TOXICOLOGY AND APPLIED PHARMACOLOGY 35,491-499 (1976)

Microsomal Drug Metabolism during a=Naphthylisothiocyanate- Induced Cholestasis’

ROGER DREW AND BRIAN G. PRIESTLY

Department of Human Physiology and Pharmacology, The University of Adelaide, Adelaide, South Australia 5001

Received August 4,1975; accepted November 4,1975

Microsomal Drug Metabolism during a-Naphthylisothiocyanate- Induced Cholestasis. DREW, R. AND PRIESTLY, B. G. (1976). Toxicol. Appl. Phurmacol. 35, 491-499. Impaired microsomal drug metabolism has been previously reported after a-naphthylisothiocyanate (ANIT) administration to rats. Our aim was to study the time course of changes in microsomal metabolism in relation to the time course of cholestasis produced by oral administration of a single 200-mg/kg dose of ANIT and to determine whether hypoactivity and hypertrophy of the smooth endoplasmic reticulum (SER) precedes the onset of cholestasis. During the first 12 hr after ANIT, bile flow remained unchanged, hexobarbital sleeping time was prolonged, and in vitro oxidation of aminopyrine and aniline was impaired in 10,000 g supernatants of liver homogenates from ANIT-treated rats, but not in 105,000 g microsomes from the same animals. Washout of inhibitory con- centrations of ANIT or its metabolites during the preparation of the micro- somes probably explains the discrepancy between the two in vitro prepara- tions used to assess SER metabolic activity. After the onset of cholestasis drug oxidation by 105,000 g microsomes was also impaired and remained so until normal bile flow was reestablished. Decreased microsomal cyto- chrome P-450 paralleled the changes in (105,000 g) microsomal activity, but at no time was microsomal protein yield per gram of liver significantly altered. The data suggest that pre-cholestatic impairment of microsomal drug metabolism was not associated with a hypertrophic, hypoactive SER. Subsequent reversible SER hypoactivity was the result of bile stasis. Eleva- tion of plasma alanine aminotransaminase provided an early index of ANIT hepatotoxicity, while elevation of plasma bilirubin and 5’-nucleotidase was associated with cholestasis and correlated with the diminution and recovery of bile flow.

The hepatotoxicity of cc-naphthylisothiocyanate (ANIT) has been extensively studied in various experimental animal species. Chronic administration of ANIT produces hepatic lesions which in some respects resemble those of human biliary cirrhosis. After acute administration of ANIT, the progressive inhibition of bile flow and the accompanying morphological disturbances have stimulated interest in ANIT as a useful experimental tool for the elucidation of mechanisms of drug-induced chole- stasis. Evidence for dose-related disturbances of the metabolic and excretory function of the hepatocytes can be detected as early as 2 hr after administration of ANIT, and these changes precede the cholestatic response (Plaa, 1969).

1 This paper was presented in part at the Australian Society of Clinical and Experimental Pharma- cologists Meeting, Melbourne, 1973. CopyrigbtO 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. 491 Printed in Great Britain

492 DREW AND PRIESTLY

We have been interested in the function of the cytochrome P-450-dependent micro- somal mixed function oxidase (MMFO) system during experimental cholestasis. It has been our aim to determine whether a hypoactive, hypertrophic smooth endo- plasmic reticulum (SER) is a causative factor in producing cholestasis, as proposed by Schaffner and Popper (1969) and elaborated by Hutterer et al. (1972), or whether it is a response which results from cholestasis. Evidence has been presented that inhibition of the in vivo metabolism of barbiturates and zoxazolamine and inhibition of the in vitro activity of microsomal N-demethylases and hydroxylases can be detected as early as 2-3 hr after administration of ANIT (Plaa et al., 1965; Derr et al., 1967; Denk, 1972; Buxton et al., 1973; Schaffner et al., 1973).

The question of whether ANIT produces hypertrophy of the SER remains unresolved. Hypertrophic SER seen in electron micrographs after ANIT (Schaffner et al., 1973; Czygan et al., 1974) may have occurred either as a direct response to ANIT or as a response to the cholestasis produced. Furthermore, other data show that microsomal protein and phospholipid, when expressed on a per gram of liver basis, were not altered at a time when MMFO activity was reported to be depressed (Denk, 1972).

