TOXICOLOGY
AND
APPLIED
The Effects
PHARMACOLOGY,
‘&441-452
of Dantrolene the Isolated
(1978)
Sodium on Excretory Perfused Rat Liver’
CHARLESO.ABERNATHY,RICCARDOUTILI,*HYMAN EZEKIEL Liver
Research
Unit, Received
Veterans April
Administration 22,1977;
Function
ANDMILDRED
J.~IMMERMAN,
Hospital,
accepted
Washington,
October
in
D.C. 20422
25,1977
The Effects
of Dantrolene Sodium on Excretory Function in the Isolated Perfused Rat Liver. C. O., UTILI, R., ZIMMERMAN, H. J., AND EZEKIEL, M. (1978). Toxicol. Appl. Pharmacol. 44, 44 l-452. Perfusion of the isolated rat liver with dantrolene sodium led to inhibition of bile secretion and of sulfobromophthalein (BSP) excretion. The [ Wlerythritol clearance and bile acid excretion data suggested that the inhibitory effect of dantrolene was on the bile acidindependent rather than the bile acid-dependent fraction of bile. Inhibition by the drug of excretion of both indocyanine green and BSP suggests that interference with hepatic conjugative processes was not the mechanism for the reduction in organic anion excretion. Analysis of the BSP ‘transfer rates’ suggested that dantrolene exerted its effects on hepatic dye clearance at the excretory level. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene enhanced the adverse effects of dantrolene, suggesting that a cytochrome P-450.mediated metabolite led to the impairment of hepatic function. ABERNATHY,
Dantrolene sodium is a hydantoin derivative used to control muscle spasticity in patients suffering from a number of neurological disorders (Dykes, 1975). The drug is a peripherally acting muscle relaxant, which exerts its effects by blocking the release of calcium from the sarcoplasmic reticulum, thereby uncoupling excitation and contraction (Ellis and Bryant, 1972). There have been several reports of hepatic dysfunction associated with dantrolene therapy, ranging from moderate transient elevations of serum aspartate aminotransferase (GOT), alanine aminotransferase, alkaline phosphatase, and bilirubin (Mayer et al., 1973; Chyatte and Basmajian, 1973; Gelenberg and Poskanzer, 1973; Chipman et al., 1974) to overt hepatic injury (Ogburn et al., 1976; Schneider and Mitchell, 1976; Utili et al., 1977~). Several therapeutic drugs which have been reported to produce hepatic injury as an idiosyncratic reaction of patients have also been shown to produce adverse effects on in vitro model systems. For example, certain phenothiazines, tricyclic antidepressants, anesthetics, and erythromycin estolate have been reported to cause an e&x of cytoplasmic and lysosomal enzymes from cultured liver cells and isolated hepatocytes I This work was supported research funds. 2 Present address: Clinica Italy.
in part by a grant delle Malattie
from Eaton
Infettive,
Facolta 441
Laboratories di Medicina
and by Veterans I, Via Cotugna
Administration 1, 80135
Napoli,
004 1-008X/78/0443-0441 $02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
442
ABERNATHY
ET AL.
(Zimmerman and Kendler, 1970; Abernathy et al., 1975; Dujovne, 1975; Goto et al., 1976). Furthermore, many of these same drugs have also been reported to affect adversely liver function of the perfused rat liver (Kendler et al., 1971, 1972; Ros et al., 1975). Accordingly, the studies reported herein were designed to test the effects of dantrolene on isolated hepatocytes and on the isolated perfused rat liver (LPRL). METHODS
Animals. Male CD rats (350 to 450 g) were obtained from Charles River Laboratories (North Wilmington, Massachusetts) and maintained in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. They were permitted free access to food and water and were kept in the animal facility for at least 1 week prior to use. In the phenobarbital (PB) studies, the rats were given PB in saline (100 mg/kg, ip) daily for 5 days and sacrificed on the sixth. For the 3-methylcholanthrene (3-MC) studies, the compound was dissolved in corn oil and the animals were treated for 2 consecutive days (20 mg/kg, ip) and used for experimentation on the third day. Control animals received either saline or corn oil. Reagents. Sodium taurocholate was purchased from Calbiochem (La Jolla, California). Sulfobromophthalein (BSP) and indocyanine green (ICG) were obtained from Hynson, Westcott and Dunning (Baltimore, Maryland). Uniformly labeled [14Cl erythritol (2.7 mCi/mmol) and hydroxysteroid dehydrogenase were purchased from Amersham/Searle (Arlington Heights, Illinois), and Worthington Biochemicals (Freehold, New Jersey), respectively. The sodium salt of dantrolene was generously provided by Eaton Laboratories. All other chemicals were reagent grade and obtained from commercial sources. Methods. Isolated hepatocytes were prepared as described previously (Abernathy et al., 1975). Dantrolene sodium at various concentrations was added to the cells and the suspensions were incubated in test tubes without shaking for 