlin) were used. The adrenal-

* Preliminary data were presented In Jerusalem. 19X0.

at the FEBS

Mee!i,l
After application of u-galactosamine-HCI no change can bc observed in the NAD + NADH, content in the liver of normal and adrenalectomized animals over a period of 6 hr (Fig. I). It is well known that nicotinamide and tryptophan lead to an increase of the NAD content. As can be seen from Fig. 2 the amount of NAD after application of nicotinamide is higher in adrenalectomized animals in comparison to normal ones. r,-galactosamine reduces the NAD synthesis initiated by nicotinamide. The NAD synthesis after injection of tryptophan is lower than that caused by nicotinamide (Fig. 3 and Tables la and I b). In adrenalectomized rats, the NAD synthesis is rather diminished which is in contrast to the results with nicotinamide. Here D-galactosamine decreases the NAD content in normal animals too. In adrenalectomized animals. however, the NAD content over a pericd of 2 hr is rather increasing.

.-.

Normoi

~.....a

Adrenaiectomszed

i

o......o

0

I I

Adrenolectoma?d (3 animals each

I 2

I 3

I 4

0 point

I 5

6 hr

I 6

\)

hr Fig. I. Content or NAD + NADH? in rat liver under influence of TV-yrrlacto.srrnli~f~-ofC! (375 mg, kg i.p.). The galactosamine was administered to the animals at time 0.

In adrenalectomized animals L-methionine given ;LS described causes a small rise of the NAD content (Fig. 4). This increase and the NAD content in normal animals is not influenced bv I,-gnlactosnmine, The NAD-synthesis initiated bv nicotinamide is slightly enhanced by methionine (Fig. 5). r)-galnctosamine applicated at times 0 has an inhibit~~ry effect. too. In normal animals the same picture can be seen in the case of DL-tryptophan 3 hr after application of galactosamine (Fig. 6). In adrenalectomized animals on the other hand D-galactosamine leads to a higher NAD content over ;t longer period contrary to normal animals (see also Fig. 3). I-Methylnicotinamide enhances the NAD in adrenalectomized rats (Fig. 7). There is some but no drastic change in the NAD content under I>-gaiactosamine. o-galactosamine causes a more hcnvy hepntitis in adrenalectomized animals than in normal ones (Kroger er (II., 198 I). These results suppose that steroid hormones have ;I protective e&x3. We were interested to learn what is going to happen to the NAD content under influence of prednisolonc. No dccisive changes can be observed (Fig. 8). Administering

Fig. 3. Content of NAD + NADH, in rat liver under intluence of I,t.-rr!,/~roi’/ttrll (300 mg ‘kg i.p. at time 0 and 12. 23. 36 hr hcfore) xs b\cII :IS of I,-g~ll;lctos:lminc HCI (375 mg kg i.p. at time 0). Number of animals in brackets.

prednisolone alone, the NAD content shows no changes (Table 2).

hr

Fig. 2. Content of NAD + NADHz in rat liver under infiuence of ~zj~~f~~zu~~ii~~ (500 mgkg i.p. at time 0 and 12. 24. 36 hr before) as well as of wgalactosamine-HCI (375 mg;kgi.p. at time 0). Number of animals in brackets.

of the liver also

We found no ch;uiges of the activity of the ADPR transferase in normal animals under the influence of I,-galactosaminr (Table 3). The activity of the enzyme is higher in adrenalectomized animals. t)-galactosamine leads to a clear decrease of this enzyme. This effect is p~~t~nti~~tedby a sirn~llt~~n~~~us~~dnliilistr~~tion of nicotinamide or trgptophan. respectively.

There are different substances with inhibitory effects on the ADPR transferase (Hilz & Stone. 1970; Purnell c’r cri.. 1980). From Table 4 it can be seen that very effective irlllibitors of the ADPR transferase as bromdcsoxyuridine, S-methyl nicotinamide and benzamide are protecting the liver against the inlhtence of

T NoCl

.---s

0

hr

3

1

I-

3

6

12

hr

o-

hr

Fig. 4. C‘ontent of NAD + NADHz in rat liver under influence of L-tnctlliol~ifl~ (500 mg:kg i.p. at time 0 and I?. 24. 36 hr before) x well as
NAD-metabolism Table

1133

in rat liver

I(a). Content of NAD and NHDH, in rat liver of normal animals inlluence of I,-g~tlnctosamine--HCI and uL-tryptophan

4 hr after time 0 NAD (htmol.‘g liver)

2 hr after time 0 NAD (/nnolEg liver)

Application

under

Time 0: iwtryxophan

(500 mg,,kg)

(5)

I. I864 * 0.0405

(5)

1.2159 + - 0.1228

Time 0: Galacrosamine

(375 mg kg)

(5)

