ELSEVIER Chemico-Biological Interactions 99 (1996) 179- 192

Alpha-naphthylisothiocyanate-induced elevation of serum bile acids: lack of causative effect on bile

acid transport ’ t2

Masoud Neghab, Neil1 H. Stacey*

Toxicology Unit, National Institute of Occupational Health and Safety (Worksafe Australia).

The University of Sydney, G. P. 0 Box 58. Sydney, NS W 2001, Australia

Received I5 June 1995; revision received 2 October 1995; accepted 9 October 1995

Abstract

In recent years chemicals including chlorinated solvents have been found to interfere with the transport of bile acids (BA) by hepatocytes, which probably accounts for the raised serum bile acids (SBA) after exposure. However, the known cholestatic agent, cr-naphthyliso- thiocyanate (ANIT) has never been fully examined for its effect on these processes. Accord- ingly, the direct effects in vitro and the effects of in vivo treatment on bile acid transport have been investigated in this study. Direct addition of ANIT (5 100 PM) to hepatocytes isolated from untreated rats did not result in any change in uptake or efflux of taurocholic acid (TC), one of the most obviously elevated SBA in ANIT-treated rats. Additionally, accumulation of TC over an extended incubation period was not affected by ANIT. In vivo treatment with ANIT (50 pmol/kg i.p. on each of 3 consecutive days) resulted in a marked elevation of total serum bile acids (TSBA) and a slight increase in the activity of serum alkaline phosphatase (ALP) and a very mild hyperbilirubinemia, while other markers of liver injury were unaltered. In hepatocytes isolated from these rats, K,,, and V,,,,, for uptake and V0 for efflux were no different between ANIT and vehicle-treated animals. In conclusion, ANIT showed no effects

Abbreviations: ALP, alkaline phosphatase; ALT, alanine amino transferase; ANIT, a-naphthyl-

isothiocyanate; AST, aspartate aminotransferase; BA, bile acids; DMSO, dimethyl sulfoxide; HEPES, 4-

(2-hydroxyethyl)-I-piperazineethanesulphonic acid; LDH, lactate dehydrogenase; SBA, serum bile acids;

TC, taurocholic acid; TSBA, total serum bile acids.

l Corresponding author, Tel.: 61 2 5659297; Fax: 61 2 5659300.

’ Presented orally in part at the 28th Annual Meeting of the Australasian Society of Clinical and

Experimental Pharmacologists and Toxicologists (ASCEPT), Auckland, New Zealand, December 1994.

’ The scientific views expressed in this paper are those of the authors and do not necessarily reflect

those of the National Occupational Health and Safety Commission.

0009-2797/96/Sl5.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0009-2797(95)03668-C

180 M. Neghab. N.H. Stacey / Chemico-Biologicul fn~eractions YY ( 1996) I79- 192

on transport of BA on in vitro exposure or after treatment in vivo where SBA were clearly elevated. The lack of effects of ANIT on transport of bile acids is consistent with other postulated mechanisms of action. Furthermore, this indicates that the effects noted with sol- vents are not necessarily replicated by substances known to cause histopathological cholestasis.

Keywor& cr-Naphthylisothiocyanate; Cholestasis; Bile acids; Uptake; Eftlux

1. Introduction

Chlorinated solvents have been well recognised as potential hepatotoxic chemicals [ 11. Raised concentrations of serum bile acids (SBA) have been reported on exposure

to these chemicals [2-51. Interestingly, increased concentrations of SBA after expo- sure to low levels of a variety of organic solvents was evident in the absence of any other biochemical or histological alterations [5,6]. Similarly, the known cholestatic agent, o-naphthylisothiocyanate (ANIT), was found to increase the total serum bile

acids (TSBA) and some individual bile acids at doses below those at which other par- ameters of liver function were affected, with taurocholic acid (TC) being elevated at a dose lower than any of the other bile acids [2]. SBA have been suggested as a sensi- tive test to detect early changes of hepatic function [2,3,5]. Therefore, it is of poten-

tial concern when substances are found to increase SBA. However, there is evidence to show that elevated SBA after exposure to chlorinated solvents may be due to the ability of these substances to interfere with the bile acid transport by hepatocytes [7].