30 min at 37OC. Then the cells were sedimented and the supernatant, designated as the medium, was decanted. The cells were lysed by alternatively freezing (dry ice in ethanol) and thawing (37’C water bath) three times. The level of GOT in each sample was measured (Zimmerman and Mao, 1965). Each test was run in triplicate and seven separate experiments were run. With minor modifications (Utili et al., 1976), the technique of Penhos et al. (1966) was used to prepare the liver for perfusion. Briefly, the rat was anesthesized with pentobarbital sodium (50 mg/kg) and the bile duct and portal vein were cannulated. The liver was transferred to a perfusion chamber (Metalloglass, Boston, Massachusetts) at 37 & 1“C. The perfusion fluid was wholly synthetic and consisted of a Krebs-Henseleit buffer (pH 7.45) augmented with 2500 U of heparin, 240 mg of glucose, and 2 g of bovine serum albumin per 100 ml and the total perfusate volume was 200 ml. The hydrostatic pressure on the portal vein was adjusted to 17 cm of water. To prevent organ anoxia, a mixture of 0, : CO, (95 : 5) was bubbled into the recirculating perfusate throughout the entire experimental period. Except where noted, taurocholate (0.5 pmol/min) was infused into the perfusate to replace the bile saltsnormally present in the enterohepatic circulation.
DANTROLENE
EFFECTS
ON HEPATIC
FUNCTION
443
After hepatectomy, each preparation was permitted a 30-min equilibration period, during which time the bile and perfusate flow rates were monitored (Utili et al., 1976) to assess the viability of the liver. At 30 min, dantrolene sodium, in various concentrations, dissolved in propylene glycol (solvent only in controls) was added to the perfusate. Fifteen minutes later, the dye, BSP (20 mg) or ICG (4 mg), was added and the perfusion was continued for an additional 45 min. Aliquots of bile (20 ~1) and perfusate (50 ~1) were taken every 5 min to measure the dye concentration in each sample. For each bile sample, the time needed to collect the 20 ~1 was measured and used as an estimate of bile flow rate. Measurement of dye concentration. To determine the concentration of BSP, the bile samples were diluted 500-fold with distilled water and perfusate samples were diluted 50-fold in 0.3 M sodiump-toluenesulfonate prior to alkalinization with 0.2 N NaOH. The amount of BSP in each sample was determined at 575 nm using the appropriate standards and the concentration mode of a Gilford 300 N spectrophotometer. For the ICG experiments, bile and perfusate samples were diluted 250- and 5-fold, respectively, with distilled water. Then the ICG content was assayed directly at 805 nm with a Gilford 2400 spectrophotometer. An ICG standard curve was prepared and three ICG standards were run with each series of determinations. BSP transfer rates. The rates of BSP transfer from the perfusate to the liver and from the liver to the bile were calculated using the model of Richards (1965). The uptake of BSP (a) was calculated from the formula a = (Ak,) + (BQ/(A
+ B),
where k, and k, are the slopes of the asymptotes of the two parts of the perfusate disappearance curve and A and B are the concentrations in the perfusate when the two lines cross the y axis at Time 0. The excretion of BSP (h) was calculated from the formula (k,kJ/a. Canalicular bile formation studies. The clearance of [14C]erythritol served as an estimate of canalicular bile secretion (Forker et al., 1967; Boyer, 1971). Radioactive erythritol (4 ,uCi) was diluted with 40 mg of the “cold” compound and added at the beginning of the perfusion. Taurocholate was also infused from the beginning of the perfusion at a rate constant for each experiment. The infusion rates were 0 (saline only), 0.25, 0.33, and 0.50 pmol/min in the separate experiments. The bile flow achieved a steady state rate in approximately 30 min, as estimated by three consecutive similar samples at 5-min intervals (Utili et al., 1977a). At this time, three bile (20 ~1) and perfusate (50 ~1) samples, collected 5 min apart, were taken. An additional bile sample was taken at each interval for the determination of bile acids. At 50 min, dantrolene (10e4 M) was added to the perfusate. Then, at 70, 75, and 80 min, additional bile and perfusate samples were obtained to measure radioactivity and bile acid concentrations after administration of the drug. Earlier work has demonstrated that the IPRL is viable for this period and that administration of the carrier did not affect erythritol clearance in this model (Utili et al., 1977a,b). [‘4C1Erythritol determination. Bile and perfusate samples were placed in scintillation vials and 10 ml of Aquafluor (New England Nuclear, Boston) was added. The vials were counted using an Intertechnique SL-4000 liquid scintillation counter and quenching was corrected using an external standard. The counting efficiency was 80%
444
ABERNATHY
ET AL.