0.8425 i_ 0.0959

IS)

0.7577 i_ 0.0759

Time 0: Ix.-tryptophan Gnlactosamine

(500 mg k& (375 m&/kg)

(5)

0.7713 k 0.1530

(5)

0.9713 _t 0.1563

(5)

0.8939 * 0.1613

(5)

0.7840 I: OS68

Control : 0.9”,, NaCI (IO mgikg)

Table

l(b). Content animals under

of NAD and NADH, in rat liver of adrenalectomized influence of I~-g~ll~ctos~lrnine-HCl and uL-tryptophnn 4 hr after time 0 NAD (pmol;g liver)

2 hr after time 0 NAD (Itmol:g liver)

Application Time 0: Lx-tryptophan

(500 mg: kg)

(-5)

I.1395 i: 0.0410

(4)

1.0572k 0.1977

Time 0: Galactosamine

(375 mgkg)

(5)

0.8279 + 0.0774

(4)

0.7775 t: 0.0960

Time 0: rx-tryptophan (500 mg:kg) G~ll~lctos~Imine (375 mg:kg)

(5)

0.9643 f 0.1307

(3)

I.0338 f. 0.2724

Control : 0.9”,, NaCI (IO mg’kg)

(5)

0.x939 * 0.1613

(4)

0.7451 -i_ 0.1453

galactosamine. Substances with lower activity against the ADPR transferax, i.e. nicotinamide and caffeine

are less effective against the galactosamine hepatitis as \vell. The inhibition provoked by caffeine and erotic acid is ~otel~ti~lted bv the new substances nicotinamide-cofl’einate and &otinamide-orotat.

.

-

.

o.....~

f

DISCX’SSION

The hepatitis induced by D-galactosamine may be prevented by different substances (for review see Kriiger t-‘rtrl., 198 1). Tryptophan also has this effect. This one was the reason to analyze the NAD metabolism of the liver under influence of D-galactosamine.

Normol Admnol-

+ O-golactosomine 0

3

6 hr

12

0

I

1

1

3

6

12

_

hr

Fig. 5. Content of NAD + NADHz in rat liver under influence of tlicotinunlitlr (500 mg:kg i.p. at time 0 and 12, 24. 36 hr before) + L-jwtkinrliw (SO0 mg;kg i.p. at time 0 and 12. 24. 36 hr before) as well as of I,-g:llactosamine-HCI (375 mg:kg i.p. at time 0). Number of animals in brackets.

1134

H. KKijwK

rt trl

a

05

z

+

hr

t

hr

0

Fig. 6. Content of NAD + NADH? in rat hver under influence of IIL-~~~~~O$IOII (300 mg;kg i.p. at time 0 and Il. 24, 36 hr before) and I.-wthrminr (500 mg. kg i.p. at time 0 and 12. 74. 36 hr before) as well as of r,-gal;~ctosamine~HCl (375 mp kg i.p. at time 0). Number of animals in brackets.

T

3

6

hr Fig. X. Infuence NAD + NADH,

of prc~tlrrisolof~c~ tr,ld ytr/clclo.stri,ll,lr on content. t 0: normal animals; the animals received IOmg,kg prednisolonc 12hr before time 0 and 5 mg kg prednisolone and 375 mg kg wgalactosamine HCI at time 0. 0. .o: adl-cnalectomircd animals: the anim;lls recel\ed IO mg kg prednisvlonc I2 hr before time 0 and 5 mg kg prcdnisolonc and 375 mg kg 1).galactosmllinc HC‘I at tlmc 0. Number of animals -1 each point.

13) 7

adenoribos!

lated.

proccsszs

Presumabl!.

of regulation transfcrase

\\hich

adrenalectotni7ed effect the

+

The NAD

hr

cxpcriments

ever Fig. 7. Content of NAD + NADH, in rat liver under influence of I-J,I~,~/I!,/JII~~~;~~[JI~I;[/~ (500 mg kg i.p. at time 0 and 12. 24, 36 hr before) as well as of wgalactoaamine HCI at time 0). •--~~- 0: Galactos~lmine: (375 mg,‘kg i.p. LI----LI: 0.15 M NaCI. Number of animals in brackets.

tophan ADPR

together is based

NAD

due

animals tryptophan

the

studies strate nell by

to the results in

have

oxydation shown

that

for adenoribos~lation et I//..

the

1980).

ADPR

NAD

reactions. also

reacts

(Hi17 & Stone,

In this

process.

transferwe

Table

(1935). NAD

of Warburg reduction

and

2. C‘ontent

NAD

is

this

;I sub-

mide

as

1976:

Pur-

IS decomposed

spccilic

(Kriiger.