To gain further insight into the possibility that the effects noted might be a general effect of solvents due to their high lipid solubility and consequently their ability to dissolve in hepatocyte membrane (the site of bile acid uptake), it was decided to in- vestigate the effects of a non-solvent, with known cholestatic properties, on the same

processes. ANIT was considered to satisfy these criteria as it has been used as an experimental cholestatic hepatotoxicant for more than 3 decades. It causes cholestasis and hyperbilirubinemia in rats and mice in a dose dependent and

reproducible manner [S]. Acute administration of a single oral dose of ANIT to rats results in an intrahepatic cholestasis within 16-24 h which is marked by necrosis of biliary epithelial cells, focal injury to hepatocytes primarily in the periportal area, reduced biliary excretion of bilirubin and BA, hyperbilirubinemia and elevated SBA

[8-lo]. The mechanisms of toxicity of ANIT have not been clearly established. In recent

years, several attempts have been made to elucidate the pathogenesis of ANIT- induced hepatic injury. Depletion of liver cell glutathione (GSH) and release of cyto-

toxic mediators such as superoxide have been implicated as factors which may be linked to toxicity of ANIT to hepatocytes. However, the mechanism of action re- mains to be clarified [ 11,121. It has been postulated that ANIT-induced cholestasis may be mediated by increased permeability of hepatic tight junctions [ 13- 191. More

recently it has been suggested that the process may be biphasic with changes in tight junctions associated with an earlier phase, while a later phase is linked to bile duct obstruction [20].

M. Neghab. N. H. Stacey / Chemico-Biological lnreractions 99 (1996) 179-192 181

As the mechanism of ANIT-induced cholestasis remains uncertain, our studies were undertaken to investigate any possible role of an inhibition of bile acid trans- port in the ANIT-induced elevation of serum bile acids.

2. Materials and methods

2.1. Chemicals

Taurocholic acid (TC) [3H(G)] (2 Ci/mmol) was purchased from Du Pont (Sydney, Australia). ANIT (>95% purity), corn oil and DMSO were purchased from Sigma Chemical Co. (St Louis, MO, USA). Percoll was obtained from Phar- macia Biotech AB (Uppsala, Sweden). Collagenase (144 U/mg solid, Type CLS2) was supplied by Worthington Biochemical Corporation, Freehold, NJ, USA. HEPES was purchased from Calbiochem-Behring, San Diego, CA, USA. All other chemicals were of the highest purity commercially available which were purchased from local agents and distributors.

2.2. Animals and treatment

Male Sprague-Dawley rats weighing 250-350 g (9-12 weeks of age) from the University of Sydney Animal House were used as experimental animals. They were fed with normal laboratory diet (Rodent cubes, Allied Stock Feeds, Sydney) and water ad libitum. Animals were housed in plastic cages with a stainless steel cover in an environmentally controlled room which was automatically maintained at a temperature of about 25°C and a relative humidity of 50 f 10% and a daily cycle of 12 h light/12 h dark. For in vivo experiments, ANIT (50 pmol/kg i.p.) suspended in corn oil in a final volume of 1 ml/kg was administered to rats at about 09:OO h on each of 3 consecutive days. The dose was selected on the basis of a previous study [2]. All of the control rats received the same amount of vehicle in the same manner. Immediately after the last treatment, food was removed from the animals and 4 h later under halothane anaesthesia either blood samples were taken from the abdom- inal aorta for liver function tests or in situ perfusion of liver for isolation of hepato- cytes was performed. In vitro experiments were conducted with cells from both untreated and treated animals.

2.3. Isolation of hepatocytes Hepatocytes were isolated from untreated and treated rats by the collagenase

digestion method as originally described by Berry and Friend [21], with a few modifications [22] plus an additional percoll centrifugation step [23]. After isolation, hepatocytes were resuspended in Hanks-Hepes buffer at a concentration of 1.4 x lo6 cells/ml for uptake and 1 x 10’ cells/ml for eMux. Initial viability, as determined by the Trypan blue exclusion test, was in the range of 90-95%.