as calculated using a quenching curve. The biliary clearance was calculated by multiplying bile flow times the bile to perfusate ratio of the [i4C]erythritol activity. Bile acid determination. The concentration of bile acids in the collected bile was measured using a slight modification (Paumgartner et al., 1971) of the method of Hurlock and Talalay (1957). A methanolic bile solution was prepared by diluting 20 ~1 of bile with 180 d of absolute methanol. Twenty microliters of this solution (50 ,ul when no taurochdate was infused) was added to 1.0 ml of 1 M glycine buffer (pH 9.4) containing 5.6 pm01 of EDTA and 0.4 mmol of hydrazine sulfate, 0.1 ml of a NAD solution (5.4 mmol/ml), and 0.1 ml of the hydroxysteroid dehydrogenase (0.7 U/ml). This solution was incubated for 45 min at 26OC. Then the absorbance of the medium was determined at 340 nm using a Gilford 2400 spectrophotometer and corrected by using a control containing no bile acid. Four taurocholate standards, ranging from 0.375 to O.l50,~mol, were run with each series of determinations. Liver weight. At the termination of each experiment, all nonhepatic tissue was carefully removed. Then the liver was blotted on gauze pads to remove excess moisture and weighed. Afterward, the liver was placed in an oven (80°C) and the dry weight was determined. None of the treatment or pretreatment regimens had any effect on the percentage dry weight. Statistics. Student’s t test was used to determine differences between means. Regression lines were calculated by the least squares method and compared for differences in slope and intercept using analysis of covariance (Snedecor and Cochran, 1967). The 5% level was taken to be significant and all data are expressed as means 2 SE. RESULTS Nonpretreated animals. Dantrolene at 10ds M caused a 17% decrease (p < 0.05) in bile flow in the IPRL and a 46% reduction at 10e4 M (p < O.Ol), (Fig. 1; Table 1). The drug at 1O-5 M also significantly decreased total BSP excretion (3 1%; p < 0.05), but except for the lo- to 15-min interval following dye administration, the drug did not affect the ability of the liver to concentrate BSP in the bile. At a higher dose ( low4 M), dantrolene caused a 74% reduction (p < 0.01) in biliary BSP excretion and also decreased the concentration of the dye in the bile over the entire experimental period. At lows M, dantrolene reduced h but not a, while at a higher dose (lop4 M), it decreased both a and h. At a concentration of 5 x 10V6 M, dantrolene did not affect hepatic clearance of BSP (Table 1, Figs. 2 and 3). Dantrolene (1O-5 M) signif?cantly reduced (35%; p < 0.05) the excretion of ICG by the IPRL. However, at this concentration, the drug did not affect the clearance of ICG from the perfusate (Table 2). Phenobarbital-pretreated rats. Pretreatment of rats with PB before sacrifice did not affect the clearance of BSP. However, pretreatment resulted in a choleretic effect when the data were expressed as total bile flow. Within this pretreatment group, dantrolene caused reductions in bile secretion at 5 x lop6 M (32%;~ < 0.05) and at 1O-5 M (38%;~ < 0.01). The 5 x low6 M concentration also caused a 45% decrease (p < 0.01) in BSP excretion and at 1O-5 M a 51% reduction (p < 0.01). In this pretreatment group, h was decreased by dantrolene at 5 x 10e6 M (p < 0.05); a higher concentration (lOes M) decreased both a and h (Table 1).
DANTROLENE
EFFECTS
ON
HEPATIC
445
FUNCTION
344ethylcholanthrene-pretreated rats. Pretreatment with 3-MC had no effect on bile flow or excretion of BSP in the IPRL. Addition of 10e5 M dantrolene to the perfusate caused a 26% reduction (p < 0.05) in bile flow and a 27% decrease (p < 0.05) in total BSP excretion, while 5 x 1O-6 M dantrolene had no effect on either parameter (Table 1). Hepatic GOT release. At concentrations of 5 x low6 and 1O-5 M, dantrolene did not lead to any increases in the amounts of GOT in the perfusate. However, at 10e4 M, the drug caused a slight but significant rise in the activity of GOT in the medium (Table 3).
