Effect of colchicine on collagen, albumin and transferrin synthesis by cirrhotic rat liver slices

415

Biochimica et Biophysica Acta, 378 ( 1 9 7 5 ) 4 1 5 - - 4 2 3 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA 98214

E F F E C T OF COLCHICINE ON COLLAGEN, ALBUMIN AND T R A N S F E R R I N SYNTHESIS BY C I R R H O T I C R A T L I V E R SLICES

MARCOS ROJKIND a and DAVID KERSHENOBICH b

aDepartamento de Biologia Celular, Centro de Investigacion y de Estudios, Avanzados del I. P.N., Apartado Postal 14- 740, M#xico 14, D.F. and bDepartam ento de Gastroenterologia, Instituto Nacional de la NutriciOn, Mdxico 22, D.F. (Mexico) ( R e c e i v e d J u l y 1 9 t h , 1974}

Summary Collagen synthesis was found to be increased in liver slices of rats made cirrhotic by chronic administration of CC14. The liver function was impaired, as determined by an increased retention of conjugated bilirubin and low serum albumin values. However, when animals received colchicine simultaneously with CCI 4, collagen synthesis and deposition were inhibited, and the liver function appeared normal. When a group of rats was made cirrhotic by chronic administration of CC14, and then kept for 30 days w i t h o u t further treatment, fibrosis persisted and collagen synthesis was very low. However, the liver function was severely impaired. When similar rats received L-azetidine-2-carboxylic acid during the 30-days period following CCI 4 administration, there was a slight b u t not significant improvement in liver function. The collagen synthesis and the extent of fibrosis were similar to the controls. However, if similar rats received colchicine during the 30 days period, collagen synthesis was almost negligible, there was a slight decrease in fibrosis and there was a great improvement in liver function. In all the cirrhotic animals studied, transferrin biosynthesis remained constant.

Introduction It has been shown in our laboratory that L-azetidine-2-carboxylic acid (AZC), a proline analog that competes with proline for transport and acylation of proline~specific tRNA, inhibits liver fibrogenesis when administered to rats during the induction of CC14 cirrhosis of the liver [1]. Modifications in the extent of collagen synthesis and deposition are accompanied b y variations in

* Abbreviation: AZC, L-azetidine-2-carboxylic acid.

416 the synthesis of albumin but not of transferrin [2]. Although the albumin content in the sera of cirrhotic rats is low, the livers have the capability of synthesizing albumin in amounts larger than normal [2,3]. Upon AZC administration, both the synthesis of albumin by the liver and the serum albumin values return to normal [2]. Recently, it has been suggested that the transfer of collagen from the fibroblast to the extracellular space is a microtubule-mediated process [4,5]. Furthermore, it was shown that colchicine, an agent that disrupts microtubule formation, inhibits collagen transport and also collagen biosynthesis [4,5]. Since colchicine is known also to enhance the production of mammalian collagenase in different tissues [ 6 ] , this c o m p o u n d could be of use in preventing experimental liver fibrosis. Preliminary results from our laboratory have shown that colchicine indeed inhibits collagen biosynthesis and deposition in the experimental animal and also improves some of the abnormal liver clinical values of human patients with liver cirrhosis [7]. The present communication describes the results obtained after treatment of rats with colchicine during the induction of liver cirrhosis with CCl~ and also the effect of colchicine and AZC on an established liver cirrhosis. Material and Methods Preparation of antisera Transferrin and albumin were isolated from rat serum and purified by gel filtration and ion-exchange chromatography as previously reported [2]. The purity of the proteins was determined by immunoelectrophoresis and by Laurell two-dimensional electrophoresis [8]. In both cases a single band of precipitation was obtained. Rabbits were immunized by intramuscular injection of the purified proteins suspended in complete Freund's adjuvant. The dose, schedule and the methods used to judge the purity of the antibodies obtained have been already published [2]. Induction o f liver cirrhosis Liver cirrhosis was produced in rats by chronic intraperitoneal administration 3 times per week (total 20 injections), of 0.15 ml of CCI4 in mineral oil (1 : 7, v/v) [9]. The animals, each weighing 50 to 100 g initially were weighed every other day throughout the experiment and were divided into two groups. The first group, a control series, received only 20 injections of CC14. A second group consisted of rats which, in addition to an identical CC14 treatment, received 10 t~g per day, 5 days a week of colchicine dissolved in water. Colchicine was delivered into the stomach with a canula attached to a syringe. Between 24 to 48 h after the last injection of CC14, the animals were exsanguinated by heart puncture and then killed by a blow to the head. Blood samples were used to measure serum iron and total iron-binding capacity [ 1 0 ] , albumin [11] and conjugated and unconjugated serum bilirubin [ 1 2 ] . Post-CCl 4 administration period A group of 30 rats was made cirrhotic by chronic administration of CC14