2.4. Determination of intracellular potassium ion (K+) and hepatocyte enzyme

activities Twenty min after incubation of the cells with vehicle (4 ~1 DMSO) or ANIT (final

concentration in the flask 5- 100 PM) dissolved in 4 ~1 DMSO at 37°C intracellular potassium ion content and leakage of cytosolic enzymes, as indices of cell viability,

182 M. Neghah, N. H. Stacey 1 Chemico-Biological lnteracrions 99 ( 1996) I79- I92

were determined. Aliquots of 200 ~1 of cell suspension were centrifuged for 5 s in a Beckman microfuge (Fullerton, CA) for separation of the cells from incubation medium using the silicone oil layer method [24]. Assays for ALT, AST and LDH were carried out on the supernatants with a Roche Centrilichem (Model 400) and appropriate kits (Roche Diagnostic System for ALT and AST and Trace Scientific Pty. Ltd. Melbourne, Australia for LDH). The bottom part was used for the deter- mination of intracellular potassium ion, employing a Corning Flame-Photometer (Mode1 405, Essex, England), as previously described [22].

2.5. Total serum bile acids and total bilirubin

These parameters were determined with the enzymatic method using a PYE Unicam PU 8800 UVNis spectrophotometer and the appropriate kits. TSBA were assayed using an Enzabile Kit supplied by Nycomed AS (Oslo, Norway). Total serum bilirubin was assayed using Unimate 5 from F-Hoffmann-La Roche Ltd. (Basel, Switzerland).

2.6. Serum enzyme assays ALT, AST, ALP and LDH were assayed enzymatically with a Roche Centrilichem

(Mode1 400) and appropriate kits (Roche Diagnostic System for ALT, AST and ALP and Trace Scientific Pty. Ltd. Melbourne, Australia for LDH).

2.7. In vitro uptake of TC

TC was selected as the mode1 substrate as it is known to enter hepatocytes by ac- tive transport across the sinusoidal membrane and it is secreted by hepatocytes across the canalicular membrane in the process of eMux [25,26] and because it is the major elevated bile acid in the serum of ANIT-treated rats [2,27]. To study the in vitro uptake of this substrate, 1.99 ml of a suspension of freshly isolated hepatocytes (1.4 x lo6 cells/ml) were preincubated (in 25-ml capped Erlenmeyer flasks at 37°C with shaking at 80 oscillations/min) for 20 min with DMSO (4 ~1) or ANIT (final concentration, 5-100 PM), dissolved in 4 ~1 DMSO. Preliminary experiments and other studies [28-311 showed that these amounts of chemical or vehicle had no cyto- toxic effects. After preincubation, 10 ~1 of radiolabelled substrate in physiological saline was added to each flask (final concentration 10 PM) and quickly mixed. The amount of radioactivity in each flask was -80 nCi/ml. Sampling for uptake deter- mination was carried out by the silicone oil centrifugation method [24] at appropri- ate times. After the pellet was dissolved in 3 M potassium hydroxide overnight, the samples containing [3H] were placed in scintillation vials containing 50 ~1 of 3 M hydrochloric acid to prevent chemiluminescence being produced by potassium hydroxide. After addition of 5 ml scintillation fluid to the vials and vortexing for 10 s, the amount of radioactivity in the supernatant and pellet was quantified using a Tri-Carb 4430 liquid scintillation counter (Packard Instrument Co., Downers Grove, IL). Determination of the amount of radiolabel and protein concentration [32] allowed calculation of uptake per mg of protein. Initial rate of uptake (V,) was determined from the slope of the lines in the linear range (20-80 s).

M. Neghab, N. H. Sracey / Chemico-Biological Interactions 99 (19%) 179- 192 183

2.8. In vitro efflux of TC For these studies, freshly isolated hepatocytes were preloaded with radiolabelled

TC by incubation of 2 x lo7 cells/2 ml for 20 min at 37°C with shaking at 80 oscillations/mm. The final concentration of TC was 25 PM and the amount of radioactivity was 160 nCi/ml. After incubation, an aliquot (100 ~1) of this concen- trated cell suspension was added to 1.9 ml of fresh incubation medium which had been preincubated with DMSO (4 ~1) or various concentrations (5-100 PM) of ANIT (dissolved in 4 ~1 of DMSO). Samples were then taken at appropriate intervals for determination of eftlux of TC as described and referenced for the uptake studies. Initial rate of efllux (V,) was determined from the slope of the lines in the linear portion (l-5 min).