25-
20x c 3
15-
s t
z
lo
-
m
-
Controls
----
Dantroiene
5~16%
- -- --
Dantrolene Dantrokme
16% 16%
5DW
0
I
I
lo
20
BSP
I I I 30 40 45 50 60 Time of Perfusion (min)
I 70
I 80
I 90
FIG. 1. Effects of different concentrationsof dantroleneon bile flow in the isolatedperfused rat liver. Dantrolenewasgiven at 30 min and sulfobromophthalein (BSP)at 45 min. Therewere sevenanimalsin eachtreatmentgroup.(*), p < 0.05. (+), p < 0.01.
Canalicular bile formation and bile acid excretion. The biliary excretion of [14Clerythritol was utilized to estimate canalicular bile flow during the infusion of varying amounts of taurocholate. After equilibration of the preparation, the bile to perfusate ratio of erythritol was 1.05 k 0.02, and after the addition of dantrolene (1O-4 M) it was 1.01 + 0.01. Moreover, there was a linear relationship between bile flow and [‘4Clerythritol clearance over a wide range of bile secretion rates (Fig. 4). Plotting the clearance of [14Clerythritol versus bile salt excretion before and after the addition of dantrolene resulted in two parallel lines, indicating that the addition of the drug did not affect the volume of water secreted per mole of bile acid excreted. However, their intercepts on they axis are significantly different (p < 0.05). The control intercept of 0.935 ,ul/min/g of liver was decreasedto 0.729 by the drug (Fig. 5). Isolated hepatocyte study. Incubation of hepatocytes with dantrolene at concentrations ranging from 10e5to 10e3M did not result in any efIlux of GOT from the
446
ABERNATHY
ET AL.
TABLE
1
EFFECTS OF DANTROLENE ON BILE FLOW AND SULFOBROMOPHTHALEIN (BSP) CLEARANCE IN THE ISOLATED PERFUSED RAT LIVER FROM NONTREATED, PHENOBARBITAL- AND 3-METHYLCHOLANTHRENE-PRETREATEDANIMALS' Total bile flow (ml)
Dantrolene (Molar Concn.)
Total BSP excreted bs)
ab (min-I)
Nontreated None 5 x lop6 1 x IO-5 I x 1o-4
0.134 0.722 0.606 0.393
* 0.04 + 0.05 & o.04c * 0.03d
4.964 5.554 3.420 1.349
2 0.54 _+ 0.36 & 0.41’ + 0.28d
rats
8.75 9.21 7.81 4.27
+ + + +
0.39 0.40 0.20 0.26d
Phenobarbital-pretreated None 5 x 10m6
0.883 IO.03’ 0.593 + 0.03’ 0.572 k 0.04f
1 x 10-s
4.974 & 0.29 2.690 i 0.19f 2.414 + 0.31f
0.721 k 0.04 0.722 & 0.05 0.537 i 0.07
7.47 7.92 5.72 3.28
8.69 f 0.47 8.45 +_ 0.36 6.09 + 0.43’
4.834 + 0.36 5.123 5 0.29 3.5 14 + 0.34g
+ + + k
8.88 & 0.27 8.195 & 0.25 7.53 & 0.42
7.11 k 0.38 5.80 2 0.22’ 4.41 f 0.41’ rats 7.17 k 0.26 7.68 + 0.32 5.76 f. 0.49
a Seven rats in each group. All data are given as the means & SE. ‘a and h are the BSP transfer rates for uptake and excretion, respectively. “p < 0.05 compared to nontreated controls. dp < 0.01 compared to nontreated controls. ‘p < 0.05 compared to phenobarbital-pretreated controls. ‘p < 0.01 compared to phenobarbital-pretreated controls. pp < 0.05 compared to 3-methylcholanthrene-pretreated controls.