417

as previously described. After completing the 20 injections of CCI 4 the animals were divided into four groups. The first group consisted of rats killed 24 to 48 h after the last dose of CC14 and were used to determine the extent of fibrosis produced. The second group consisted of rats kept for 30 days without any further treatment. The third group c o n s i s t e d of animals treated with 10 pmoles of AZC every other day for a total of 12 doses. Azetidine was administered orally (vide supra). The fourth group consisted of rats treated with 10 pg a day (5 days a week) of colchicine during 4 weeks. Colchicine was administered as described. The animals of the last three groups were fed ad libitum and did not receive any further injections of CC14. The animals were sacrificed as described above. In vitro incorporation of 14 C-labeled proline into collagen and non-collagenous proteins 1 g (wet wt) portions of liver slices were placed in a 25-ml Erlenmeyer flask containing 3 ml of Ringer's bicarbonate solution and 1 pCi of U-x4Clabeled proline (spec. act., 1 Ci/mole). The samples were incubated for 60 min in a D u b n o f f incubator at 37°C under an atmosphere of 95% O: and 5% CO2. The reaction was stopped b y rapidly chilling the flasks; 3 ml of 10% ice-cold trichloroacetic acid was added, and the mixture was then homogenized. The precipitated protein was collected by centrifugation at 750 )< g for 15 min, washed twice in succession with 6 ml of cold 5% trichloroacetic acid, and collagen extracted in its denatured form with hot 5% trichloroacetic acid as described previously [1]. The extract was dialyzed to remove the acid and hydrolyzed in 6 M HCI. The residual protein after acid extraction (mainly non-collagenous proteins) was also hydrolyzed in 6 M HC1. Proline and hydroxyproline as well as the incorporation of radioactive proline into proline or collagen hydroxyproline were analyzed by the method of Rojkind and Gonza]ez [13]. In vitro incorporation o f ~4 C-labeled proline into albumin and transferrin Approx. 1 g of liver slices (wet wt) was placed in a 25-ml Erlenmeyer flask containing 10 ml of a medium of the following composition: 105/zmoles NaCl, 10 /lmoles KC1, 10 pmoles CaCl:, 40 #moles NaHCO3 and 1/~Ci of [U -~ 4C]proline (1 Ci/mole). Incubations were carried out for 2.5 h at 37°C in a Dubnoff metabolic water bath with a continuous atmosphere of O2--CO2 (95 : 5,

v/v). The reaction was terminated by homogenization of the flask contents and centrifugation at 20 000 × g for 20 min. The supernatant was dialyzed for 24 h with tap water and 48 h with two changes of distilled water. An aliquot of I ml of the supernatant was "cleared" twice with 4/zl of human serum and 0.15 ml of whole human antiserum [14] and then used for the precipitation of transferrin and albumin with the corresponding antibodies [2]. The immunoprecipitates were dissolved in 1 ml of " S o l u e n e " TMI o0 (Packard) and quantitatively transferred to low-background scintillation vials containing 16 ml of scintillation fluid. The a m o u n t of albumin present in the "cleared" supernatant was determined b y the m e t h o d of Fernandez et al. [15] and transferrin by the m e t h o d of Beale et al. [10].

418

All determinations were performed in duplicate with the livers from three to six rats in each experimental group. The values reported are means +- S.D. Results

