- Email: [email protected]
Toxic effect of streptozotocin on the biliary secretion of nicotinamide-treated rats
Toxicology Letters, 36 (1987) 259-265
259
Elsevier
TXL 01776
TOXIC EFFECT OF STREPTOZOTOCIN OF NICOTINAMIDE-TREATED RATS
(Streptozotocin;
C.E.
nicotinamide;
CARNOVALE,
Institute
R.A.
ON THE BILIARY SECRETION
bile flow; biliary excretion)
MARINELLI
de Fisiologia Experimental,
and E.A. Consejo
RODRIGUEZ
National
GARAY
de Investigaciones
Cientificas y TPcnicas,
Universidad National de Rosario, Rosario (Argentina) (Received
3 September
(Revision
received
(Accepted
1986)
15 December
5 January
1986)
1987)
SUMMARY The effect studied
of streptozotocin
(SZ) on bile flow (BF) and on protein
in rats with bile fistula.
was administrated Decreases SZ-treated
(500 mg/kg
Nicotinamide mediated
body
in BF and in biliary rats; conversely,
to the diabetic
excretion
proteins
and acid phosphatase
of cholesterol
the hyperglycemia
and phospholipid
biliary
state. The results also demonstrated
and phospholipids
were
induced outputs,
were observed
by SZ and also suppressed suggesting
in
was increased. the SZ-
that they could be related
a direct effect of SZ on BF and on the biliary
Since SZ is used clinically,
by this drug on the rat liver should
outputs
body wt.). Nicotinamide
10 min prior to SZ.
of bile acids,
prevented
of cholesterol
tion of bile acids and proteins. duced
wt., i.p.)
outputs
the biliary
pretreatment
increase
and lipid biliary
SZ was given i.v. as a single dose (50 mg/kg
and in experimental
diabetes,
excre-
the effects pro-
be considered.
INTRODUCTION
Streptozotocin (SZ) is a glucosamine-containing nitrosourea antibiotic [ 11, which given intravenously in the rat as a single dose causes a permanent diabetic state [2].
Correspondence: Ciencias
Dr. Emilio
Bioquimicas
Abbreviations: PI, phospholipids;
0378-4274/87/$
A. Rodriguez
y Farmactuticas,
SZ, streptozotocin; Pr, proteins;
03.50
0 Elsevier
Garay,
Suipacha
NICO,
lnstituto
de Fisiologia
570, 2000 Rosario,
nicotinamide;
Experimental,
Publishers
de
BF, bile flow; BA, bile acids; Cho, cholesterol;
AP, acid phosphatase.
Science
Fact&ad
Argentina.
B.V. (Biomedical
Division)
260
In addition, this drug also possesses antitumour activity as well as carcinogenic property [3]. SZ is a &cell of Langerhans’ islets toxin used to produce a model of diabetes in rats. Extrapancreatic toxic responses are also induced by this agent. In this connection, previous studies from this laboratory demonstrated that SZ-treated rats presented a decrease in bile flow (BF) and an alteration of the biliary excretion of bile acids (BA), proteins (Pr) and lipids [4]; however, it remains unclear if the impaired liver function observed in SZ-treated rats is a consequence of the diabetic state or results of a direct effect of SZ on the hepatobiliary system. Several studies demonstrated that a single administration (i.p.) of nicotinamide (NICO) prior to SZ prevented the diabetogenic effect of this latter without concomitant loss of its antitumour activity [5]. The purpose of this study was to investigate if the hepatic alterations observed in SZ-treated rats are the result of a direct toxic effect of SZ on the liver rather than a manifestation of its diabetogenic action. MATERIALS
AND METHODS
Animals and treatment Adult male Wistar rats weighing 310-370 g were used. All the animals were allowed free access to a standard diet commercial and tap water. SZ was dissolved in 0.05 M citrate buffer, pH 4.5, and used within 5 min of preparation 161. NICO was dissolved in distilled water [S]. Two series of four experimental groups were studied: (i) rats injected with a single i.v. dose of SZ (50 mg/kg body wt.), (ii) animals which received NICO (500 mg/kg body wt, i.p.); 10 min before SZ; (iii) rats injected i.p. with NICO alone; (iv) control animals which only received an i.v. injection of citrate buffer. Experimental procedures On a first series of experiments, rats were anaesthetized with sodium pentobarbital (50 mglkg body wt., i.p.) 24 hr after the iv. injection and the bile duct and the femoral vein were cannulated for bile and blood sampling, respectively. Immediately after cannulation, the bile was collected on ice into preweighed tubes for 60 min. The BF was measured and the concentrations of bile acids (BA), cholesterol (Cho), phospholipids (PI) and proteins (Pr) as well as the lysosomal acid phosphatase activity (AP) were determined. Rectal temperature was monitored for all rats and m~ntained at 38.0 t 05°C throughout the experiments to prevent hypothermic alterations of BF, as stated elsewhere [7]. At the end of bile collection a venous blood sample (50 ~1) was obtained for serum glucose quantitation and the liver was removed and weighed. In a second series of experiments, rats were anaesthetized, blood was withdrawn by heart puncture, and the liver was promptly removed. The largest lobe of the liver was homogenized in 9 volumes of ice-chilled Sorensen buffer (pH 7.4), using a Potter-Elvehjem tissue homogenizer equipped with a Teflon pestle. Homogenates