This report presents data which show that the interpretation of ANIT effects on in vitro microsomal metabolism in the pre-cholestatic stage depends upon the type of microsomal preparation used to assess metabolic activity. Furthermore, we report on the time course of altered microsomal metabolism during the course of and recovery from ANIT-induced cholestasis.

METHODS

Materials. Male Wistar-derived rats (250-300 g) were bred at the University of Adelaide Central Animal House. They were allowed food and water ad libitum. ANIT was obtained from K&K Laboratories and Tokyo Kasei (Japan), and biochemical reagents were obtained from Sigma Chemical Co. and Calbiochem. ANIT was dissolved in peanut oil and administered by gavage (200 mg/kg). Control animals received the appropriate dose of peanut oil (1 ml/kg).

Bileflow experiments. At 2-24 hr and 3 or 7 days after ANIT administration, rats were anesthetized with sodium pentobarbital(50 mg/kg). The bile duct was cannulated, and, after a 15-min equilibration period during which the body temperature was main- tained at 33°C by placing the rat on a warming table, bile flow was measured gravi- metrically over a lo-min interval. A blood sample was then taken for determination of plasma bilirubin (Nosslin, 1960), plasma alanine aminotransferase (AlAT) (Reitman and Frankel, 1957) and plasma 5’-nucleotidase (Campbell, 1962).

Hexobarbitalsleeping time. At 2 and 12 hr and l-10 days after ANIT administration, the duration of hexobarbital-induced loss of righting reflex was measured after injec- tion of hexobarbital, 125 mg/kg ip.

Microsomal metabolism in vitro. At 2 and 12 hr and l-10 days after ANIT admini- stration, rats were killed by stunning and rapid exsanguination. The livers were per- fused with ice-cold saline, minced and homogenized in 20 ml of 0.25 M sucrose/O.05 M

Tris (pH 7.4) using a Potter-Elvehjem homogenizer and a motor-driven Teflon pestle. The homogenate was centrifuged for 20 min at 10,000 g. Part of the supernatant was retained and the remainder centrifuged for 1 hr at 105,000 g. The microsomal pellet was resuspended in cold 0.15 % KC1 (buffered with 0.01 ht phosphate buffer, pH 7.4)

ANIT CHOLESTASIS AND DRUG METABOLISM 493

and recentrifuged for 1 hr at 105,000 g. The washed microsomes were resuspended with cold 1.15 % KCI. Microsomal protein was determined by the method of Lowry et al. (1951). Microsomal cytochrome P-450 was determined from thecarbon monoxide difference spectrum of dithionite-reduced microsomes assuming a molar extinction coefficient of 91 cm-’ mr+-’ between 450 and 490 nm (Omura and Sate, 1964).

Aminopyrine N-demethylase and aniline p-hydroxylase activities were determined from 15-min incubations of substrate (5 mM) in incubation media described by Schenk- man et al. (1967), except that semicarbazide (4.1 mM) was added to trap formaldehyde produced during aminopyrine demethylation. Either washed microsomes (3 mg of microsomal protein/3 ml of incubate) or 10,000 g supernatant (equivalent to approx 250 mg of liver wet wt/3 ml of incubate) were used as a source of the enzyme. Formalde- hyde was estimated by the method of Nash (1953) and p-aminophenol by a modified method of Imai et al. (1966). Mercuric chloride (500 pg) was added prior to color development when the 10,000 g supernatant was used in order to prevent sulfhydryl group interference with the estimation of p-aminophenol (Chhabra et al., 1972).

RESULTS

The onset and time course of cholestasis determined by measurement of bile flow after 200 mg/kg of ANIT (Table 1) was similar to that reported by Indacochea-Redmond and Plaa (1971), who used an indirect fluorescein method (Plaa and Becker, 1965) to establish the time course of cholestasis after 300 mg/kg of ANIT. Bile flow was not significantly diminished during the first 12 hr, but by 24 hr complete bile stasis was established and bile flow had returned to the control value after 7 days. Plasma AIAT was elevated prior to the onset of cholestasis and returned to control values as bile Bow recovered. Neither plasma bilirubin nor 5’-nucleotidase, an enzyme localized in the plasma and canalicular membranes of hepatocytes, was elevated prior to the onset of cholestasis. These two parameters also became elevated when cholestasis became established, and returned toward control values with the return of bile flow. The main purpose of this experiment was to establish the time course of changes in hepatocyte excretory function and cell integrity as a framework for the comparison of the time course of changes in MMFO activity. Mild liver enlargement was noted 2 days after ANIT, following the period of maximal bile stasis (Table 2). There was no evidence for hypertrophy of the SER, since at no time was the microsomal protein yield significantly different from controls. Hexobarbital sleeping time (Table 3) was prolonged during the first 12 hr after ANIT. These data corroborate the prolongation of pentobarbital sleeping time observed by Buxton et al. (1973) and suggest that in uivo barbiturate metabolism is inhibited prior to the onset of cholestasis.

