Effect of malotilate on chronic liver injury induced by carbon tetrachloride in the rat

Journal of Hepatology, 1986;3:260-268

260

Elsevier HEP 213

Effect of Malotilate on Chronic Liver Injury Induced by Carbon Tetrachloride in the Rat

J . - M . D u m o n t , M . - F . M a i g n a n , B. J a n i n , D . H e r b a g e a n d D . P e r r i s s o u d Research Department, Zyma SA, Nyon (Switzerland) and Centre de Recherche AppliquOe de Dermobiochimie and UniversitO Claude Bernard, Lyon I, UA CNRS 244, Lyon (France)

(Received 5 November, 1985) (Accepted 27 March, 1986)

Summary The effect of malotilate, a new drug proposed for the treatment of chronic liver diseases, was studied in carbon tetrachloride (CCl4)-induced chronic liver injury in the rat. Treatment with CC! 4 (0.5 ml/kg twice per week, intraperitoneally for 6 or 9 weeks) led to marked necrosis, steatosis and fibrosis, as shown by both biochemical and histological examinations, and a significant decrease of the bromosulfophtaleine (BSP) clearance test. Malotilate (50 mg/kg p.o., 5 days per week given simultaneously with.CCI 4 for 6 weeks), suppressed the increase of plasma aminotransferase activity and decreased significantly the accumulation of lipid and collagen in the liver; histology confirmed this protective effect of malotilate. The BSP clearance test returned to normal values and the rise in hepatic collagen synthesis activity in the malotilate-treated and intoxicated rats was reduced as compared with intoxicated control rats. The same effect was found when malotilate (100 mg/kg, p.o., 5 days per week), was given for 3 weeks to rats already intoxicated during the 6 previous weeks. Malotilate was able to prevent the increase of hepatic alterations that appeared during the last 3 weeks of CC14 intoxication, These results show clearly that malotilate can markedly reduce the hepatic disorders induced by a chronic CCI 4 intoxication in the rat.

Introduction Malotilate (diisopropyl-l.3-dithiol-2-ylidene malonate) is a new drug proposed for the treatment of chronic liver disease. It has been reported that malotilate protects against acute liver injury in laboratory animals induced by allylalcohol, bromobenzene, car-

bon tetrachloride (CCI4), chloroform, dimethylnitrosamine, thioacetamide [1] and paracetamol [2]. This compound has a protective and therapeutic effect on fatty liver induced by CCI 4 [3]. The aim of this study was to test the influence of malotilate on another experimental model of chronic liver disease, the CC14 induced hepatic fibrosis.

Correspondence and reprint requests to: J.-M. Dumont, Zyma SA, CH - 1260Nyon, Switzerland, Tel. (22) 63 11 11. 0168-8278/86/$03.50© 1986Elsevier SciencePublishers B.V. (BiomedicalDivision)

MALOTILATE IN CHRONIC CCI4 INJURY Since, in this model, fibrosis is largely due to de novo collagen synthesis [4,5], the influence of malotilate treatment on hepatic collagen synthesis was also studied

261 Weeks ol t r e a t m e n t Groups

Animals Male RA 25 rats (obtained from Ciba-Geigy, Basel, Switzerland) of 250-280 g were used, They were fed a standard maintenance diet (890-899 OANI NAFAG, Gossau, Switzerland) ad libitum during the whole experiment and fasted 24 h before sacrifice. Chemicals Maiotilate (diisopropyl-l.3-dithiol-2-ylidene-malonate) was produced by Nihon Nohyaku Ltd (Japan), its purity was greater than 99%. CCI 4 (analytical grade) was obtained from Merck (Darmstadt, F.R.G.). Treatment of animals CCI 4 (0.5 ml/kg dissolved in liquid paraffin), was given by intraperitoneal (i.p.) route twice weekly (Tuesday and Friday at 10 am). Malotilate (50 or 100 mg/kg), suspended in a 5% arabic gum solution, was administered orally 5 days per week (Monday to Friday at 10 am). Control animals received an equal volume of vehicles alone. 104 rats were randomized into 8 groups with different durations of treatment with CCI 4 (6 or 9 weeks) according to the schedule reported in Fig. 1. Malotilate was given at two different dosages (50 and 100 mg/kg) and with two different periods of therapy: 50 mg/kg during 6 weeks of CC14 intoxication (experiment A) and 100 mg/kg from the 6th to the 9th week of CCI 4 intoxication (experiment B). The animals were killed 3 days after the last administration of G E l 4. They were anaesthetized with ether; blood from the abdominal aorta was collected in the presence of heparin. The liver was rinsed with saline and stored at -20 °C until processed for lipid and hydroxyproline content measurements.