261
were centrifuged in a refrigerated centrifuge (3000 rpm, 10 min), and the supernatants were used for determination of Cho, PI, Pr and AP. Analytical methods Bile volume was measured by gravimetry, assuming a density for bile of 1.O g/ml; BF was expressed as ,ul/min/g of liver. BA concentration in bile was assayed with 3ol-hydroxysteroid dehydrogenase according to Talalay’s method modified by Berthelot et al. [8]. AP activity (EC 3.1.3.2), Pr, Cho, and Pl were measured in bile samples and liver homogenates. One unit of AP activity [9] corresponded to the hydrolysis of 1 (*mol of ~-naphthylphosphate/min at 37°C; Pr concentration was measured in bile samples [lo] and liver homogenates [I I], using bovine albumin as standard; Cho concentration was determined by the enzymatic esterase-oxidase methods [12]; Pl concentration was measured as phosphorus by the Bartlett’s method [13]. The biliary outputs of the bile constituents were calculated as the product of BF times the bile concentration or activity. Serum glucose was determined enzymatically [ 141. Chemicals All the chemicals used were of reagent grade quality. SZ, NICO, bovine albumin (fraction V) and 3c-u-hydroxysteroid dehydrogenase, were purchased from Sigma Chemical Company, U.S.A. GOD-PAP test was from Boehringer Mannheim, F.R.G., Colestat and FacP tests (for Cho and AP determination, respectively) were from Wiener Lab., Argentina. Statistics The results are expressed as mean values f S.E.M. Statistical significance of the differences between groups was assessed by the Kruskal-Wallis one-way analysis of variance; P values of 0.05 or less were considered significant. RESULTS
BF and biiiary outperts of lipids and proteins Data are shown in Table I. NICO pretreatment prevented the SZ-mediated increase in blood glucose levels, while it did not influence the SZ-induced decrease in BF and BA output. The outputs of Cho and PI in SZ-treated rats were significantly increased from controls, whereas they exhibited a significant decrease in NICOpretreated rats. The biliary excretion of Pr and AP were decreased in both SZ and SZ+ NICO-treated rats from controls. None of the parameters measured was affected by the pretreatment of NICO alone (P > 0.05 in comparison to control rats).
262
TABLE
I
SERUM
GLUCOSE
COMPONENTS
LEVELS,
BILE FLOW
IN CONTROL
Data are mean
+ S.E.M.
AND BILIARY
AND TREATED
from
EXCRETION
OF ENDOGENOUS
BILE
RATS
5-9 rats.
Rat treatment Controls
Parameter Serum
glucose
BF (al/min/g
(mg/dl)
121
of liver)
2.5
NICO f 10
107
* 0.04
2.5
SZ + NICO
SZ k 10 + 0.04
480
+9+
1.6+0.1+
129
f 12++
1.6 k
0.2+
BA output (nmol/min/g
of liver)
55.9
rt 2.4
51.6
k
5.2
34.2 k 3.3+
0.1
2.1+0.3+
41.2
k
3.4+
Cho output (nmol/min/g
of liver)
1.24f
0.1
0.95+
0.621
O.ll+
Pl output (nmol P/mitt/g
of liver)
9.5
*
1.3
8.7
+ 0.9
13.4+ 1.2+
25.1
k
4.6
22.0
+ 4.8 k54.4
5.6
k
0.9{+
12.1+2.6+
12.5
k
3.1+
92.4 + 4.9+
98.8
z!z36.1+
Pr output (pg/min/g
of liver)
AP output (JJ/min/g
of liver)
+Significantly
different
++Significantly
different
Determination
181.2
k36.0
213.2
from controls. from
SZ-treated
of bile components
group.
in liver homogenates
Data on hepatic Cho, Pl, Pr and AP are presented in Fig. 1. The hepatic content of Cho was increased in SZ-treated rats while it did not show difference in SZ + NICO-treated rats in comparison to controls. Hepatic PI content was increased in NICO-treated and SZ + NICO-treated groups, while the SZ-treated group did not show differences. No differences were found in the hepatic Pr content for the different groups, while AP activity in the supernatants was significantly increased in both SZ-treated and SZ+NICO-treated groups in comparison to controls. Hepatic Cho, Pr and AP contents in animals treated with NICO alone did not differ
from control
values.