The growth curve of the CC14-treated animals, and the animals receiving colchicine in addition to CC14 were very similar (see Fig. 1). These results are comparable to those obtained when CC14 treated animals received in addition AZC. Histological examination revealed that the livers of animals treated with CC14 showed the well-known large increase in collagen and reticular fibers, cell necrosis, fatty infiltration and distortion of liver architecture. The livers of animals treated with colchicine in addition to CC14 showed a lesser degree of fibrosis, b u t the same degree of necrosis and fatty infiltration. Similar results have been reported for AZC-treated, cirrhotic animals [1]. The group of rats kept for 30 days after CC14 administration was ended showed the same histological appearance as the cirrhotic animals. The extent of necrosis, however, appeared to be reduced. The livers of rats treated with AZC during the 30 days of regression, did n o t show any improvement in fibrosis or fatty infiltration. In contrast to these findings, the livers of rats treated with colchicine during the post-CC1, administration period showed a lesser a m o u n t of collagen deposition and the extent of fatty infiltration was also reduced. Incorporation of radioactive proline into collagen hydroxyproline was greatly enhanced in the animals receiving CC14. However, in the animals treated with colchicine, a 50% inhibition in proline incorporation into collagen hydroxyproline was observed. Furthermore, the incorporation of radioactive proline into the non-collagenous proteins was n o t modified. These results appeared in a preliminary communication [7]. Liver function in rats receiving CC14 was impaired as judged by the high values of conjugated bilirubin, and the modifications in the serum albumin levels. In the CCl4-treated animals the a m o u n t of conjugated bilirubin was 1.00 -+ 0.33 mg per 100 ml. However, in the colchicine-treated animals conjugated bilirubin was 0.49 +- 0.29 mg per 100 ml. Total protein in the sera of the

2O0

ISQ Z ~.~ LOC

50

'~o

~o

F i g . 1. G r o w t h t o CC14 (=

50

60

/o

8'o

~o

AGE iN DAYS o ) and o f rats t r e a t e d w i t h c o l c h l c l n e in a d d i t i o n c u r v e s o f r a t s t r e a t e d w i t h C C l 4 (o -~). E a c h p o i n t r e p r e s e n t s the average w e i g h t o f f o u r d i f f e r e n t a n i m a l s .

419

TABLE I EFFECT

OF COLCHICINE ON SERUM ALBUMIN AND GLOBULIN LEVELS IN CIRRHOTIC

RATS

D u p l i c a t e a n a l y s e s w e r e p e r f o r m e d w i t h the sera o f three to six d i f f e r e n t a n / m a l ~ V a l u e s are m e a n s -+ S.D. T h e P v a l u e s w e r e o b t a i n e d b y S t u d e n t ' s t test. O n l y s i g n i f i c a n t P v a l u e s are s h o w n .

Control (cirrhotics) Cirrhotics + colchicine

Total protein

Albumin

Globulin

(gllO0 ml)

(gll00 ml)

(gll00 ml)

4.58 ± 0.48 5.01 ± 0 . 1 6

2.22 ± 0.73 3.03 ± 0.28

2.16 + 0 . 8 1 1.98 + 0 . 2 5

(P < 0.05)

(P < 0.025)

CCl4-treated animals was normal (see Table I). However, the serum albumin values were below normal, and the globulins were increased. The differences in globulin content in both groups were not statisticallysignificant. Albumin biosynthesis by liver slices of cirrhotic animals was increased as compared to livers of untreated animals of the same age (see Table II). The values obtained were similar to the values previously reported [2]. In the colchicine-treated animals, the extent of albumin synthesis was in general smaller than that of the cirrhotic animals. However, the values were still above normal, and the differences were not statisticallysignificant. The net amount of albumin in the slicesof the cirrhotic animals was 439 + 20.5 #g/g. This value is similar to the value previously reported for the cirrhotic animals and is above the amount reported for normal livers(355 -+ 12 pg/g) (P < 0.05) [2]. In the animals treated with colchicine in addition of CCI4, the amount of albumin in the liverwas 391 -+ 8.3 #g/g. In these experiments, the synthesis of transferrin has been used as an internal control, to rule out differences in synthesis due to changes in the amino acid pools. It was previously shown that the amount of transferrinand the incorporation of proline into transferrin remained constant in the liversof cirrhotic animals [2]. As can be seen in Table II the incorporation of radioactive proline into transferrin of both cirrhotic and colchicine liver slices was the same. Since colchicine [7] and A Z C [1] have been shown to have an inhibitory effect on fibrogenesis during the induction of CCI4 cirrhosisof the liver,it was T A B L E II E F F E C T O F C O L C H I C I N E O N A L B U M I N A N D ~ T R A N S F E R R I N B I O S Y N T H E S I S BY L I V E R S L I C E S OF CIRRHOTIC RATS D u p l i c a t e a n a l y s e s w e r e p e r f o r m e d w i t h the livers o f three to six d i f f e r e n t a n i m a l s . V a l u e s are m e a n s -+ S.D. T h e P value= w e r e o b t a i n e d b y S t u d e n t ' s t test, a n d w e r e n o n - s i g n i f i c a n t .