TABLE
2
BILE FLOW AND INDOCYANINE GREEN (ICG) CLEARANCE DURING PERFUSION OF THE ISOLATED PERFUSED RAT LIVER WITH OR WITHOUTDANTROLENE~ Total bile excreted (ml)
Total ICG excreted kid
0.67 & 0.03 0.44 +_ 0.036
2.73 & 0.21 1.75 & 0.23’
Molar concentration of dantrolene None (6) 1 x 10-5 (5) “The All data bp < ‘p
AND
APPLIED
The Effects
PHARMACOLOGY,
‘&441-452
of Dantrolene the Isolated
(1978)
Sodium on Excretory Perfused Rat Liver’
CHARLESO.ABERNATHY,RICCARDOUTILI,*HYMAN EZEKIEL Liver
Research
Unit, Received
Veterans April
Administration 22,1977;
Function
ANDMILDRED
J.~IMMERMAN,
Hospital,
accepted
Washington,
October
in
D.C. 20422
25,1977
The Effects
of Dantrolene Sodium on Excretory Function in the Isolated Perfused Rat Liver. C. O., UTILI, R., ZIMMERMAN, H. J., AND EZEKIEL, M. (1978). Toxicol. Appl. Pharmacol. 44, 44 l-452. Perfusion of the isolated rat liver with dantrolene sodium led to inhibition of bile secretion and of sulfobromophthalein (BSP) excretion. The [ Wlerythritol clearance and bile acid excretion data suggested that the inhibitory effect of dantrolene was on the bile acidindependent rather than the bile acid-dependent fraction of bile. Inhibition by the drug of excretion of both indocyanine green and BSP suggests that interference with hepatic conjugative processes was not the mechanism for the reduction in organic anion excretion. Analysis of the BSP ‘transfer rates’ suggested that dantrolene exerted its effects on hepatic dye clearance at the excretory level. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene enhanced the adverse effects of dantrolene, suggesting that a cytochrome P-450.mediated metabolite led to the impairment of hepatic function. ABERNATHY,
Dantrolene sodium is a hydantoin derivative used to control muscle spasticity in patients suffering from a number of neurological disorders (Dykes, 1975). The drug is a peripherally acting muscle relaxant, which exerts its effects by blocking the release of calcium from the sarcoplasmic reticulum, thereby uncoupling excitation and contraction (Ellis and Bryant, 1972). There have been several reports of hepatic dysfunction associated with dantrolene therapy, ranging from moderate transient elevations of serum aspartate aminotransferase (GOT), alanine aminotransferase, alkaline phosphatase, and bilirubin (Mayer et al., 1973; Chyatte and Basmajian, 1973; Gelenberg and Poskanzer, 1973; Chipman et al., 1974) to overt hepatic injury (Ogburn et al., 1976; Schneider and Mitchell, 1976; Utili et al., 1977~). Several therapeutic drugs which have been reported to produce hepatic injury as an idiosyncratic reaction of patients have also been shown to produce adverse effects on in vitro model systems. For example, certain phenothiazines, tricyclic antidepressants, anesthetics, and erythromycin estolate have been reported to cause an e&x of cytoplasmic and lysosomal enzymes from cultured liver cells and isolated hepatocytes I This work was supported research funds. 2 Present address: Clinica Italy.
in part by a grant delle Malattie
from Eaton
Infettive,
Facolta 441
Laboratories di Medicina
and by Veterans I, Via Cotugna
Administration 1, 80135
Napoli,
004 1-008X/78/0443-0441 $02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
442
ABERNATHY
ET AL.
(Zimmerman and Kendler, 1970; Abernathy et al., 1975; Dujovne, 1975; Goto et al., 1976). Furthermore, many of these same drugs have also been reported to affect adversely liver function of the perfused rat liver (Kendler et al., 1971, 1972; Ros et al., 1975). Accordingly, the studies reported herein were designed to test the effects of dantrolene on isolated hepatocytes and on the isolated perfused rat liver (LPRL). METHODS
Animals. Male CD rats (350 to 450 g) were obtained from Charles River Laboratories (North Wilmington, Massachusetts) and maintained in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. They were permitted free access to food and water and were kept in the animal facility for at least 1 week prior to use. In the phenobarbital (PB) studies, the rats were given PB in saline (100 mg/kg, ip) daily for 5 days and sacrificed on the sixth. For the 3-methylcholanthrene (3-MC) studies, the compound was dissolved in corn oil and the animals were treated for 2 consecutive days (20 mg/kg, ip) and used for experimentation on the third day. Control animals received either saline or corn oil. Reagents. Sodium taurocholate was purchased from Calbiochem (La Jolla, California). Sulfobromophthalein (BSP) and indocyanine green (ICG) were obtained from Hynson, Westcott and Dunning (Baltimore, Maryland). Uniformly labeled [14Cl erythritol (2.7 mCi/mmol) and hydroxysteroid dehydrogenase were purchased from Amersham/Searle (Arlington Heights, Illinois), and Worthington Biochemicals (Freehold, New Jersey), respectively. The sodium salt of dantrolene was generously provided by Eaton Laboratories. All other chemicals were reagent grade and obtained from commercial sources. Methods. Isolated hepatocytes were prepared as described previously (Abernathy