DISCUSSION
As expected, the results indicated that a pretreatment of NICO prevents the appearance of the hyperglycemia seen in SZ-treated rats. However, NICO pretreatment did not prevent the decrease in the BF nor the decreased biliary excretion of BA produced by SZ-treatment (see Table I). Thus, the diabetic state is unlikely to be responsible for the decrease in BF and BA output. Previous data from this laboratory showed that SZ administration to rats produced a decrease in BF, by affecting both bile acid-independent (BIAF) and bile acid-dependent flow of bile (BADF) [4]. The diminished BA output observed in
263
this study is consistent with a decreased BADF. This may be due to an alteration of the enterohepatic circulation of these compounds (the main mechanism which support the hepatic pool of BA). Such an effect might be produced either by a direct effect of SZ on the intestinal cells, or by the antibiotic activity of SZ exerted on the intestinal bacteria impairing enzymatic biotransformation of bile salts in the intestinal lumen [ 151. It has been shown that some proteins within the hepatocyte are transported in secretory vesicles which move across the cell to the bile canalicular region; proteins thus transported are degraded within the hepatocyte and the vesicles containing them, fuse with lysosomes previously to the discharge into bile [16]. BA probably favour the uptake and intrahepatic transport of Pr [16] and the biliary secretion of lysosomal vesicles including enzymes like AP [17]. In this study, we observed an increase of the hepatic AP activity in SZ-treated and SZ + NICO-treated rats (see Fig. l), probably resulting from a lysosomal enzyme accumulation produced by its diminished biliary excretion. The decrease in Pr biliary excretion associated with the lack of modification of hepatic protein content (see Table I and Fig. l), indicates a diminution in their uptake and intrahepatic transport. In agreement with previous studies [17,18], these results could be related to the decrease in BA biliary secretion. Increases in hepatic and biliary Cho observed in SZ treated rats (see Fig. 1 and Table I) were probably related to the development of the diabetic state in agreement with other studies [ 191. In this regard, in SZ + NICO-treated animals (absence of
cl Controls NICO
z T G
sz
m
SZ+NlCO
++
B
1 5oo
q
2500 z
g g_ 2
1000
.Lz T 1500
500
P 2
500
z 6
Fig.
1. Hepatic cholesterol
trol and treated 3 %-treated
(A), phospholipid
tt
and Methods)
slgmfxant
(B), protein
Data are mean values
rats and 4 SZ + NICO-treated
ed in Materials controls;
rats.
+ S.E.M.
rats. Homogenates
using the supernatants
difference
from
(C) and acid phosphatase for 4 controls,
SZ-treated
were prepared
for determinations. groups.
(D) contents
4 rats treated
with NICO
and centrifuged t Significant
in conalone,
(as describ-
difference
from
264
hyperglycemia) the hepatic Cho did not differ from controls, while the Cho output was decreased (see Figure 1 and Table I). This latter might be due to diminished BA secretion because the rate of BA output is the major driving force for Cho secretion in bile [20]. Several studies have demonstrated the relationship between Cho and Pl outputs [21-231. In agreement to this view, the changes in Cho biliary output described in this study were accompanied by parallel variations in Pl secretion in both SZtreated rats and SZ + NICO-treated animals. SZ did not modify the hepatic Pl content; the increase observed in SZ+NICO-treated rats could be due at least in part by an effect of NICO itself, as may be suggested by the results in NICO-treated group (see Fig. 1). In conclusion, SZ decreases BF and alters the outputs of some bile components by a mechanism non dependent on its diabetogenic action. The effects induced by SZ should be considered in hepatic metabolism studies in experimental diabetes induced shortly after the administration of this compound. Furthermore, extrapolation of animal data to humans suggests that physicians should take in account that the liver function of SZ-treated patients could be modified. ACKNOWLEDGMENTS
This work was supported by a research grant from the Cr. sejo National de Investigaciones Cientrficas y Tecnicas (CONICET), ReptibLa Argentina. The valuable technical assistance of Mr. Raul TrbojeEich is gratefully acknowledged. REFERENCES 1 J.J. Vavra,
C. De Boer, A. Dietz, L.J. Hanka
antibiotic, 2 L.M.