6_

N E xpemment A 6 w e e k s CCI 4 0 5 ml/kg twice a week

Materials and Methods

,3

o

Malohlate 50 mg/kg p,, I~r 6 weeks

E ~perlment 8 9 ~ e e ~ s CCI4 0 5 m l / k 9 t w i c e a,,~,eek Malolllate 100 rng/kg p o f r o m the 6 t h up to the 9 t h w e e k

10

[

]

12

[

J

15

L~

]

15

[${'k~J,x,N.~Sx."~Xx.~- ::,t

~o

[

~2

[

15

[-

I

I

L::

I

Molotllate

I.

I

CCI 4

I

'

,

D¢{<<<.'<.]

Vehicle

CCt4 • Malotllate

Fig. 1. Experimental schedule.

Parameters Investigated The animals were weighed once per week during the whole experiment. Body weight and liver weight were measured at the time of killing. Aspartate aminotransferase (ASAT) activity, alanine aminotransferase (ALAT) activity, glutamate dehydrogenase (GIDH) activity, alkaline phosphatase activity, total bilirubin, total proteins, albumin, triglycerides and total cholesterol levels were determined in the plasma automatically with a centrifuge analyzer 'Cobas' using commercial reagent kit from Roche Diagnostica, Switzerland. A bromosulfophtaleine (BSP) clearance test was carried out under light ether anaesthesia 4 days before the killing of the animals [6]. Hepatic total lipid content was determined by the method of Foich et al. [7]. Hepatic hydroxyproline, as an index of hepatic collagen, was measured according to Prockop and Udenfriend [8]. Results are expressed as hepatic collagen content in mg/g delipidated dry liver. Hepatic protein synthesis activity was determined by incorporation of labelled proline into collagen and non-colla-

262

J.-M. DUMONT et al.

gen proteins [9]. Twenty-four h before killing, the rats were injected i.p. with 10/~Ci/100 g of [14C]proline (proline, L-[14C(u)], New England Nuclear). After killing, the liver samples were taken, washed by saline, delipidated by a solution of chloroform/methanol (2:1), absolute ethanol and dried by ether. Sample portions (about 100 mg) were submitted to hydrolyse with 6N HCI for 24 h at 110 °C. From the liver samples, Proline (Pro) and hydroxyproline (HyPro) were then separated by modification of the chromatographic method described by Etherington et al. [10]. The hydrolysate sample was passed through a column (1 cm x 25 cm) of Duolite SRC16 equilibrated with 0.067 M sodium citrate buffer, p H 3.25. The elution was developed at 30 °C with the same buffer. The radioactivity of Pro and HyPro was measured by counting an aliquot of each fraction (scintillation fluid: aqualyte Baker liquid; scintillation counter: Intertechnique SL4000). The quantification of the apparent collagen synthesis rate was carried out by measuring the conversion of [14C]Pro

to protein bound [14C]HyPro. The Pro radioactivity corresponds to total protein synthesis activity and the HyPro radioactivity to collagenous protein synthesi~ activity. Then the ratio Pro/HyPro which represents the activity of total proteins versus that of collagen was calculated. This investigation was performed with 5 animals per group in both experiments. Histological examinations were carried out on liver specimens, and fixed in Bouin's fluid. Each liver section was stained with hematoxylin-eosin and Masson's trichrome. Hepatic tissue was assessed, without knowledge of the biochemical analysis, for histological features of necrosis, inflammation, steatosis, fibrosis and ductular proliferation. The degree of alteration was noted on an arbitrary scale from A (normal) to D (severe). In each group of rats, the animals were classified according to their degree of pathology.