Control (cirrhotics) Cirrhotics + c o l c h i c i n e Normals * T a k e n f r o m ref. 2.

Albumin (dpm//~g)

Transferrin (dpm/#g)

2.94 ± 0.56 2.46 ± 0.30 1.94 ± 0.08*

1 . 6 6 -+ 0 . 0 8 1 . 5 8 -+ 0 . 1 8 1 . 6 0 -* 0 . 1 2 "

420

T A B L E Ill E F F E C T O F AZC OR C O L C H I C I N E D U R I N G T H E POST-CCI 4 A D M I N I S T R A T I O N P E R I O D * ON C O L L A G E N B I O S Y N T H E S I S BY L I V E R S L I C E S O F C I R R H O T I C R A T S D u p l i c a t e a n a l y s e s w e r e p e r f o r m e d w i t h the livers o f t h r e e to six d i f f e r e n t animals. V a l u e s ate m e a n s ± S.D. T h e P values w e r e o b t a i n e d b y S t u d e n t ' s t test. Only significant P values axe s h o w n . Group

Total protein proline (dpm/~mole)

Collagen hydtox~,proline (dpm//~mole)

I

Control (cirrhotics)

2 2 4 0 0 0 +- 4 9 3 3 5

1138

II

Post-CC14 c o n t r o l

69700 ± 24194 (P < 0 . 0 0 2 5 ) * *

199 ± 44 (P < 0 . 0 0 2 5 ) * *

l I I Post-CCl 4 A Z C - t r e a t e d group

94100-+ 13985 (P < 0 . 0 0 5 ) * *

321 ± 165 (P < 0 . 0 0 5 ) * *

I V Post-CC14 c o l e h i c m e - t r e a t e d g r o u p

46875 ± 21918 (P < 0 . 0 0 5 ) * *

77.5 ± 29.3 (P < 0 . 0 0 2 5 ) * * ' * * *

+- 3 5 9

* A f t e r 2 0 i n j e c t i o n s o f CCI 4 the t o x i c agent w a s d i s c o n t i n u e d a n d t h e rats k e p t o n a n o r m a l diet for 30 days. ** C o m p a r e d to Group I. *** C o m p a r e d to G r o u p If.

important to determine whether the two c o m p o u n d s are also effective in modifying the course of the fibrotic process after cirrhosis has been established by 20 injections of CCL. A new group of cirrhotic rats was prepared as described under Materials and Methods, and the incorporation of radioactive proline into collagen hydroxyproline and into proline of non-collagenous proteins was determined. As can be seen in Table III, collagen hydroxyproline contained 1138 +- 359 dpm/pmole. The incorporation of radioactive proline into proline of non-collagenous proteins was very high in this particular groups of rats as compared with the values obtained in several other experiments. In the cirrhotic animals kept for 30 days w i t h o u t any further treatment of CC14, the incorporation of radioactive proline into collagen hydroxyproline was greatly diminished (P < 0.0025). The incorporation of proline into the non-collagenous proteins was similar to the values previously reported [1]. The specific activity of collagen hydroxyproline in the group of rats treated with colchicine for 30 days after termination of CC14 administration was very low (P < 0.0025). Similar results were obtained with the group of animals treated with AZC during the post-CC14 administratior~ period. The a m o u n t of hydroxyproline (collagen) in the four groups is shown in Table IV. As can be seen, the animals kept for 30 days after CC14 treatment was ended, have in general lower collagen values than the control cirrhotic group. However, the differences are n o t statistically significant. Liver function was n o t greatly impaired in these cirrhotic animals. As can be seen in Table V, the a m o u n t of serum albumin is close to normal values. However, in the cirrhotic animals kept for 30 days w i t h o u t any further treatment the total protein and serum albumin decreased (P < 0.005} and the globulins increased (P < 0.025). In the post-CCl4 colchicine-treated group the total protein was very close to normal values as well as the serum albumin.