et al., 1975). Dantrolene sodium at various concentrations was added to the cells and the suspensions were incubated in test tubes without shaking for 30 min at 37OC. Then the cells were sedimented and the supernatant, designated as the medium, was decanted. The cells were lysed by alternatively freezing (dry ice in ethanol) and thawing (37’C water bath) three times. The level of GOT in each sample was measured (Zimmerman and Mao, 1965). Each test was run in triplicate and seven separate experiments were run. With minor modifications (Utili et al., 1976), the technique of Penhos et al. (1966) was used to prepare the liver for perfusion. Briefly, the rat was anesthesized with pentobarbital sodium (50 mg/kg) and the bile duct and portal vein were cannulated. The liver was transferred to a perfusion chamber (Metalloglass, Boston, Massachusetts) at 37 & 1“C. The perfusion fluid was wholly synthetic and consisted of a Krebs-Henseleit buffer (pH 7.45) augmented with 2500 U of heparin, 240 mg of glucose, and 2 g of bovine serum albumin per 100 ml and the total perfusate volume was 200 ml. The hydrostatic pressure on the portal vein was adjusted to 17 cm of water. To prevent organ anoxia, a mixture of 0, : CO, (95 : 5) was bubbled into the recirculating perfusate throughout the entire experimental period. Except where noted, taurocholate (0.5 pmol/min) was infused into the perfusate to replace the bile saltsnormally present in the enterohepatic circulation.
DANTROLENE
EFFECTS
ON HEPATIC
FUNCTION
443
After hepatectomy, each preparation was permitted a 30-min equilibration period, during which time the bile and perfusate flow rates were monitored (Utili et al., 1976) to assess the viability of the liver. At 30 min, dantrolene sodium, in various concentrations, dissolved in propylene glycol (solvent only in controls) was added to the perfusate. Fifteen minutes later, the dye, BSP (20 mg) or ICG (4 mg), was added and the perfusion was continued for an additional 45 min. Aliquots of bile (20 ~1) and perfusate (50 ~1) were taken every 5 min to measure the dye concentration in each sample. For each bile sample, the time needed to collect the 20 ~1 was measured and used as an estimate of bile flow rate. Measurement of dye concentration. To determine the concentration of BSP, the bile samples were diluted 500-fold with distilled water and perfusate samples were diluted 50-fold in 0.3 M sodiump-toluenesulfonate prior to alkalinization with 0.2 N NaOH. The amount of BSP in each sample was determined at 575 nm using the appropriate standards and the concentration mode of a Gilford 300 N spectrophotometer. For the ICG experiments, bile and perfusate samples were diluted 250- and 5-fold, respectively, with distilled water. Then the ICG content was assayed directly at 805 nm with a Gilford 2400 spectrophotometer. An ICG standard curve was prepared and three ICG standards were run with each series of determinations. BSP transfer rates. The rates of BSP transfer from the perfusate to the liver and from the liver to the bile were calculated using the model of Richards (1965). The uptake of BSP (a) was calculated from the formula a = (Ak,) + (BQ/(A
+ B),
where k, and k, are the slopes of the asymptotes of the two parts of the perfusate disappearance curve and A and B are the concentrations in the perfusate when the two lines cross the y axis at Time 0. The excretion of BSP (h) was calculated from the formula (k,kJ/a. Canalicular bile formation studies. The clearance of [14C]erythritol served as an estimate of canalicular bile secretion (Forker et al., 1967; Boyer, 1971). Radioactive erythritol (4 ,uCi) was diluted with 40 mg of the “cold” compound and added at the beginning of the perfusion. Taurocholate was also infused from the beginning of the perfusion at a rate constant for each experiment. The infusion rates were 0 (saline only), 0.25, 0.33, and 0.50 pmol/min in the separate experiments. The bile flow achieved a steady state rate in approximately 30 min, as estimated by three consecutive similar samples at 5-min intervals (Utili et al., 1977a). At this time, three bile (20 ~1) and perfusate (50 ~1) samples, collected 5 min apart, were taken. An additional bile sample was taken at each interval for the determination of bile acids. At 50 min, dantrolene (10e4 M) was added to the perfusate. Then, at 70, 75, and 80 min, additional bile and perfusate samples were obtained to measure radioactivity and bile acid concentrations after administration of the drug. Earlier work has demonstrated that the IPRL is viable for this period and that administration of the carrier did not affect erythritol clearance in this model (Utili et al., 1977a,b). [‘4C1Erythritol determination. Bile and perfusate samples were placed in scintillation vials and 10 ml of Aquafluor (New England Nuclear, Boston) was added. The vials were counted using an Intertechnique SL-4000 liquid scintillation counter and quenching was corrected using an external standard. The counting efficiency was 80%
444
ABERNATHY
ET AL.