A further prolongation of hexobarbital sleeping time occurred after cholestasis had been established, and this effect persisted for l-7 days after ANIT administration.

The rate of in vitro metabolism of aminopyrine (type I substrate) and aniline (type II substrate) by microsomal preparations from ANIT-treated rats differed according to the microsomal preparation used (Table 4). Inhibition of both aminopyrine N-de- methylation and aniline p-hydroxylation occurred in 10,000 g supernatants of liver homogenates at all times after ANIT up to the recovery of normal bile flow. These data are consistent with the hexobarbital sleeping time data. In washed microsomal

TABL

E 1

EFFE

CT

OF

ANIT

ON

LI

VER

FUNC

TION

IN

RA

TS’

Tim

e nb

Bile

flo

w

(mg/

mW

g)

- -

Con

trol

ANIT

2 hr

12

hr

18 h

r 24

hr

2 da

ys

3 da

ys

4 da

ys

7 da

ys

IO d

ays

Plas

ma

AIAT

(IU

/lite

r)

Con

trol

ANIT

5 5 4 4 (3

) 4 4

(5)

4 4 4

86.8

+

1.5

78.0

+

3.6

13.0

+ 0

.6

86.8

f

1.5

72.7

+

12.3

13

.0 +

0.6

77

.0

zk 3

.4

28.5

+

18.5

’ N

D

77.0

+

3.4

oc,d

13

.0 r

f: 0.

6 N

D

ND

12

.7 +

1.

4 67

.7

+ 1.

9 32

.0

f 5.

3’

10.6

f.

0.8

ND

N

D

11.5

+

1.0

91.1

&

1.8

92.1

+

6.1

9.7

+ 1.

6 N

D

ND

7.

2 +

0.6

30+

1’

0.3

f 0.

03

0.4

L- 0

.02

66 +

3”

0.

3 *

0.03

0.

3 +

0.04

N

D

ND

N

D

107

* 3’

0.

3 *

0.03

2.

6 f

0.3’

16

3 f

16’

0.6

k 0.

1 6.

8 +

0.9’

10

8 f

23’

0.5

f 0.

03

4.7

* 0.

9’

22.5

&-

1.5

’ 0.

6 f

0.07

0.

9 *

0.05

’ 8.

3 k

0.19

N

D

ND

8.

8 +

0.4

0.15

f

0.03

0.

3 +

0.05

’

Plas

ma

bilir

ubin

C

m/d

l)

Con

trol

ANIT

u Fi

gure

s ar

e m

ean

+SE;

N

D,

not

dete

rmin

ed.

* N

umbe

r of

rat

s in

the

AN

IT

grou

p sh

own

in p

aren

thes

es

when

di

ffere

nt

from

con

trols

. c

Sign

ifica

ntly

di

ffere

nt

from

con

trols

; p

< 0.

05 (

t te

st).

d 51

5 AN

IT-tr

eate

d ra

ts w

ere

also

con

firm

ed

as c

hole

stat

ic

by th

e in

dire

ct

fluor

esce

in

met

hod

of P

laa

and

Beck

er (

1965

).

Plas

ma

5’-n

ucle

otid

ase

(II-J

/lite

r)

Con

trol

ANIT

E m

0 0 ND

0 0 0

1.2

f 1.

2 0

8.0

k 2.

8’

1.7

f 1.

7 4.