T A B L E IV E F F E C T O F A Z C O R C O L C H I C I N E D U R I N G T H E POST-CCI 4 A D M I N I S T R A T I O N P E R I O D * ON T H E HYDROXYPROLINE CONTENT OF CIRRHOTIC LIVERS D u p l i c a t e a n a l y s e s w e r e p e r f o r m e d w i t h t h e livers o f t h r e e to six d i f f e r e n t a n i m a l s . V a l u e s sxe m e a n s -+ S.D. T h e P v a l u e s w e r e o b t a i n e d b y S t u d e n t ' s t test, and w e r e n o n - s i g n i f i c a n t . Group

Hydroxyproline (~moles/g)

I

Control (ctrrhotics)

2.34 ± 0.36

II

Post-CCl 4 c o n t r o l

2.13 ± 0.25

III Post-CCl 4 A Z C - t r e a t e d g r o u p

1.87 -+ 0 . 3 0

IV Post-CCl 4 c o l c h i c i n e - t r e a t e d g r o u p

1.93 ± 0.31

* A f t e r 20 i n j e c t i o n s o f CCI 4, t h e t o x i c a g e n t w a s d i s c o n t i n u e d a n d the rats k e p t o n a n o r m a l diet for 3 0 days.

TABLE V E F F E C T O F A Z C O R C O L C H I C I N E D U R I N G T H E POST-CCI4 A D M I N I S T R A T I O N P E R I O D * ON S E R U M A L B U M I N A N D G L O B U L I N L E V E L S IN C I R R H O T I C R A T S D u p l i c a t e a n a l y s e s w e r e p e r f o r m e d w i t h the sera o f t h r e e to six d i f f e r e n t animals. V a l u e s are m e a n s -+ S.D. T h e P values w e r e o b t a i n e d b y S t u d e n t ' s t test. Only s i g n i f i c a n t P values are s h o w n . Group

Total protein

Albumin

Globulin

(g/100 ml)

(g/100 ml)

(g/lO0 m l )

I

Control (cirrhotics)

4.46 ± 0 . 2 5

2.93 ± 0 . 2 5

1.53 ± 0 . 2 5

II

Post-CCl 4 c o n t r o l

3.60 ± 0.20 (P < 0 . 0 0 5 ) * *

1.63 ~ 0 . 1 5 (P < 0 . 0 0 5 ) * *

1.96 -+ 0 . 1 5 (P < 0 . 0 2 5 ) * *

Ill P o s t ' C C l 4 A Z C - t r e a t e d g r o u p

5.33 ± 1 . 3 4 (P < 0 . 0 5 ) * * *

2.30 ± 0.55 (P < 0 . 0 5 ) * * *

3.03 -+ 1.05 (P < 0 . 0 5 ) * *

I V Post-CCl4 c o l c h i c i n e - t r e a t e d g r o u p

4.97 ± 0 . 7 b (P < 0.005)***

2.87 ± 0.37

2.17 -+ 0 . 8 5

(P < 0.005)***

* A f t e r 20 i n j e c t i o n s o f CC14, the t o x i c a g e n t w a s d i s c o n t i n u e d a n d the r a t s k e p t on a n o r m a l d i e t for 30 days. ** C o m p a r e d to G r o u p I. *** C o m p a r e d t o G r o u p lI, T A B L E VI E F F E C T O F AZC O R C O L C H I C I N E D U R I N G T H E POST-CCI 4 A D M I N I S T R A T I O N P E R I O D * ON A L B U M I N A N D T R A N S F E R R I N B I O S Y N T H E S I S BY L I V E R S L I C E S O F C I R R H O T I C R A T S D u p l i c a t e a n a l y s e s w e r e p e r f o r m e d w i t h t h e livers o f t h r e e t o six d i f f e r e n t animals. V a l u e s are m e a n s ± S.D. T h e P v a l u e s w e r e o b t a i n e d b y the S t u d e n t ' s t test. Only s i g n i f i c a n t P values are s h o w n . Group

Albumin (dpm/pg)

Transferrin ( d p m ]/,lg )

1

Control (cirrhotics)

2.53 ± 0 . 1 5

1.70 ± 0 . 2 6

II

Post-CCl 4 c o n t r o l

2.53 ± 0 . 6 6

1.46 ± 0 . 2 8

III Post-CCl 4 A Z C - t r e a t e d g r o u p

2.46 ± 0 . 5 0

1.60 ± 0 . 3 0

I V Post-CCl 4 c o l c h i c i n e - t r e a t e d g r o u p

2.05 ± 0 . 2 0 (P < 0 . 0 0 5 ) * *

1.62 -+ 0 . 2 5

* A f t e r 20 i n j e c t i o n s of CC140 t h e t o x i c a g e n t w a s d i s c o n t i n u e d a n d t h e rats k e p t on a n o r m a l d i e t for 3 0 days. ** C o m p a r e d t o G r o u p I.