Recent

proteins

arc

NAD

IO min before and at time 0: Prednisolone (5 mp kg) IO min before 2nd at time 0: Prcdnisolone (5 mg kg) Time 0: Gnlactosaminc (375 mg kg)

shown

that

inhibitors could

substrate

NAD

synthesircd

one

in the for

the

2 hr after time 0 NAD (irmol g liver)

transferasc. in

of of Other

adrenalectomized administration

the

ADPR-transferax results).

bc observed could

prcscnce

speculate

PIDPR-tr~~nsfcr~lst

animals

under

1111. after tlmc 0 NAD (~rmol g li\cr)

of Since

if nicotinathat

of trjptophan

by nicotinamide.

I~YCI- ol acir~nalectomiled HCI and prcdnisolone

the

accumulation

unpublished not

tryp-

aftcr of

and Grahn.

administered

synthesized

better

of NAD + NADHz in rat intlucnce of I)-galactosamine

Application

Griitz

is accttmu-

decomposition

ADPR

is accumulated

phenomenon U;IS

inhibited

of

how

redtws

the

the diminished

liwr

Since

galactosaminc

the

trypto-

animals NAD

that

that or

of’ t,-galuctosamine.

have and

in this

protect

in the

of tryptophnn

t~pon

NAD

the

of

further

nicotinamide

I,-~ulactos~imine

WC suppose

to

she\\

b!

with

activity.

NAD

upon bc shorn

transfcrase

adrenalectomired

influence

experiments

According

In

application

tmdcr

infucnce

Ah ;I consequence

presented hq

animals.

after

lated

inllucnce

rcspcctively.

especialI\.

of ADPR

induced

is inhibited

normal

hr

changes

&ctosaminc.

slnthcsis

phan

0

inhibitors

against

NI

could

animals.

strong liver

such

or diflerentiation,

seems to have

Galactosam~nc ADPR

I;)

involved

24

I2

than

the is a the

NAD-metabolism T:~blt 3, intluence

of I,-gal;lctosaiiline-HC1 upon the activity ase m nuclei from rat liver Normal

N>lCI wgalactosumine

1135

in rat liver

-HCl

animals

ofADPR

Adrenalectomized

nnimals

10X.29*

162.83

I 16.31

I I8.83

1I?I.lX

l&tryptophan

133.41

l~I,-tr~pt~~j~h~lll + ~)-~~tl~~~t~~~~rnineHCI

I06.60

79.78

Nicotinamidc

I %.hh

176.78

Nicotinamide + u-galactosamine-HCI

104.13

transfer-

67.23

* cpm IOh cells. The anim;lls received at time 0: 375 mg,‘kg I,-galactosamine--HCl. N:IC‘I (0. I5 %I)and 500 mg:kg of the other substzmces. The animals ficed 4 hr ;lfter the application of the substances.

10 ml;kg were sacri-

T‘able 4. influence of various substances on the increase of GOT in the rat serum after application of I,-galactosamine--HCI. The substances were injected together bcith I,-galactosamine--HCI.

Doses (mg: kg) i)-~~~l~~~t~)~~rni~~e HCI 1) -i- guanosine I> + adenosine I) -+ nicotinnmide 1) + calreine I) + No-cnffcinut 11 + erotic acid 1) + Nn-orotat I) + I,i.-tryptophan I) -t benzamide 11 + 5-methyl-NA I) + br(~mdesoxy~tridine *The cnryme wtivity I)-gal~~ctosamine~HC1.

335 350 250 250 50 2.5 50 50 500 100 25 250 wits measured

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Increase of GOT* Galactosamine treated 0.15 M NaCl treated 7.47 1.40 2.10 2.00 1.8i I.61 I.61 1.44 1.26 I.‘?. I.1 1 0.X4 14 hr after the application

of

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PURNELL

M.

R.. SIOU!-

ADP-ribosylntlon

7-ml.\8. 2I5

of

P. R. & nuclear

Wrwr protems.

W. J. D. (19X0) Bioc~l~cw. Sot,.

127. R.. WAC;I.I. S. R. 6t D1.c KI K K. (197X) Inkcrsc effects of I,-galactosamine and inorg:lnic phosphntc on glycogenolysis in isolated rat hcpiltoc! tes. fir/r. .I. L&r-

ST~RMANU

ilrtwl.

88, 79 85.

WARRUKG 0. & CHRISTIAN W. (19353 C‘o-Fermentprobicm. Biochon. z. 27s. I I2 I 13. W~GMAUN A.. C‘HAKRIAT’rt: N.. RrrNlitK H. CG DITTRICH A. (1979) Wlrkung des Lebzrschutzpr~ipar~ltes ES 914 ;luf durch wG:daktosamin ;~usgeli%te experimentelle Hepiititis bei den Ratten. -t~~tt~~i~~~itt~~/-F~~~sc~/r.:D~rc~~ Rn. 29, hS- 70.