Biometric Analysis of the Results For the parameters 'plasma enzyme activities' and

TABLE 1 RAT BODY WEIGHT AND LIVER WEIGHT AT THE TIME OF KILLING Mean + 1 SD.

• Experiment A (6 weeks) Control

Number of rats/group Body weight (g)

10 374+38

Maiotilate

12 375+41

Liver weight (g) 9.54+0.96 11.25+1.73 Liver weight/ body weight % 2.55+0.06 Gain of body weight during the experi ments (g)

70+37

Experiment B (9 weeks) EEl 4

11 307+16 +++ 10.34+2.06

3.01+0.11 +++ 3.38+0.22 +++

91+38

5+22 +++

EEl 4 + malotilate

Control

14

10

353+25*** 10.80+1.80

3.07+0.14"**

39+20*

Malotilate

CCI4

G E l 4 q-

malotilate 12

444+12

439+42

10.75+1.21

11.44+1.00

2.41+0.20

2.60+0.21

133+14

128+31

11 335+29 +++ 11.87+1.14

12 371+32" 11.79+1.25

3.55+0.36 +++ 3.19+0.40

32+21 +++

70+26***

Statistically significant differences: +++P < 0.001 as compared to control; *P < 0.05 as compared to CC14; ***P < 0.001 as compared to C C i 4

MALOTILATE IN CHRONIC

CCl 4

INJURY

plasma bilirubin level, the statistical evaluation was carried out on the logarithms of the value, since it is known that these parameters follow a log normal distribution [11], the tables indicate the value of the antilogarithm of the mean and standard deviation in parenthesis. In the case of the other parameters, the arithmetic means and standard deviation are given. Comparison of means was made by Student's t-test. Results

Five animals died in the experiment A (4 in the CC14 group and 1 in the CC14 + malotilate) and 4 in the experiment B (2 CC14 and 2 CC14 + maiotilate) during the ether anaesthesia for the determination of BSP clearance. 3 rats of experiment B (2 CCI 4 and 1 CC14 + malotilate) died 2 weeks before killing.

Body and Liver Weight In both experiments, the CCI4-intoxicated animals gained significantly less weight than control animals (Table 1). In malotilate- and CC14-treated rats, the gain of body weight was significantly higher than that of intoxicated rats. No differences concerning the liver weight were noted between the different groups. The liver weight to body weight ratio was significantly greater in the CCl4-treated groups; it was significantly decreased by malotilate treatment in experiment A. The treatment with malotilate alone (50 mg/kg for 6 weeks) increased the liver weight to body weight ratio.

263 when it was administered from the 6th up to the 9th week of CC14 intoxication. Total plasma protein and albumin levels did not change after 6 weeks of CC14, a moderate decrease was noted after 9 weeks of intoxication. In this case, malotilate failed to normalize total protein and albumin levels, although a trend to an increase was observed. A significant rise of plasma triglycerides and total cholesterol levels was observed after 6 weeks of CC14 intoxication; this alteration was much more marked after 9 weeks. Malotilate, in the experiment B, Suppressed the change of these lipid parameters. A decrease of hepatic excretory function (as indicated by respectively 34% and 48% diminution of the BSP clearance) appeared after 6 and 9 weeks of CCI 4 (Fig. 2); the BSP clearance was normalized by the malotilate treatments in the two experiments.