Srivastava,
macol. 3 C.G.
Antibiotics
Ann.
P.S.
and W.T. Sololski,
Streptozotocin
a new antibacterial
230-235.
Bora and S.D. Bhatt,
Diabetogenic
action
of streptozotocin,
Trends
Phar-
Sci., 3 (1982) 376-378. Moertel
the malignant 4 C. Carnovale, position
and J. Hanley, carcinoid
tozotocin
Combination
syndrome,
R.A. Marinelli
diabetes
Roberts,
C.D.
sulfobromophtalein Biol. Med., 8 P. Berthelot, hypercholeresis
rats,
Clin. Trials,
Rodriguez Biochem.
and M.L. Vernon,
without
6 R.R. Herr, H.K. Jahnke 89 (1967) 4808-4809.
chemotherapy.
Cancer
and E.A.
in streptozotocin-treated
5 P.S. Schein, D.A. Cooney
8 R.J.
(1959/1960)
loss of antitumor
and A.D. Argoudelis, Klaassen
during
and
G.L.
Garay,
Bile flow decrease
Pharmacol.,
activity,
carcinoid
tumor
and
Cancer
The structure
bile com-
to modify
the toxicity
of strep-
Res., 27 (1967) 2324-2332. of streptozotocin,
Maximun alteration
and altered
(1987) in press.
The use of nicotinamide
Plaa,
anaesthesia-induced
Trials in metastatic 2 (1979) 327-334.
biliary of rectal
J. Am. Chem.
excretion temperature,
of
bilirubin
Proc.
Sot.
Sot., and Exp.
125 (1967) 313-316. S. Erlinger,
D. Dhumeaux
in the rat, Am. J. Physiol.,
and A.M.
Preaux,
9 A.L. Babson, P.A. Read and G.E. Phillips, The importance phosphatase in serum Am. J. Clin. Pathol., 32 (1959) 83-87. 10 O.H. Lowry, N.J. Rosebrough, phenol reagent, J. Biol. Chem.,
Mechanism
of phenobarbial
induced
819 (1970) 809-813.
A.L. Farr and R.J. 193 (1961) 265-275.
Randall,
of the substrate Protein
measurement
in assays
of acid
with the Folin
265 11 E.M. Suranyi and Y. Avi-Dor, SweIling-contraction of mitochondria in hypotonic medium, Biochim. Biophys. Acta, 118 (1966) 445-452. 12 R.J. Henry, D.S. Cannon and J.W. Winkelmann, Clinical Chemistry Principles and Techniques, Harper & Row, 1974, 1440-1449. 13 G.R. Barlett, Phosphorus assay in column chromatography, J. Biol. Chem., 66 (1925) 375-400. 14 P. Trinder, Determination of blood glucose using an oxidase peroxidase system with a noncarhnogenic chromogen., J. Clin. Pathol., 22 (1969) 158-161. 1.5 H.P.A. Illing, Critical review: Techniques for micro~oral and associated metabolic studies in reiation to the absorption and enterohepatic circulation of drugs, Xenobiotics, 11 (1981) 815-818. 16 A.L. Jones, D.L. Schumucker, R.H. Reston and T. Murakani, The architecture of bile secretion: A morphological perspective of physiology, Dig. Dis. Sci., 25 (1980) 609-629. 17 R.A. Marinelli, M.G. Luquita and E.A. Rodriguez Garay, Bile salt-related secretion of acid phosphatase in rat bile, Can. J. Physiol. Pharmacol., (1987) in press. 18 R.A. Marinelli, E.A. Rodriguez Garay, Excrecidn biliar de enzimas en la rata, Medicina, 46 (1986) 85-90. 19 CD. Saudek and H.A. Eder, Lipid metabofism in diabetes mellitus, Am. J. Med., 66 (1979) 843-852. 20 R. Del Pozo, F. Nervi, C. Covarrubias and F. Ronco, Reversal of Progesterone-induced biliary cholesterol output by dietary cholesterol and ethynyfestradiol. Biochim. Biophys. Acta, 753 (1983) 164-172. 21 D.L. Eaton and CD. Klaassen, Effects of acute administration af taurocholic and taurochenodeoxycholic acid on biliary lipid excretion in the rat, Proc. Sot. Exp. Biol. Med., 151 (1976) 198-202. 22 W.G.M. Hardison and J.T. Apter, Micellar theory of biliary cholesterol excretion, Am. J. Physiol., 222 (1972) 61-67. 23 H.O. Wheeler, Biliary excretion of bile acids, lecithin, and cholesterol in hamsters with gallstones, Gastroenterology, 65 (1973) 92-103.