5 +

0.9

ANIT CHOLESTASIS AND DRUG METABOLISM 495

TABLE 2

EFFECT OF ANIT ON LIVER WEIGHT AND MICROSOMAL PROTEIN

YIELD IN RATS’

Time nb

Liver weight (g/kg body wt)

Control ANIT

Microsomal protein (mg/g liver)

Control ANIT

2 hr 8 48.1 2 1.4 42.5 + 2.1 14.0 + 1.4 13.8 2 1.3 12hr 8 47.8 k 1.9 52.2 I’L 2.4 13.5 + 0.9 13.0 + 1.1 24 hr 4 45.5 If: 1.6 43.3 + 1.6 13.9 + 1.0 13.5 + 1.1 2 days 4 42.4kO.9 45.9 ~~0.6’ 16.7 kO.2 15.4kO.5 3 days 3 (4) 42.1 _+ 2.4 51.4+ 2.5 14.2 k 0.7 13.3 + 0.5 7 days 4 42.3 rf: 1.1 46.2 + 2.2 13.6 + 1.3 10.5 + 0.3 11 days 3 (4) 42.8 + 0.6 43.6 t 3.4 12.7 + 0.8 12.0 + 0.7

o Figures are means &SE. ’ Number of rats in ANIT group shown in parentheses when different

from controls. c Significantly different from control; p < 0.05 (t test).

TABLE 3 EFFECT OF ANIT ON HEXOBARBITAL

SLEEPING TIME IN RATS”

Hexobarbital sleeping time (min)*

Time

2 hr 12hr 24 hr 2 days 3 days 7 days 10 days

Controlc ANIT

21 f 2 (6) 46 f 3d (5) 21 f 2 (6) 40 f 3’ (6) 21 f 2 (6) 54 + 8d (5) 34 + 3 (7) 63 f 5d (7) 34 f 2 (5) 87 f 7’ (8) 33 i 3 (7) 56 + 6d (7) 28 f 1 (7) 27 k 3 (7)

’ Figures are means *SE, and the number of rats is shown in parentheses.

b Dose of hexobarbital was 125 mg/ kg ip.

c Five separate control groups were used for the 2-24hr and 2-, 3-, 7-, and IO-day studies.

d Significantly different from con- trol; p < 0.01 (t test).

pellet suspensions prepared from the above 10,000 g supernatants by centrifugation at 105,000 g, inhibition of aminopyrine and anihne metabolism was not observed until 48 hr; that is, 24 hr after cholestasis had been established. Changes in microsomal cyto- chrome P-450 content (Table 5) correlated well with changes observed in in vitro drug metabolism by the 105,000 g liver fraction.

TABL

E 4

EFFE

CT

OF

ANlT

ON

IN

VI

TRO

DR

UG

MET

ABO

LISM

1~

RA

TS”

-..

Amin

opyr

ine

dem

ethy

lase

”

Tim

e C

ontro

l --~

~-

-

10,0

00

g su

pern

atan

ts

2 hr

10

1 f

6 (8

) 12

hr

118f

16

(4)

24 h

r 10

8 +

10 (

4)

2 da

ys

130f

12

(4)

3 da

ys

145+

22(4

) 7

days

12

7+

9(4)

11

day

s 13

6 +

15 (

8)

105,

000

g m

icro

som

es

2 hr

8.

67 f

0.

50

(8)

12hr

6.

86 L

- 0.

18

(8)

24 h

r 7.

56 +

0.3

2 (4

) 2

days

6.

75 f

0.

53

(4)

3 da

ys

5.57

*

0.31

(4

) 7

days

7.

16 +

0.

42

(4)

I 1 d

ays

6.02

k 0

.21

(4)

ANIT

73 -

t 6d

(8)

72

13

.2 f

0.

6 (8

) 6.

6 +

0.5d

(8)

50

64

+

6d (4

) 54

13

.9 +

1.

8 (4

) 5.

5 +

0.5d

(4)

39

64

?I

3’(4

) 59

12

.9 +

1.

0 (4

) 3.

9 +

O.ld

(4)

30

52

k

7’

(4)

40

14.2

+

1.1

(4)

4.7

L- 0

.4d

(3)

33

44 f

gp

(4)

30

15

.6 f

1.

5 (4

) 4.

5 f

0.4d

(4)

28

83

+

10d

(4)

65

14.7

L- 1

.4 (

4)

9.0

f 0.

6d (

4)

61

105

+ 9

(8)

77

13.8

_+ 0

.9 (

8)

12.9

+

1.1

(8)

93

7.92

+ 0

.30

(8)

91

6.42

2

0.39

(8

) 94

7.

26

L- 0

.18

(4)

96

3.89

+

0.31

d (4

) 58

1.

69 f

0.

15d

(4)

32

5.36

+

0.26

d (4

) 75

5.