Hepatic Biochemical Parameters Malotilate alone did not modify significantly any hepatic parameters in the normal rat. Hepatic lipid content (Fig. 3) was significantly higher in the 6 weeks CCI 4 group (22% rise) than in control group. No further increase was observed after continuation of the intoxication. In the two experiments, malotilate inhibited completely the CCl4-in-

*/. 25

2O

Plasma Biochemical Parameters No significant changes in all investigated parameters were observed between control and malotilate control groups (Table 2). Plasma activities of both cytoplasmic and mitochondrial enzymes (ALAT, ASAT and GIDH) were significantly increased by the CC14 intoxication. This rise was much more pronounced after 9 weeks than after 6 weeks of intoxication, respectively 7.0 and 4.2 times the control values for the ASAT. Plasma bilirubin level and phosphatase alkaline activity were also enhanced by CC14 in a time-dependent manner. These alterations were significantly alleviated by malotilate treatment, even

By

¢'315 De_

~

au'~ ~m ~E5

°"

I

10

0

I~

i

Experiment B CCl 4 9 w e e k s

Experiment A CCl4 6 w e e k s Control Malotllote CCl 4 CC[ 4 • iViolotllate

Fig. 2. BSP clearance.

-** p 0 0010S compared t o c o n t r o l s p ' 0 001 as compared to CCI 4 alone ~_.SD

xxx

264

J.-M. DUMONT et al.

TABLE 2 PLASMA BIOCHEMICAL PARAMETERS AT THE TIME OF KILLING Results are expressed as antilogarithm of the mean and SD in brackets for plasma enzyme activities and bilirubin, as arithmetic mean + SD for the other parameters. Groups

ALAT (IU/I)

ASAT (IU/I)

GIDH (IU/I)

Alkaline phosphatase (IU/I)

Bilirubin (,umol/l)

Total proteins (g/l)

Albumin (g/l)

Tryglycerides (mmol/l)

Total cholesterol (mmol/I)

22 (17-28)

37 (27-50)

18 (13-24)

57 (49-66)

0.79 (0.51-1.24)

58.7+2.8

25.0+1.9

0.80+0.20

0.57+0.26

24 (7-34)

34 (26-45)

14 (8-24)

57 (47-69)

1.21 (0.75-1.64)

61.3+5.5

25.3+3.0

0.91+0.22

0.61+0.21

91 +++ (60-138)

155+++ (79-303)

37++ (20-68)

113++ (65-198)

2.83 + (1.30-6.17)

57.8+2.7

26.0+1.4

1.06+0.19 + 1.07+0.43 ++

70** (33-127)

19 (6-57)

61"** (54-69)

1.49" (1.04-2.15)

60.4+1.9

26.7+1.0

0.96+0.28

0.84+0.19

Experiment A 6 weeks CCI4 Control Control malotilate 50 mg/kg p.o. CCI4

CCI4+ malotilate 50 44* mg/kg p.o. (20-97)

Experiment B (9 weeks CC14) Control Control malotilate 100 mg/kg p.o. CCI4

20 (17-23)

75 (63-89)

14 (10-19)

53 (41-68)

0.74 (0.29-1.86)

61.0+1.8

26.5+0.9

0.96+0.13

0.69+0.13

21 (15-29)

61 (33-112)

21 (13-34)

55 (44-70)

0.54 (0.25-1.18)

63.0+2.5

26.1+ 1.0

0.96+0.26

0.81+0.23

205 +++ (118-357)

524 +++ (290-947)

74 +++ (35-158)

175+++ (144-269)

9.53 +++ 57.8+2.8 + 24.4+2.2 + 1.76+0.89 + 2.14+1.45 ++ (4.60-19.70)

95*** (77-117)

20*** (13-33)

62*** (54-72)

1.52 I** (0.84-2.70)

CCI4+ malotilate 100 52*** mg/kg p.o. (29-92)

59.4+2.0

25.6+1.1

0.89_+0.30** 1.06+0.22"