259
Elsevier
TXL 01776
TOXIC EFFECT OF STREPTOZOTOCIN OF NICOTINAMIDE-TREATED RATS
(Streptozotocin;
C.E.
nicotinamide;
CARNOVALE,
Institute
R.A.
ON THE BILIARY SECRETION
bile flow; biliary excretion)
MARINELLI
de Fisiologia Experimental,
and E.A. Consejo
RODRIGUEZ
National
GARAY
de Investigaciones
Cientificas y TPcnicas,
Universidad National de Rosario, Rosario (Argentina) (Received
3 September
(Revision
received
(Accepted
1986)
15 December
5 January
1986)
1987)
SUMMARY The effect studied
of streptozotocin
(SZ) on bile flow (BF) and on protein
in rats with bile fistula.
was administrated Decreases SZ-treated
(500 mg/kg
Nicotinamide mediated
body
in BF and in biliary rats; conversely,
to the diabetic
excretion
proteins
and acid phosphatase
of cholesterol
the hyperglycemia
and phospholipid
biliary
state. The results also demonstrated
and phospholipids
were
induced outputs,
were observed
by SZ and also suppressed suggesting
in
was increased. the SZ-
that they could be related
a direct effect of SZ on BF and on the biliary
Since SZ is used clinically,
by this drug on the rat liver should
outputs
body wt.). Nicotinamide
10 min prior to SZ.
of bile acids,
prevented
of cholesterol
tion of bile acids and proteins. duced
wt., i.p.)
outputs
the biliary
pretreatment
increase
and lipid biliary
SZ was given i.v. as a single dose (50 mg/kg
and in experimental
diabetes,
excre-
the effects pro-
be considered.
INTRODUCTION
Streptozotocin (SZ) is a glucosamine-containing nitrosourea antibiotic [ 11, which given intravenously in the rat as a single dose causes a permanent diabetic state [2].
Correspondence: Ciencias
Dr. Emilio
Bioquimicas
Abbreviations: PI, phospholipids;
0378-4274/87/$
A. Rodriguez
y Farmactuticas,
SZ, streptozotocin; Pr, proteins;
03.50
0 Elsevier
Garay,
Suipacha
NICO,
lnstituto
de Fisiologia
570, 2000 Rosario,
nicotinamide;
Experimental,
Publishers
de
BF, bile flow; BA, bile acids; Cho, cholesterol;
AP, acid phosphatase.
Science
Fact&ad
Argentina.
B.V. (Biomedical
Division)
260
In addition, this drug also possesses antitumour activity as well as carcinogenic property [3]. SZ is a &cell of Langerhans’ islets toxin used to produce a model of diabetes in rats. Extrapancreatic toxic responses are also induced by this agent. In this connection, previous studies from this laboratory demonstrated that SZ-treated rats presented a decrease in bile flow (BF) and an alteration of the biliary excretion of bile acids (BA), proteins (Pr) and lipids [4]; however, it remains unclear if the impaired liver function observed in SZ-treated rats is a consequence of the diabetic state or results of a direct effect of SZ on the hepatobiliary system. Several studies demonstrated that a single administration (i.p.) of nicotinamide (NICO) prior to SZ prevented the diabetogenic effect of this latter without concomitant loss of its antitumour activity [5]. The purpose of this study was to investigate if the hepatic alterations observed in SZ-treated rats are the result of a direct toxic effect of SZ on the liver rather than a manifestation of its diabetogenic action. MATERIALS
AND METHODS
Animals and treatment Adult male Wistar rats weighing 310-370 g were used. All the animals were allowed free access to a standard diet commercial and tap water. SZ was dissolved in 0.05 M citrate buffer, pH 4.5, and used within 5 min of preparation 161. NICO was dissolved in distilled water [S]. Two series of four experimental groups were studied: (i) rats injected with a single i.v. dose of SZ (50 mg/kg body wt.), (ii) animals which received NICO (500 mg/kg body wt, i.p.); 10 min before SZ; (iii) rats injected i.p. with NICO alone; (iv) control animals which only received an i.v. injection of citrate buffer. Experimental procedures On a first series of experiments, rats were anaesthetized with sodium pentobarbital (50 mglkg body wt., i.p.) 24 hr after the iv. injection and the bile duct and the femoral vein were cannulated for bile and blood sampling, respectively. Immediately after cannulation, the bile was collected on ice into preweighed tubes for 60 min. The BF was measured and the concentrations of bile acids (BA), cholesterol (Cho), phospholipids (PI) and proteins (Pr) as well as the lysosomal acid phosphatase activity (AP) were determined. Rectal temperature was monitored for all rats and m~ntained at 38.0 t 05°C throughout the experiments to prevent hypothermic alterations of BF, as stated elsewhere [7]. At the end of bile collection a venous blood sample (50 ~1) was obtained for serum glucose quantitation and the liver was removed and weighed. In a second series of experiments, rats were anaesthetized, blood was withdrawn by heart puncture, and the liver was promptly removed. The largest lobe of the liver was homogenized in 9 volumes of ice-chilled Sorensen buffer (pH 7.4), using a Potter-Elvehjem tissue homogenizer equipped with a Teflon pestle. Homogenates