as calculated using a quenching curve. The biliary clearance was calculated by multiplying bile flow times the bile to perfusate ratio of the [i4C]erythritol activity. Bile acid determination. The concentration of bile acids in the collected bile was measured using a slight modification (Paumgartner et al., 1971) of the method of Hurlock and Talalay (1957). A methanolic bile solution was prepared by diluting 20 ~1 of bile with 180 d of absolute methanol. Twenty microliters of this solution (50 ,ul when no taurochdate was infused) was added to 1.0 ml of 1 M glycine buffer (pH 9.4) containing 5.6 pm01 of EDTA and 0.4 mmol of hydrazine sulfate, 0.1 ml of a NAD solution (5.4 mmol/ml), and 0.1 ml of the hydroxysteroid dehydrogenase (0.7 U/ml). This solution was incubated for 45 min at 26OC. Then the absorbance of the medium was determined at 340 nm using a Gilford 2400 spectrophotometer and corrected by using a control containing no bile acid. Four taurocholate standards, ranging from 0.375 to O.l50,~mol, were run with each series of determinations. Liver weight. At the termination of each experiment, all nonhepatic tissue was carefully removed. Then the liver was blotted on gauze pads to remove excess moisture and weighed. Afterward, the liver was placed in an oven (80°C) and the dry weight was determined. None of the treatment or pretreatment regimens had any effect on the percentage dry weight. Statistics. Student’s t test was used to determine differences between means. Regression lines were calculated by the least squares method and compared for differences in slope and intercept using analysis of covariance (Snedecor and Cochran, 1967). The 5% level was taken to be significant and all data are expressed as means 2 SE. RESULTS Nonpretreated animals. Dantrolene at 10ds M caused a 17% decrease (p < 0.05) in bile flow in the IPRL and a 46% reduction at 10e4 M (p < O.Ol), (Fig. 1; Table 1). The drug at 1O-5 M also significantly decreased total BSP excretion (3 1%; p < 0.05), but except for the lo- to 15-min interval following dye administration, the drug did not affect the ability of the liver to concentrate BSP in the bile. At a higher dose ( low4 M), dantrolene caused a 74% reduction (p < 0.01) in biliary BSP excretion and also decreased the concentration of the dye in the bile over the entire experimental period. At lows M, dantrolene reduced h but not a, while at a higher dose (lop4 M), it decreased both a and h. At a concentration of 5 x 10V6 M, dantrolene did not affect hepatic clearance of BSP (Table 1, Figs. 2 and 3). Dantrolene (1O-5 M) signif?cantly reduced (35%; p < 0.05) the excretion of ICG by the IPRL. However, at this concentration, the drug did not affect the clearance of ICG from the perfusate (Table 2). Phenobarbital-pretreated rats. Pretreatment of rats with PB before sacrifice did not affect the clearance of BSP. However, pretreatment resulted in a choleretic effect when the data were expressed as total bile flow. Within this pretreatment group, dantrolene caused reductions in bile secretion at 5 x lop6 M (32%;~ < 0.05) and at 1O-5 M (38%;~ < 0.01). The 5 x low6 M concentration also caused a 45% decrease (p < 0.01) in BSP excretion and at 1O-5 M a 51% reduction (p < 0.01). In this pretreatment group, h was decreased by dantrolene at 5 x 10e6 M (p < 0.05); a higher concentration (lOes M) decreased both a and h (Table 1).
DANTROLENE
EFFECTS
ON
HEPATIC
445
FUNCTION
344ethylcholanthrene-pretreated rats. Pretreatment with 3-MC had no effect on bile flow or excretion of BSP in the IPRL. Addition of 10e5 M dantrolene to the perfusate caused a 26% reduction (p < 0.05) in bile flow and a 27% decrease (p < 0.05) in total BSP excretion, while 5 x 1O-6 M dantrolene had no effect on either parameter (Table 1). Hepatic GOT release. At concentrations of 5 x low6 and 1O-5 M, dantrolene did not lead to any increases in the amounts of GOT in the perfusate. However, at 10e4 M, the drug caused a slight but significant rise in the activity of GOT in the medium (Table 3).