.t22

However, the globulins remained increased. In the post-CCl4 AZC-treated group the total proteins were higher than normal, due to a great increase in the globulin fraction. Synthesis of albumin and transferrin by liver slices was also determined in the four groups. As can be seen in Table VI, the incorporation of proline into transferrin was very similar in all the groups. In the cirrhotic animals, the incorporation of proline into albumin was increased and the same was found in all groups except for the colchicine treated animals in which albumin biosynthesis was normal (P < 0.005). Discussion The results obtained in this and other communications [1,2,7] demonstrate that collagen biosynthesis in the liver "in vivo" can be inhibited with drugs that modify collagen biosynthesis "in vitro" [ 1 6 - - 1 9 ] . The specificity of these drugs may depend, among other factors, on the rate of collagen synthesis in a particular tissue, and also, on the actual concentration of the drug. In rats with CCl 4-induced cirrhosis, the liver synthesizes collagen very actively [9] and also concentrates AZC and colchicine. At the dose used, both c o m p o u n d s inhibit collagen synthesis in the liver and do not show detectable side effects. The growth of the animals is normal, as well as the red blood cell count, serum iron and total iron-binding capacity. However, different proline analogues, have different degrees of toxicity depending on the animal species used. 3,4-Dehydroproline is very toxic for mice when administered "in vivo" [20]. Collagen biosynthesis in the cirrhotic animals kept for 30 days w i t h o u t any treatment was very low. However, the extent of fibrosis determined by histological and chemical methods was the same as in the cirrhotic animals. These results could indicate that collagen synthesis is turned off when CCI4 administration is discontinued. Furthermore, the presence of identical amounts of collagen in the liver of cirrhotic animals and in livers of rats kept for 30 days after discontinuation of CCl4 suggest that the mechanisms needed for collagen degradation are not actively working at this time. A direct measurement of collagenase [21]. should provide an answer to this very important question. In the colchicine-treated animals, collagen synthesis was even more diminished than in the untreated rats and a lesser a m o u n t of collagen was seen in the histological sections. In the AZC-treated animals, collagen synthesis was similar to the untreated group. These results are to be expected if AZC interferes with transport of proline, and acylation of t R N A by proline [22,23]. Thus, AZC is more effective in a tissue that is actively synthesizing collagen. Since discontinuation of CC14 administration is accompanied by a decrease in collagen synthesis, AZC is less effective; however, colchicine has an effect on the biosynthesis, and could also influence collagen degradation. If colchicine enhances the biosynthesis of tissue collagenase as has been shown "in vitro" for synovial collagenase [6], collagen reabsorption in the liver could take place at a faster rate than in the other two groups. Administration of colchicine is also accompanied by an improvement in the liver function of the animals. These findings are similar to the results obtained in cirrhotic rats treated with AZC, and suggest the possibility that at

423 least some abnormalities in CC14-induced liver cirrhosis may be secondary to scar formation, and perhaps independent of cell necrosis. It is possible that a small percentage of viable cells could perform a normal liver function. As already shown, CC14-treated animals have abnormal liver function tests. However, in the animals kept for 30 days without any further administration of CCI4, the function of the liver was even worse, in spite of the observed decrease in collagen synthesis. This finding is also in agreement with the role of fibrogenesis in modifying the function of liver cells, in the absence of any further cell damage. Whether this fact is due only to a modification of collagen biosynthesis and degradation, or if colchicine has a direct effect on liver function, is currently under investigation. Colchicine has been shown to induce a marked increase in fecal sterols and bile acids, which is accompanied by a decrease in serum cholesterol [ 2 4 ] . The albumin content in the liver of CC14treated rats which received colchicine reached normal values. In the untreated cirrhotic liver, however, albumin is increased. These and other similar effects of colchicine are perhaps partially responsible for the improvement of liver function in the cirrhotic liver. The results obtained in the colchicine-treated animal have been reproduced in human patients with liver cirrhosis [ 7 ] , providing a new and perhaps useful approach in the treatment of this disease.