261
were centrifuged in a refrigerated centrifuge (3000 rpm, 10 min), and the supernatants were used for determination of Cho, PI, Pr and AP. Analytical methods Bile volume was measured by gravimetry, assuming a density for bile of 1.O g/ml; BF was expressed as ,ul/min/g of liver. BA concentration in bile was assayed with 3ol-hydroxysteroid dehydrogenase according to Talalay’s method modified by Berthelot et al. [8]. AP activity (EC 3.1.3.2), Pr, Cho, and Pl were measured in bile samples and liver homogenates. One unit of AP activity [9] corresponded to the hydrolysis of 1 (*mol of ~-naphthylphosphate/min at 37°C; Pr concentration was measured in bile samples [lo] and liver homogenates [I I], using bovine albumin as standard; Cho concentration was determined by the enzymatic esterase-oxidase methods [12]; Pl concentration was measured as phosphorus by the Bartlett’s method [13]. The biliary outputs of the bile constituents were calculated as the product of BF times the bile concentration or activity. Serum glucose was determined enzymatically [ 141. Chemicals All the chemicals used were of reagent grade quality. SZ, NICO, bovine albumin (fraction V) and 3c-u-hydroxysteroid dehydrogenase, were purchased from Sigma Chemical Company, U.S.A. GOD-PAP test was from Boehringer Mannheim, F.R.G., Colestat and FacP tests (for Cho and AP determination, respectively) were from Wiener Lab., Argentina. Statistics The results are expressed as mean values f S.E.M. Statistical significance of the differences between groups was assessed by the Kruskal-Wallis one-way analysis of variance; P values of 0.05 or less were considered significant. RESULTS
BF and biiiary outperts of lipids and proteins Data are shown in Table I. NICO pretreatment prevented the SZ-mediated increase in blood glucose levels, while it did not influence the SZ-induced decrease in BF and BA output. The outputs of Cho and PI in SZ-treated rats were significantly increased from controls, whereas they exhibited a significant decrease in NICOpretreated rats. The biliary excretion of Pr and AP were decreased in both SZ and SZ+ NICO-treated rats from controls. None of the parameters measured was affected by the pretreatment of NICO alone (P > 0.05 in comparison to control rats).
262
TABLE
I
SERUM
GLUCOSE
COMPONENTS
LEVELS,
BILE FLOW
IN CONTROL
Data are mean
+ S.E.M.
AND BILIARY
AND TREATED
from
EXCRETION
OF ENDOGENOUS
BILE
RATS
5-9 rats.
Rat treatment Controls
Parameter Serum
glucose
BF (al/min/g
(mg/dl)
121
of liver)
2.5
NICO f 10
107
* 0.04
2.5
SZ + NICO
SZ k 10 + 0.04
480
+9+
1.6+0.1+
129
f 12++
1.6 k
0.2+
BA output (nmol/min/g
of liver)
55.9
rt 2.4
51.6
k
5.2
34.2 k 3.3+
0.1
2.1+0.3+
41.2
k
3.4+
Cho output (nmol/min/g
of liver)
1.24f
0.1
0.95+
0.621
O.ll+
Pl output (nmol P/mitt/g
of liver)
9.5
*
1.3
8.7
+ 0.9
13.4+ 1.2+
25.1
k
4.6
22.0
+ 4.8 k54.4
5.6
k
0.9{+
12.1+2.6+
12.5
k
3.1+
92.4 + 4.9+
98.8
z!z36.1+
Pr output (pg/min/g
of liver)
AP output (JJ/min/g
of liver)
+Significantly
different
++Significantly
different
Determination
181.2
k36.0
213.2
from controls. from
SZ-treated
of bile components
group.
in liver homogenates
Data on hepatic Cho, Pl, Pr and AP are presented in Fig. 1. The hepatic content of Cho was increased in SZ-treated rats while it did not show difference in SZ + NICO-treated rats in comparison to controls. Hepatic PI content was increased in NICO-treated and SZ + NICO-treated groups, while the SZ-treated group did not show differences. No differences were found in the hepatic Pr content for the different groups, while AP activity in the supernatants was significantly increased in both SZ-treated and SZ+NICO-treated groups in comparison to controls. Hepatic Cho, Pr and AP contents in animals treated with NICO alone did not differ
from control
values.