Significant differences: Control compared with CCI4: +P < 0.05, ++P < 0.01, +++P < 0.001; CCI4 compared with CCI4 + malotilate: *P < 0.05, **P < 0.01, ***P < 0.001. duced elevation of lipid content. Table 3 presents data on collagen metabolism: CC14 administration for 6 and 9 weeks p r o v o k e d respectively a 2 and 3 times increase of the hepatic collagen content. This accumulation of collagen was related to an increased hepatic collagen synthesis activity, as reflected by the modification of the H y P r o radioactivity and the Pro/ H y P r o ratio. T h e reduction of this ratio was m o r e m a r k e d after 9 weeks (51% of diminution) than after 6 weeks (19%) of CC14. The decrease of the ratio Pro/HyPro is the consequence of an increase of the collagen synthesis activity, as indicated by a stronger stimulation of H y P r o radioactivity versus Pro radioactivity in both experiments. In the case of experiment A , malotilate inhibited c o m p l e t e l y the hepatic

collagen accumulation and the ratio P r o / H y P r o returned to normal values. In the e x p e r i m e n t B, malotilate, a d m i n i s t e r e d from the 6th to the 9th week of intoxication, suppressed totally the rise in hepatic collagen, which a p p e a r e d during the last 3 weeks of the intoxication period. M a l o t i l a t e normalized the collagen synthesis activity (expressed by the H y P r o radioactivity) and the value of P r o / H y P r o ratio was similar to that of control.

Liver Histological Examinations The results of the histological examinations of each liver specimen are p r e s e n t e d in Table 4 and illustrated in Figs. 4 and 5. A f t e r 6 weeks of intoxication, there was m o d e r a t e

265

MALOTILATE IN CHRONIC CCI4 INJURY 125

markedly

~oo

(necrosis and steatosis), and ductular proliferation. It almost completely prevented the fibrosis. W h e n

~

decreased

hepatocellular degeneration

the drug was given from the 6th to the 9th week of CCI 4 intoxication, it prevented the aggravation of liver alterations, especially fibrosis.

_LL P 5c

Discussion

25

Experiment A CCl4 6weeks EZ] Control /v~loUlate CCI 4 t3k'3~ CCI4 *Malotllote

Chronic administration of CCI 4 to rats induces a severe impairment of hepatic functions resulting from

Experiment B CCt,i 9weeks • p ~ 0 0 5 . . . p
Fig. 3. Hepatic total lipids.

necrosis, severe inflammation and fatty infiltration. The liver showed moderate fibrosis, connective tissue septa and ductular proliferation. Three weeks later, these liver alterations appeared to be more severe: many animals showed cirrhosis with thick connective tissue septa and small nodules of liver cell plates, as well as signs of regeneration, such as binucleated hepatocytes and polyploidy. Malotilate, given at 50 mg/kg during the 6 weeks of intoxication,

marked necrosis, steatosis and fibrosis [12-14]. In our experiment we observed that, except for steatosis, liver alterations were more p r o n o u n c e d after 9 weeks of intoxication than after 6 weeks. Malotilate treatment reduced most of the signs of CCl 4 chronic intoxication, namely necrosis, steatosis and fibrosis, even when the treatment started 6 weeks after the beginning of the intoxication. Correction of these alterations by malotilate was associated with a protection of liver function as demonstrated by maintenance of a normal BSP clearance test in malotilatetreated rats. It has been reported previously that malotilate treatment protected against hepatic necrosis and

TABLE 3 COLLAGEN AND NON-COLLAGEN PROTEIN SYNTHESIS IN THE LIVER Mean + 1 SD, 5 animals per group for determination of Pro and HyPro radioactivity. Groups

Experiment A (6 weeks CC14) Control Control malotilate 50 mg/kg p.o. CC14 CCI4 + malotilate 50 mg/kg p.o. Experiment B (9 weeks EEl4) Control Control malotilate 100 mg/kg p.o. CCI 4

EEl 4 + malotilate 100 mg/kg p.o.