2.9. Uptake and efflux of TC after in vivo exposure All the procedures and protocols for these experiments were basically the same as

for the in vitro exposure experiments, except that the hepatocytes isolated from ANIT- or corn oil-pretreated rats were incubated with various concentrations (2-100 PM) of radiolabelled substrate.

2.10. Data analysis and statistical procedures The data were statistically evaluated using Student’s t-test, analysis of variance

and Duncan’s test with a preset probability of P < 0.05. Each value is a mean which has been obtained from 3-8 experiments using hepatocytes from different rats.

1 .o 1 T

ANIT Concentrations (PM)

Fig. 1. In vitro effect of ANIT on initial rate of uptake (V,) of taurocholic acid (10 pM). Each column represents the mean value and bars the S.E. (n = 7). None of the columns were significantly different to any other (P < 0.05).

184 M. Neghab, N. H. Slacey / Chemico- Biologicul Inrerucrions 99 ( 1996) I79- I92

3. Results

3.1. Cell viability Preincubation of the hepatocytes with various concentrations of ANJT for 20 min

did not result in any decrease in intracellular potassium ion content or increase in leakage of cytosolic enzymes as compared with control. The mean levels (obtained from 6 separate experiments) of ALT, AST and LDH leakage for control cells when compared with their homogenate, were 1 l%, 5% and 1 l%, respectively. Relative values for the highest dose of ANIT (100 FM) were lo%, 6% and 1 l%, respectively. Furthermore, intracellular potassium ion content, as a sensitive index of cell viabili- ty, did not show any significant decrease in ANIT exposed cells as compared with the control cells. Mean levels of this parameter (obtained from 6 separate experi- ments) for control cells, 20 min after the incubation of the cells with DMSO (vehicle) and at the end of experiments were 77 and 73 pmol/g of cell, respectively, whereas, these values for the highest dose of ANIT (100 PM) were 76 and 68 pmollg of cell, respectively.

2.5-

2.0-

T

ij

5i

F 3 1.5-

5

E

F 6 l.O-

t

P

0.5-

0 CONTROL

I lO/JMANIT

tB IOOUM ANIT

Incubation Time (mm)

Fig. 2. In vitro effect of ANIT on accumulation of taurocholic acid (IO PM) over time. Each column

represents the mean value and bars the S.E. (n = 7). Columns with the same capital letters are not significantly different (P < 0.05).

M. Neghab, N. H. Stacey / Chemico-Biological Interactions 99 ( 19%) 179-l 92 185

3.2. Uptake of TC in vitro

Effects of various concentrations of ANIT on uptake of 10 PM TC were exam- ined. As shown in Figs. 1 and 2, ANIT did not show any significant inhibitory effect on initial rate of uptake (Vs) or accumulation of bile acid.

3.3. Efflux of TC in vitro There were no effects of ANIT on TC eMux from cells preloaded with 25 PM

[3H]TC for either I’,, (Fig. 3) or continuous efflux of TC in the presence of any dose of ANIT (Fig. 4).

3.4. Total serum bile acids and the other liver function tests In vivo treatment with ANIT (50 pmol/kg i.p. on each of 3 consecutive days)

resulted in a highly significant rise in total serum bile acids. In addition, there was a slight increase in the activity of serum ALP and a very mild hyperbilirubinemia. None of the other enzyme liver tests was affected by ANIT (Table 1).

3.5. Uptake of TC after in vivo administration of ANIT Uptake of various concentrations of TC (2-100 PM), using hepatocytes isolated

from ANIT- and corn oil-pretreated rats, was not significantly different for either V0 (Fig. 5) or accumulation of TC over time (Fig. 6).

3.6. Kinetic parameters of TC uptake after in vivo administration of ANIT K,,, and I’,,, for uptake after in vivo treatment of rats with ANIT or vehicle were

calculated from Eadie-Hofstee plots. These values were no different for ANIT or ve-

ANIT Concentrations (uM)

Fig. 3. In vitro effect of ANIT on the initial rate of emux (I’,) of taurocholic acid (25 PM). Each column represents the mean value and bars the SE. (n = 5). None of the columns were significantly different to any other (P c 0.05).