DISCUSSION
As expected, the results indicated that a pretreatment of NICO prevents the appearance of the hyperglycemia seen in SZ-treated rats. However, NICO pretreatment did not prevent the decrease in the BF nor the decreased biliary excretion of BA produced by SZ-treatment (see Table I). Thus, the diabetic state is unlikely to be responsible for the decrease in BF and BA output. Previous data from this laboratory showed that SZ administration to rats produced a decrease in BF, by affecting both bile acid-independent (BIAF) and bile acid-dependent flow of bile (BADF) [4]. The diminished BA output observed in
263
this study is consistent with a decreased BADF. This may be due to an alteration of the enterohepatic circulation of these compounds (the main mechanism which support the hepatic pool of BA). Such an effect might be produced either by a direct effect of SZ on the intestinal cells, or by the antibiotic activity of SZ exerted on the intestinal bacteria impairing enzymatic biotransformation of bile salts in the intestinal lumen [ 151. It has been shown that some proteins within the hepatocyte are transported in secretory vesicles which move across the cell to the bile canalicular region; proteins thus transported are degraded within the hepatocyte and the vesicles containing them, fuse with lysosomes previously to the discharge into bile [16]. BA probably favour the uptake and intrahepatic transport of Pr [16] and the biliary secretion of lysosomal vesicles including enzymes like AP [17]. In this study, we observed an increase of the hepatic AP activity in SZ-treated and SZ + NICO-treated rats (see Fig. l), probably resulting from a lysosomal enzyme accumulation produced by its diminished biliary excretion. The decrease in Pr biliary excretion associated with the lack of modification of hepatic protein content (see Table I and Fig. l), indicates a diminution in their uptake and intrahepatic transport. In agreement with previous studies [17,18], these results could be related to the decrease in BA biliary secretion. Increases in hepatic and biliary Cho observed in SZ treated rats (see Fig. 1 and Table I) were probably related to the development of the diabetic state in agreement with other studies [ 191. In this regard, in SZ + NICO-treated animals (absence of
cl Controls NICO
z T G
sz
m
SZ+NlCO
++
B
1 5oo
q
2500 z
g g_ 2
1000
.Lz T 1500
500
P 2
500
z 6
Fig.
1. Hepatic cholesterol
trol and treated 3 %-treated
(A), phospholipid
tt
and Methods)
slgmfxant
(B), protein
Data are mean values
rats and 4 SZ + NICO-treated
ed in Materials controls;
rats.
+ S.E.M.
rats. Homogenates
using the supernatants
difference
from
(C) and acid phosphatase for 4 controls,
SZ-treated
were prepared
for determinations. groups.
(D) contents
4 rats treated
with NICO
and centrifuged t Significant
in conalone,
(as describ-
difference
from
264
hyperglycemia) the hepatic Cho did not differ from controls, while the Cho output was decreased (see Figure 1 and Table I). This latter might be due to diminished BA secretion because the rate of BA output is the major driving force for Cho secretion in bile [20]. Several studies have demonstrated the relationship between Cho and Pl outputs [21-231. In agreement to this view, the changes in Cho biliary output described in this study were accompanied by parallel variations in Pl secretion in both SZtreated rats and SZ + NICO-treated animals. SZ did not modify the hepatic Pl content; the increase observed in SZ+NICO-treated rats could be due at least in part by an effect of NICO itself, as may be suggested by the results in NICO-treated group (see Fig. 1). In conclusion, SZ decreases BF and alters the outputs of some bile components by a mechanism non dependent on its diabetogenic action. The effects induced by SZ should be considered in hepatic metabolism studies in experimental diabetes induced shortly after the administration of this compound. Furthermore, extrapolation of animal data to humans suggests that physicians should take in account that the liver function of SZ-treated patients could be modified. ACKNOWLEDGMENTS
This work was supported by a research grant from the Cr. sejo National de Investigaciones Cientrficas y Tecnicas (CONICET), ReptibLa Argentina. The valuable technical assistance of Mr. Raul TrbojeEich is gratefully acknowledged. REFERENCES 1 J.J. Vavra,
C. De Boer, A. Dietz, L.J. Hanka
antibiotic, 2 L.M.
Srivastava,
macol. 3 C.G.