65

zk 0

.43

(4)

94

Perc

ent

of

cont

rol

Anilin

e hy

drox

ylas

e”

-__

-

Con

trol

ANIT

Perc

ent

of

cont

rol

0.74

f

0.04

(8

) 0.

65

+ 0.

03

(8)

88

0.90

+

0.05

(8

) 0.

80

f 0.

05

(8)

89

0.74

+

0.03

(4

) 0.

72

i- 0.

02

(4)

97

1 .O

l f

0.07

(4

) 0.

57

+ 0.

05d

(4)

56

0.81

+_

0.01

(4

) 0.

26

+ 0.

02d

(4)

32

0.98

_+

0.07

(8

) 0.

69

+ 0.

05d

(8)

70

0.73

f

0.02

(8

) 0.

64

xk 0

.04

(8)

88

’ Fi

gure

s ar

e m

eans

*SE

, w

ith

the

num

ber

of ra

ts u

sed

in p

aren

thes

es.

b N

anom

oles

of

for

mal

dehy

de

form

ed

per

min

ute

per

gram

of

live

r by

10,

OO

Og

supe

rnat

ants

or

per

milli

gram

of

mic

ro-

som

al p

rote

in

by 1

05,0

00 g

sup

erna

tant

s.

c Nan

omol

es

ofp-

amin

ophe

nol

form

ed

per

min

ute

per

gram

of

liver

by

10,O

OO

g su

pern

atan

ts

or p

er m

illigr

am

of m

icro

- so

mal

pro

tein

by

105

,000

g m

icro

som

es.

d Si

gnifi

cant

ly

diffe

rent

fro

m c

ontro

ls;

p -c

0.0

5 (t

test

).

ANIT CHOLESTASIS AND DRUG METABOLISM 497

TABLE 5

EFFECT OF ANIT ON MICROSOMAL CYTOCHROME P-450 CONTENT IN RAT LIVER

Cytochrome P-450 (nmol/mg microsomal protein)

Time Control ANIT

2 hr 1.09 f 0.05 (8) 1.02 f 0.04 (8) 12hr 1.05 f 0.04 (8) 0.98 + 0.03 (4) 24 hr 1.14 + 0.04 (4) 1.11 + 0.08 (4) 2 day5 1.18 f 0.06 (4) 0.81 + 0.04’ (4) 3 days 1 .lO f 0.06 (4) 0.37 f 0.01” (4) 7 days 1.02 + 0.06 (8) 0.77 + 0.05’ (7) 11 days 1.14 f 0.03 (8) 1.11 & 0.04 (7)

p Significantly different from controls;p < 0.05 (t test).

DISCUSSION

A number of previous studies have established that microsomal drug metabolism is inhibited during the early stages of ANIT-induced cholestasis. One point which has not been clearly established is whether the inhibition of microsomal metabolism is an early manifestation of the hepatocyte dysfunction which leads eventually to cholestasis or whether it might be due to an interaction between ANIT and substrates of the MMFO system.

Our data suggest that the latter hypothesis is probably more correct. Using hexo- barbital sleeping time and metabolism of aminopyrine and aniline by the 10,000 g liver fraction as criteria for the activity of the MMFO system, inhibition was demon- strated during both the pre-cholestatic phase (2, 12 hr) and after cholestasis was estab- lished (24 hr). However, the specific metabolic activity of washed and resuspended 105,OOOg microsomal pellets and their cytochrome P-450 content was not altered during this time. The subsequent impairment of metabolism in both types of microsomal prep arations, which began at 48 hr and persisted until cholestasis had been relieved, probably reflects damage to the microsomal membranes and elements of the cytochrome P-450 electron transport mechanism due to accumulation of bile within the hepatocytes.

Other studies which report inhibition of MMFO activity during the first 2-3 hr after ANIT administration used 10,000 g supernatants as the source of the enzyme or an in vivo index such as barbiturate sleeping time (Plaa et al., 1965; Buxton et al., 1973). An exception to this was Denk (1972), who used 105,000 g microsomes and reported inhibition of aniline, but not aminopyrine metabolism, 3 hr after 200 mg/kg of ANIT. In Denk’s experiments the concentration of aniline used in vitro (0.06 mM) was much lower than the 5 mM used in our experiments. Derr et al. (1967) reported inhibition of puromycin N-demethylase 2 hr after ANIT, 80 mg/kg, but it is not clear whether the microsomal preparation was a 9000 g supernatant of liver homogenates or a 105,000 g microsomal pellet suspension fortified with supernatant fraction.