186 M. Neghab. N. H. Stacey / Chemico-Biological Interacrions 99 (1996) 179- 192

0.9

0.8 H ANIT io0p.M

A ANlTYy.M

0 Control

I 1 1

10 15 20 25 30 35

Incubation time (min)

Fig. 4. In vitro effect of ANIT on continuous elllux of taurocholic acid (25 pM) from hepatocytes, over

time. Symbols represent the mean value and bars the SE. (n = 5). Data for the other concentrations of

ANIT were the same and, for the sake of clarity, they were excluded.

hicle treated animals. Mean values (obtained from 3-4 separate experiments) of K,,, for control and treated cells were 17.7 and 16.9 PM, respectively, whereas mean

values for V,,, in control and treated cells were 2.53 and 2.49 nmoliminlmg protein, respectively.

Table 1 Mean levels of serum total bile acids, bilirubin, ALP, ALT, AST and LDH in control and ANIT-

pretreated rats

Treatment Total bile acids Total bilirubin ALP ALT AST LDH

(pmolfl) (W

ControY 14.7 zt 2.1b (6)= I.5 zt 0.1 (4) 130*14(4) 40+3(4) 42*2(4) 79*8(4) ANIT* 74.1 f 9.1’ (7) 2.1 f 0.1* (7) 173 f 6* (7) 44 f 6 (6) 37 f 5 (6) 95 f 18 (6)

‘Corn oil-treated rats (I ml/kg i.p. for 3 consecutive days).

bValues are means f S.E.

%I value. *Dose (50 Fmol/kg i.p. for 3 consecutive days).

‘Significantly different from the respective controls (Student’s f-test, P < 0.05)

M. Neghab. N. H. Sracey / Chemico-Biological Interactions 99 (1996) 179-l 92 187

2.5

1

C

0 CONTROL -TREATED T

2.0 1

-11 2

Taurocholate Concentrations ( uM )

Fig. 5. Initial rate of uptake (Va) of various concentrations of taurocholic acid by hepatocytes from ANIT-pretreated rats (50 pmollkg i.p. on each of 3 consecutive days). Each column represents the mean value and bars the S.E. (n = 3 or 4). Columns with the same capital letters are not significantly different (P < 0.05). I’e for the other concentrations of taurocholic acid showed the same pattern and the data were not included for the sake of clarity.

3.7. Efflux of TC after in vivo administration of ANIT After in vivo administration of ANIT and subsequent isolation of bepatocytes

from pretreated animals, efflux of TC from the cells which were preloaded with 25 PM [3H]TC was studied. Initial velocity of efflux (V,,) of TC was not significantly different between ANIT- and vehicle-treated rats (0.12 f 0.01 vs. 0.11 f 0.01, respectively). Continuous efllux of TC was not significantly different either (Fig. 7).

4. Discussion

The data presented here show that isolated rat hepatocytes do not lose their ability to transport bile acid in the presence of different concentrations of ANIT up to 100 PM. Lack of any inhibitory effects on the uptake and efIIux of bile acid by ANIT indicates that raised concentrations of serum bile acids after exposure to this hepatotoxicant are not mediated by alterations in these functions.

188 M. Neghab. N. H. Stacey / Chemico-Biological Interactions 99 (19%) 179-192

a. Substrate cont. 5pM

0.8-

n treated

0 control

0.01 . , . , I , I , 1 , I , 8 ,

0 5 10 15 20 25 30 35

Incubation time (min)

b. Substrate cont. 1OOkM

OI I, 1,. 1 - 9 - I. I - I

0 5 10 15 20 25 30 35

Incubation time (min)

Fig. 6. Accumulation of taurocholic acid by hepatocytes isolated from ANIT-pretreated rats (50 pmollkg

i.p. on each of 3 consecutive days). Symbols represent the mean value and bars the S.E. (n = 3 or 4).

These results are consistent with previous studies, where no change in initial up- take rate of TC by isolated perfused rat liver was observed [13,33], and the release of bile acids from hepatocytes exposed to doses of ANIT up to 1000 PM in vitro was

normal [30].