Antibiotics
Ann.
P.S.
and W.T. Sololski,
Streptozotocin
a new antibacterial
230-235.
Bora and S.D. Bhatt,
Diabetogenic
action
of streptozotocin,
Trends
Phar-
Sci., 3 (1982) 376-378. Moertel
the malignant 4 C. Carnovale, position
and J. Hanley, carcinoid
tozotocin
Combination
syndrome,
R.A. Marinelli
diabetes
Roberts,
C.D.
sulfobromophtalein Biol. Med., 8 P. Berthelot, hypercholeresis
rats,
Clin. Trials,
Rodriguez Biochem.
and M.L. Vernon,
without
6 R.R. Herr, H.K. Jahnke 89 (1967) 4808-4809.
chemotherapy.
Cancer
and E.A.
in streptozotocin-treated
5 P.S. Schein, D.A. Cooney
8 R.J.
(1959/1960)
loss of antitumor
and A.D. Argoudelis, Klaassen
during
and
G.L.
Garay,
Bile flow decrease
Pharmacol.,
activity,
carcinoid
tumor
and
Cancer
The structure
bile com-
to modify
the toxicity
of strep-
Res., 27 (1967) 2324-2332. of streptozotocin,
Maximun alteration
and altered
(1987) in press.
The use of nicotinamide
Plaa,
anaesthesia-induced
Trials in metastatic 2 (1979) 327-334.
biliary of rectal
J. Am. Chem.
excretion temperature,
of
bilirubin
Proc.
Sot.
Sot., and Exp.
125 (1967) 313-316. S. Erlinger,
D. Dhumeaux
in the rat, Am. J. Physiol.,
and A.M.
Preaux,
9 A.L. Babson, P.A. Read and G.E. Phillips, The importance phosphatase in serum Am. J. Clin. Pathol., 32 (1959) 83-87. 10 O.H. Lowry, N.J. Rosebrough, phenol reagent, J. Biol. Chem.,
Mechanism
of phenobarbial
induced
819 (1970) 809-813.
A.L. Farr and R.J. 193 (1961) 265-275.
Randall,
of the substrate Protein
measurement
in assays
of acid
with the Folin
265 11 E.M. Suranyi and Y. Avi-Dor, SweIling-contraction of mitochondria in hypotonic medium, Biochim. Biophys. Acta, 118 (1966) 445-452. 12 R.J. Henry, D.S. Cannon and J.W. Winkelmann, Clinical Chemistry Principles and Techniques, Harper & Row, 1974, 1440-1449. 13 G.R. Barlett, Phosphorus assay in column chromatography, J. Biol. Chem., 66 (1925) 375-400. 14 P. Trinder, Determination of blood glucose using an oxidase peroxidase system with a noncarhnogenic chromogen., J. Clin. Pathol., 22 (1969) 158-161. 1.5 H.P.A. Illing, Critical review: Techniques for micro~oral and associated metabolic studies in reiation to the absorption and enterohepatic circulation of drugs, Xenobiotics, 11 (1981) 815-818. 16 A.L. Jones, D.L. Schumucker, R.H. Reston and T. Murakani, The architecture of bile secretion: A morphological perspective of physiology, Dig. Dis. Sci., 25 (1980) 609-629. 17 R.A. Marinelli, M.G. Luquita and E.A. Rodriguez Garay, Bile salt-related secretion of acid phosphatase in rat bile, Can. J. Physiol. Pharmacol., (1987) in press. 18 R.A. Marinelli, E.A. Rodriguez Garay, Excrecidn biliar de enzimas en la rata, Medicina, 46 (1986) 85-90. 19 CD. Saudek and H.A. Eder, Lipid metabofism in diabetes mellitus, Am. J. Med., 66 (1979) 843-852. 20 R. Del Pozo, F. Nervi, C. Covarrubias and F. Ronco, Reversal of Progesterone-induced biliary cholesterol output by dietary cholesterol and ethynyfestradiol. Biochim. Biophys. Acta, 753 (1983) 164-172. 21 D.L. Eaton and CD. Klaassen, Effects of acute administration af taurocholic and taurochenodeoxycholic acid on biliary lipid excretion in the rat, Proc. Sot. Exp. Biol. Med., 151 (1976) 198-202. 22 W.G.M. Hardison and J.T. Apter, Micellar theory of biliary cholesterol excretion, Am. J. Physiol., 222 (1972) 61-67. 23 H.O. Wheeler, Biliary excretion of bile acids, lecithin, and cholesterol in hamsters with gallstones, Gastroenterology, 65 (1973) 92-103.