The problem in using a 10,000 g supernatant is that this preparation is likely to contain substantial amounts of ANIT or ANIT metabolites. ANIT has been shown

498 DREW AND PRIESTLY

to be a potent inhibitor of in vitro drug metabolism when added to this system irz vitro (Plaa er al., 1965), but it is not known whether the inhibition of metabolism in 10,000 g supernatants after in viuo administration is due to ANIT itself or a metabolite. There is increasing evidence that ANIT is metabolized by the MMFO system (Roberts, 1973) and that its biotransformation is crucial for the cholestatic effects of ANlT (Indacochea-Redmond et al., 1973).

Capizzo and Roberts (1970) detected radioactivity in the liver 472 hr after oral administration of [‘“ClANIT, and showed that radioactivity was evenly distributed between the unwashed microsomal and 105,000 g-supernatant fractions. The nature of the binding to microsomes is important, and, although Roberts (1973) suggests that the protein binding of ANIT is irreversible, our data indicate that the ANIT can be removed, or at least depleted to less than inhibitory concentrations, during the prep- aration of the washed microsomes. We did perform an experiment in which we attempted to dialyze the inhibitor out of the 10,000 g supernatants. Although the experiment was not conclusive, due to a decrease in activity of controls, the trend of the results suggested that the inhibitor might be partially dialyzable.

Our experiments do not support the hypothesis that hypertrophic, hypoactive SER is an important precursor to ANlT-induced cholestasis. Since we did not detect any change in microsomal protein yield, we could not conclude that hypertrophy of the SER occurred. Neither would we classify the SER as hypoactive until after cholestasis had become established. Schaffner ef al. (1973) also concluded that a hypertrophic, hypoactive SER was not an initiating factor in ANIT-induced cholestasis. They com- pared the SER hypoactivity after ANIT treatment with that seen after ligation of the common bile duct and surmised that the former was due to ANIT or ANIT metabolites. interfering with substrate binding to cytochrome P-450.

While hypoactivity of the SER occurred after the onset of cholestasis, it is clear that release of alanine aminotransferase (AIAT) from the hepatocyte was an early sign of the effects of ANIT and clearly preceded the onset of cholestasis. Hyperbilirubinemia and the elevation of plasma 5’-nucleotidase followed a time course very similar to the changes in bile flow. It is suggested that these last two parameters would be most useful in studies on the time course of ANIT-induced cholestasis in rats where direct bile flow determination is not feasible. Estimates of the activity of the MMFO system, particularly those using an in vitro technique such as hexobarbital sleeping time, do not provide information useful in determining the onset of cholestasis but might be useful to signal the resumption of normal hepatocyte function.

REFERENCES

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CAMPBELL, D. M. (1962). Determination of 5’-nucleotidase in blood serum. Biochem. J. 82, 34P.

CAPIZZO, F. AND ROBERTS, R. J. (1970). Disposition of the hepatotoxin a-naphthylisothio- cyanate (ANIT) in the rat. Toxicol. Appl. Pharmacol. 17, 262-271.

CZYGAN, P., GREIM, H., HUTTERER, F., SCHAFFNER, F. AND POPPER, J. (1974). Comparison of two types of intrahepatic jaundice in rats with bile duct ligation. Acta Hepato-Gastroenterol 681, 339-345.

ANIT CHOLESTASIS AND DRUG METABOLISM 499

DENK, H. (1972). Die chemische Struktur des endoplasmatischen Retikulums und die Funktion des mikrosomalen Biotransformationssystems der Leberzelle der Ratte bei experimenteller Cholestase. Pathol. Eur. 7,43-65.

DERR, R. F., LOECHLER, D. K., ALEXANDER, C. S. AND NAGASAWA, H. T. (1967). Inhibition of rat liver microsomal N-demethylase by a-naphthylisothiocyanate: Studies with puro- mycin amino nucleotide. Proc. Sot. Exp. Bioi. Med. 126,844-845.

HUTTERER, F., GREIM, H., TRULZCH, D., CZYGAN, P. AND SCHENKMAN, J. B. (1972). Micro- somal biotransformation in cholestasis. In Progress in Liver Diseases, (H. Popper and F. Schaffner, Eds.) Vol. 4 Grune and Stratton, New York pp. 151-171.

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