M. Neghab, N. H. Slacey / Chemico-Biological Interactions 99 (1996) I79- 192 I89

0.9-

0.8-

0.7-

0.6-

0.5-

0.4-

0.3-

0.2-

O.l-

0 Control

0.0 f ! 1 I I I 1 I

0 5 10 15 20 25 30 35

Incubation time (min)

Fig. 7. Continuous ef?lux of taurocholic acid (25 PM) by hepatocytes isolated from ANIT-pretreated rats (50 pm&kg i.p. on each of 3 consecutive days). Symbols represent the mean value and bars the SE. (n = 6 or 8).

As it is possible that the effects do not necessarily occur directly in this in vitro exposure situation, the transport of bile acid by hepatocytes isolated from ANIT- pretreated rats was further studied. Initial rate of uptake of various concentrations

of model substrate (TC, 2-100 PM) and accumulation of TC by hepatocytes isolated from ANIT-pretreated rats did not show any significant difference when compared with the vehicle-treated rats. Additionally K, and V,,, were no different between ANIT- and vehicle-exposed animals. Similarly, in vivo treatment with ANIT had no

effect on the process of bile acid eMux. These results are again in accord with the observation of Lavigne et al. [30] that functions of bile acid transport were preserved in hepatocytes isolated from ANIT-pretreated rats.

Theoretically, an increased SBA after ANIT treatment may be as a result of

decreased hepatocellular uptake of bile acids, reduced canalicular transport of bile acids, regurgitation of bile acids across tight junctions and/or bile duct obstruction [13,20,33]. Bile duct obstruction is perhaps unlikely to be the cause of ANIT-induced elevation of serum bile acids observed here, given that the dose of ANIT used in our

experiments did not cause any histological abnormalities in the liver sections as examined under light microscopy [2,34] and this dose is less than those (40-200 mg/kg) [12,20,35] causing profound cholestasis associated with evidence of degene- ration and exfoliation of biliary epithelial cells, formation of bile plugs and bile duct

occlusion. Kossor et al. [35] recently reported no changes in serum total bile acids

190 M. Neghab, N. H. Stacey / Chemico-Biological Interactions 99 (19%) I79- 192

after a single oral dose of 25 mg/kg of ANIT, which would seem inconsistent with our data and data of other studies [2,34]. However, it is difficult to make a direct comparison because of the different protocols adopted (our treatment regime used 3 i.p. doses of 9.26 mg/kg on each of 3 consecutive days). Furthermore, it has been demonstrated that increased SBA and onset of cholestasis at 16 h after ANIT treat- ment precede bile duct obstruction which occurs 48-72 h after ANIT administration [20,35]. Our data would suggest that either decreased hepatocellular uptake or hepatocanalicular transport of TC is also an unlikely reason for ANIT-induced elevation of SBA observed here, given the lack of any in vitro or in vivo effect on transport of bile acids by ANIT. Therefore, increased permeability of hepatic tight junctions which has been documented as an early event in ANIT-induced cholestasis [ 13- 191 is perhaps more likely to be causally linked to an elevation of SBA observed under our experimental conditions.

The raised ALP after ANIT treatment in this study deserves comment. Since ALP is found in bile [36,37], the increased plasma activity of this enzyme may result from regurgitation into serum of hepatic ALP from damaged and disrupted tight junc- tions [38]. However, the extent of increase in serum activity of ALP after ANIT treatment is less than for bile acids presumably due to the greater molecular size (M, 130 000) and hence a lower diffusion rate across tight junctions.

In conclusion, the data indicate that, unlike the case with solvents, ANIT-induced elevation of serum bile acids is not a consequence of its interference with bile acid transport. This is not inconsistent with the postulated mechanism of action of an in- creased permeability of hepatic tight junctions. Neither would it be inconsistent with bile duct obstruction as a mechanism at higher doses. Furthermore, the increased SBA after ANIT treatment in the absence of any alteration in bile acid transport (as evidenced by normal uptake function by cells) or hepatocyte integrity (as indicated by normal values for serum enzymes) may further substantiate the importance of these sensitive indicators of hepatobiliary dysfunction in response to hepatotoxicants.

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