Collagen content ( m g / g dry liver)

Hydroxyproline radioactivity (cpm/mg tissue)

Proline radioactivity (cpm/mgtissue)

Pro/HyPro

6.5 + 1.3 6.5 + 1.1 15.5 + 5.1 +++ 7.2 + 1.7"**

28.8 + 1.0 29.5 + 2.0 52.8 + 14.0++ 31.7 +__8.0*

1513 + 25 1426 + 73 2005 + 290++ 1406 + 196"*

52.5 + 48.5 + 39.3 + 46.2 +

0.9 1.0+++ 7.9 ++ 9.0

5.5 + 0.7 5.9 + 0.8 21.1 + 6.5 +++ 11.9 + 4.2***

40.5 + 2.0 32.7 + 5.0 + 89.5 + 34.0+ 36.0 + 4.5**

1710 _ 20 1506 + 159 2086 + 293+ 1409 + 56***

41.5 + 46.7 + 25.0 + 39.7 +

6 7.0 1.5+++ 6.0***

Statistically significant differences: +P < 0.05, ++P < 0.01, +++P < 0.001 as compared to control group; *P < 0.05, **P < 0.01, ***P < 0.001 as compared to CCI4group.

266

Fig. 4. A: Control malotilate 50 mg/kg. Portal tract free of pathological changes. Note the regular size of liver cell nuclei. Gomori stain, x 160. B: CCI 4 6 weeks. Irregular bundles of collagen fibers are salient in the centrolobular area at upper left side. The liver cell nuclei is enlarged. Note steatosis at lower left side. Gomori stain, x 160. C: CCI 4 + malotilate 100 mg/kg from 6th to 9th week. Only delicate collagen fibers are visible . The cell and the nuclei are mainly of normal size. Steatosis is not obvious. Gomori stain, x 160.

J.-M. D U M O N T et al.

Fig. 5. A: Control malotilate 100 mg/kg from 6th to 9th week. Part of liver Iobule with a portal tract in the upper left side. The liver cells are arranged in normal plates. Gomori stain, x 160. B: Bundles of collagen fibers in a large portal-central septurn. Note an extreme atrophy of the liver parenchyma and many cellular infiltrates. Gomori stain, x 160. C: CCI 4 9 weeks + malotilate 100 mg/kg from 6th to 9th week. The collagen bundles, partly along a septum, partly surrounding small groups of liver cells are relatively fine. Necrosis is less developed. Gomori stain, x 160.

MALOTILATE IN CHRONIC CCI4 INJURY

267

TABLE 4 LIVER HISTOLOGICAL EXAMINATIONS Groups

n

Necrosis

Inflammation

Steatosis

Fibrosis

Ductular proliferation

A B C D

A B C D

A B C D

A B C D

A B C D

4 6 0 0 11 1 0 0 0 0 2 9 3 11 0 0

10 12 0 6

0 0 0 8

0 0 9 0

0 0 2 0

4 8 0 0

5 4 0 8

0 0 8 4

0 0 3 0

10 12 0 0

0 0 0 2

0 0 2 8

0 0 9 2

4 8 0 0

3 4 0 6

3 0 2 6

0 0 9 0

Experiment A (6 weeks CCI4) Control Control malotilate 50 mg/kg p.o. CCI4 CCl4+malotilate50mg/kgp.o.

10 12 I1 14

3 11 0 1

7 1 0 9

0 0 7 4

0 0 4 0

1 9 0 0 0 12 0 0 0 0 3 8 0 9 5 0

Experiment B (9 weeks CC14) Control Controlmalotilatel00mg/kgp.o. CCI4 CCI4+malotilatel00mg/kgp.o.

10 12 11 12

2 10 0 0

8 2 0 3

0 0 3 9

0 0 8 0

0 0 0 0

10 12 0 2

0 0 3 10

0 0 8 0

4 7 0 0

5 5 0 6

1 0 6 6

0 0 5 0

n = number of animals per group. Score: A = normal, B = slight, C = moderate, D = severe.