25-
20x c 3
15-
s t
z
lo
-
m
-
Controls
----
Dantroiene
5~16%
- -- --
Dantrolene Dantrokme
16% 16%
5DW
0
I
I
lo
20
BSP
I I I 30 40 45 50 60 Time of Perfusion (min)
I 70
I 80
I 90
FIG. 1. Effects of different concentrationsof dantroleneon bile flow in the isolatedperfused rat liver. Dantrolenewasgiven at 30 min and sulfobromophthalein (BSP)at 45 min. Therewere sevenanimalsin eachtreatmentgroup.(*), p < 0.05. (+), p < 0.01.
Canalicular bile formation and bile acid excretion. The biliary excretion of [14Clerythritol was utilized to estimate canalicular bile flow during the infusion of varying amounts of taurocholate. After equilibration of the preparation, the bile to perfusate ratio of erythritol was 1.05 k 0.02, and after the addition of dantrolene (1O-4 M) it was 1.01 + 0.01. Moreover, there was a linear relationship between bile flow and [‘4Clerythritol clearance over a wide range of bile secretion rates (Fig. 4). Plotting the clearance of [14Clerythritol versus bile salt excretion before and after the addition of dantrolene resulted in two parallel lines, indicating that the addition of the drug did not affect the volume of water secreted per mole of bile acid excreted. However, their intercepts on they axis are significantly different (p < 0.05). The control intercept of 0.935 ,ul/min/g of liver was decreasedto 0.729 by the drug (Fig. 5). Isolated hepatocyte study. Incubation of hepatocytes with dantrolene at concentrations ranging from 10e5to 10e3M did not result in any efIlux of GOT from the
446
ABERNATHY
ET AL.
TABLE
1
EFFECTS OF DANTROLENE ON BILE FLOW AND SULFOBROMOPHTHALEIN (BSP) CLEARANCE IN THE ISOLATED PERFUSED RAT LIVER FROM NONTREATED, PHENOBARBITAL- AND 3-METHYLCHOLANTHRENE-PRETREATEDANIMALS' Total bile flow (ml)
Dantrolene (Molar Concn.)
Total BSP excreted bs)
ab (min-I)
Nontreated None 5 x lop6 1 x IO-5 I x 1o-4
0.134 0.722 0.606 0.393
* 0.04 + 0.05 & o.04c * 0.03d
4.964 5.554 3.420 1.349
2 0.54 _+ 0.36 & 0.41’ + 0.28d
rats
8.75 9.21 7.81 4.27
+ + + +
0.39 0.40 0.20 0.26d
Phenobarbital-pretreated None 5 x 10m6
0.883 IO.03’ 0.593 + 0.03’ 0.572 k 0.04f
1 x 10-s
4.974 & 0.29 2.690 i 0.19f 2.414 + 0.31f
0.721 k 0.04 0.722 & 0.05 0.537 i 0.07
7.47 7.92 5.72 3.28
8.69 f 0.47 8.45 +_ 0.36 6.09 + 0.43’
4.834 + 0.36 5.123 5 0.29 3.5 14 + 0.34g
+ + + k
8.88 & 0.27 8.195 & 0.25 7.53 & 0.42
7.11 k 0.38 5.80 2 0.22’ 4.41 f 0.41’ rats 7.17 k 0.26 7.68 + 0.32 5.76 f. 0.49
a Seven rats in each group. All data are given as the means & SE. ‘a and h are the BSP transfer rates for uptake and excretion, respectively. “p < 0.05 compared to nontreated controls. dp < 0.01 compared to nontreated controls. ‘p < 0.05 compared to phenobarbital-pretreated controls. ‘p < 0.01 compared to phenobarbital-pretreated controls. pp < 0.05 compared to 3-methylcholanthrene-pretreated controls.
TABLE
2
BILE FLOW AND INDOCYANINE GREEN (ICG) CLEARANCE DURING PERFUSION OF THE ISOLATED PERFUSED RAT LIVER WITH OR WITHOUTDANTROLENE~ Total bile excreted (ml)
Total ICG excreted kid
0.67 & 0.03 0.44 +_ 0.036
2.73 & 0.21 1.75 & 0.23’
Molar concentration of dantrolene None (6) 1 x 10-5 (5) “The All data bp < ‘p