Acknowledgements The authors are indebted to Professor Ruy Pdrez-Tamayo, for his help in preparing the manuscript. This investigation was supported in part with a grant from C~nara Nacional de la Industria de Laboratorios Quimfco-Farrnacduticos, M6xico, D.F. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

R o j k i n d , M. ( 1 9 7 3 ) J. Clin. Invest. 52, 2 4 5 1 - - 2 4 5 6 K e r s h e n o b i c h , D. a n d R o | k i n d , M. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 9 , 2 1 6 - - 2 2 2 H u b e r m a n , A. and S o b e r d n , G. ( 1 9 7 0 ) Clin. Chim. A c t a 29, 1 2 1 - - 1 2 7 Ehrlich, H.P. and Bornstein, P. ( 1 9 7 2 ) Nat. N e w Biol. 2 3 8 , 2 5 7 - - 2 6 0 D i e g e l m a n n , R . F . a n d P e t e r k o f s k y , B. ( 1 9 7 2 ) Proc. Natl. A c a d . Sci. U.S. 6 9 , 8 9 2 . - 8 9 6 Harris, E.D. and Krane, S.M. ( 1 9 7 1 ) Arthritis R h e u m . 14, 6 6 9 - - 6 8 4 R o j k i n d , M., Uribe, M. and K e r s h e n o b i c h , D. ( 1 9 7 3 ) L a n c e t 7 7 9 3 , 3 8 - - 3 9 Clarke, H.G.M. and F r e e m a n , T. ( 1 9 6 7 ) in Proc. 1 4 t h Coll. Burges, 1966. Protides o f the Biological Fluids (Peeters, H., ed.), pp. 5 0 3 - - 5 0 6 , Elsevier, A m s t e r d a m H u b e r m a n , A., Recio, A. a n d R o i k i n d , M. ( 1 9 6 9 ) Proc. Soc. Exp. Biol. Med. 1 3 1 , 2 0 0 - - 2 0 3 Beale, R.N., B o s t r o m , J.O. and T a y l o r , R.S. ( 1 9 6 2 ) J. Clin. Pathol. 15, 1 5 6 - - 1 6 3 G o r n a l l , A.G., Bardawill, C.J. and David, M.M. ( 1 9 4 9 ) J. Biol. C h e m . 1 7 7 , 7 5 1 - - 7 6 6 S e p u l v e d a , B. and Osterberg, A.E. ( 1 9 4 3 ) J. Lab. Clin. Med. 28, 1 3 5 - - 1 3 9 R o j k i n d , M. a n d Gonz~lez, E. ( 1 9 7 4 ) Anal. B i o c h e m . 57, 1--7 C a m p b e l l , P.N. and S t o n e , N.E. ( 1 9 5 7 ) B i o c h e m . J. 6 6 , 19--31 F e r n a n d e z , A., Soben° C. and G o l d e n b e r g , H. ( 1 9 6 6 ) Clin. C h e m . 12° 1 9 4 - - 2 0 5 T a k e u c h i , T. a n d P r n c k o p , D.J. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 175, 1 4 2 - - 1 5 5 T a k e u c h i , T., R o s e n b l o o m , J. a n d P r o c k o p , D.J. ( 1 9 6 9 ) Biochim. B i o p h y s . A c t a 1 7 5 , 1 5 6 - - 1 6 4 R o s e n b l o o m , J. a n d P r o c k o p , D.J. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 3 3 6 1 - - 3 3 6 8 Uitto, J. a n d P r o c k o p , D.J. ( 1 9 7 4 ) ' B i o c h i m . Biophys. A c t a 3 3 6 , 2 3 4 - - 2 5 1 M a d d e n , J.W., Chvapil, M., Carlson, E.C. and R y a n , J.N. ( 1 9 7 3 ) Toxicol. Appl. P h a r m a c o L 26, 426--437 F u j i w a r a , K., Sakai, T., O d a , T. and lgarashi, S. ( 1 9 7 3 ) Biochem. B i o p h y s . Res. C o m m u n . 54, 5 3 1 - 537 F o w d e n , L. and R i c h m o n d , M.H. ( 1 9 6 3 ) B i o c h i m . B i o p h y s . A c t a 71, 4 5 9 - - 4 6 1 Papas, T.S. and Mehler, A.H. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 1 5 8 8 - - 1 5 9 5 Rubulis, A., R u b e r t , M. a n d F a l o o n , W.N. ( 1 9 7 0 ) Am. J. CHn. Nutr. 23, 1 2 5 1 - - 1 2 5 9