steatosis induced by different hepatotoxic agents [1-3]. This protection was associated with an enh a n c e m e n t of liver protein synthesis by malotilate [15,16]. In addition to the confirmation of the antinecrotic and antisteatosic effects of the c o m p o u n d , the present study brings new data showing an antifibrotic effect of malotilate. The m e a s u r e m e n t of collagen content in the liver, as confirmed by histological examination, showed a highly significant decrease of the fibrotic process in the m a l o t i l a t e - t r e a t e d animals. The chronic CCi4 intoxication m o d e l is a very severe experimental model and few c o m p o u n d s (colchicine [17,18], D-penicillamine [19], zinc [20, 21], L-azetidine-2-carboxylic acid [22] have been shown to decrease or stabilize hepatic collagen accumulation under these conditions. It is worth noting that malotilate exerted its antifibrotic effect u n d e r 2 schedules of administration: 50 mg/kg/day p.o. given since the beginning of the intoxication p e r i o d (experiment A ) and 100 mg/kg/day orally given from the 6th to the 9th week of intoxication ( e x p e r i m e n t B). It is thought that the latter condition mimics better than the former the clinical situations where therapy is started in patients with already established liver fibrosis and still subjected to an ongoing liver injury (alcohol or virus). A n antifibrotic effect in the m o d e l of chronic CCI 4 intoxication may be elicited by different mecha-

nisms: (a) inhibition of the bioactivation of CC! 4 to its toxic metabolites; (b) decrease of the cell necrosis; (c) suppression of the fibrogenic stimuli; (d) inhibition of the collagen biosynthesis or increase of the collagen degradation. Previous experiments [23,24] d e m o n s t r a t e d that malotilate is not an inhibitor, but rather an activator of the liver microsomal drug oxidation sytem. Part of this system is involved in CCI 4 metabolism. It is therefore unlikely that the protective effect of malotilate resulted from an inhibition of CCI4 bioactivation. Since malotilate protects hepatocytes against necrosis, acutely or chronically induced by CC14, it may be that the antifibrotic effect results from the antinecrotic effect and hence from a reduction in the level of the pathological fibrogenic stimuli. O u r experiment supports this view since we observed a parallel evolution of necrosis and fibrosis assessed by histological or biochemical examinations. It was however surprising to see that the level of hepatic collagen of animals treated with malotilate from the 6th to the 9th week (experiment B) was stabilized, although the drug treatment started at a time when the necrotic fibrogenic process was fully activated. In our e x p e r i m e n t we observed that malotilate did not modify the hepatic collagen biosynthesis of normal control rats, as measured by the incorporation of

268 [14C]proline into proteins. In another study, Ryhanen et al. [25] reported that malotilate did not modify the hydroxyproline content of tissues (liver, lung, skin) in young rats and did not inhibit the collagen biosynthesis of h u m a n skin fibroblasts in cultures. It is therefore excluded that malotilate behaves as a direct inhibitor of the normal collagen biosynthetic pathways. Malotilate, although it does not modify

J.-M. DUMONT et al. inhibit the collagen production of pathological connective tissue cells. Experiments along this line of research are presently in progress and preliminary resuits suggest such an action of malotilate [26]. Whatever the mechanism involved, this experim e n t shows that malotilate is able to reduce liver injury induced by CC14 in the rat even when administered 6 weeks after the beginning of the intoxication.

the collagen metabolism of normal fibroblasts, could

References 1 Kitagawa H, Saito H, Sugimoto T, et al. Effects of diisopropyl-l.3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs and heavy metals. J. Toxicol Sci 1982; 7: 123-134. 2 Younes M, Siegers CP. Effect of malotilate on paracetamol induced hepatotoxicity. Tox Lett 1985; 25: 143-146. 3 Imaizumi Y, Sugimoto T, Kasai T. Effect of diisopropyl1.3-dithiol-2-ylidenemalonate on fatty liver induced by carbon tetrachloride. Jap J Pharmacol 1981;31: 15-21. 4 Tamayo RP. Is cirrhosis of the liver experimentally produced by CCI4 an adequate model of human cirrhosis. Hepatology 1983; 3: 112-120. 5 Huberman A, Recio A, Rojkind M. Collagen biosynthesis in normal and cirrhotic rat liver slices. Proc Soc Exp Biol Med 1969; 131: 200-209. 6 Perrissoud D. L'61ectrophor~se en gel de polyacrylamide. In: A Bertelli (Ed.), New Trends in the Therapy of Liver Diseases. S. Karger, Basel, 1975: 38-54. 7 Folch J, Lees, M, Stanley, GH. A simple method for the isolation and purification of total lipids from animals tissues. J Biol Chem, 1957; 226: 497-510. 8 Prockop DJ, Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1960; 1: 228-239. 9 Flandin F, Buffevent C, Herbage D. Age-related changes in the biochemical and physico-chemical properties of rat skin. Collagen synthesis and maturation and mechanical parameters (an axial tension). Cell Mol Biol 1986; In press. 10 Etherington DJ. Stability of rat skin collagen during recovery from under-nutrition.Biochem J 1977; 168: 579-581. 11 Heath DF. Normal or log normal: Appropriate distributions. Nature (Lond) 1967;213: 1159-1160. 12 Popper H, Udenfriend S. Hepatic fibrosis. Correlation of biochemical and morphologic investigations. Amer J Med 1070; 49: 707-721. 13 Galligani L, Lonati-Galligani M, Fuller G. Collagen metabolism in the liver of normal and carbon tetrachloride treated rats. Biomed 1979; 31: 199-201. 14 Rojkind M, Rojkind MH, Cordero-Hernandez H. In vivo collagen synthesis and deposition in fibrotic and regenerat-

ing rat livers. Collagen Rel Res 1983; 3: 335-347. 15 Imaizumi Y, Katoh M. Effect of diisopropyl-l.3-dithiol-2ylidene malonate on the regeneration of partially hepatectomized rat liver. Fol Pharmacol Jap 1981; 78: 60. 16 Iamizumi Y, Katoh M, Sugimoto T, Kasai T. Effect of malotilate on the protein synthesis in rat liver. Jap J Pharmacol 1982; 32: 369-375. 17 Tanner MS, Jackson D, Mowatt DP. Hepatic collagen synthesis in a rat model of cirrhosis and its modification by colchicine. J Path 1981; 135: 179-187. 18 Rojkind M, Kersherobich D. Effect of colchicine on collagen albumin and transferrin synthesis by cirrhotic rat liver slices. Biochim Biophys Acta 1975; 378: 415-423. 19 Tugarinova VN, Zolotnistskaya RP, Lavrova LA et al. Effect of D-penicillamineon the development and induction of experimental cirrhosis of the liver. Pat Fiziol Exp Ter 1979; 6: 37-41. 20 Chrapil M, Ryan JN, Elias SL, Peng YM. Protective effect of zinc on carbon tetrachloride induced liver injury in rats. Exp Mol Path 1973; 19: 186-196. 21 Anttinen H, Ryhanen L, Puistola Arrato A, Oikarinen A. Decrease in liver collagen accumulation in carbon tetrachloride injured and normal growing rats upon administration of zinc. Gastroenterology 1984; 86: 532-539. 22 Rojkind M, Inhibition of liver fibrosis by L-azetidine-2carboxylic acid in rats treated with carbon tetrachloride. J Clin Invest 1973; 52: 2451-2456. 23 Kawata S, Sugiyama T, Seki K, et al. Stimulatory effect of cytochrome b5 induced by p-nitroanisole and diisopropyl1.3-dithiol-2-ylidine malonate on rat liver microsomal drug hydroxylations. J Biochem 1982; 92: 305-313. 24 Katoh M, Kjtaka M, Satoh T, Khagawa H, Sugimoto T, Kasai T. Further studies on the in vivo effect of diisopropyl1.3-dithiol-2-ylidine maionate (NKK-105) as the liver microsomal drug oxydation system in rats. Biochem Pharmacol 1981; 30: 2759-2768. 25 Ryhanen L, Hamalainen L. Effect of malotilate on the metabolism of collagen. (Abstract). EASL Satellite Symposium, Berne, 1984: 17. 26 Borojevic R, Vinhas SA, Domont GB, Grimaud, JA. Effect of malotilate on human connective tissue cells in vitro. (Abstract) EASL Satellite Symposium, Berne, 1984: 15.