TOXICOLOGY AND APPLIED PHARMACOLOGY 7, 680-685 (1965)

The Nature of a-Naphthylisothiocyanate-Induced Cholestasis’

BERNARD A. BECKER~ AND GABRIEL L. PLAA

Department of Pharmacology, College of Medicine, The University of Iowa, Iowa ‘City, Iowa 52241

Received August 17, 1964

Cholestasis, until recent years, has usually been considered a problem for the internist, the surgeon, or the pathologist. However, cholestasis has now become a problem for the toxicologist as well, since a causal relationship has been established between it and certain drug therapies. Popper and Schaffner (195913) state, “This question [drug-induced cholestasis] arises with increasing frequency in view of the continuing expansion of the use of drugs.”

The first case of cholestasis due to drug therapy, according to Popper and Schaffner (1959b), was reported by Hanger and Gutman (1940). The drug involved was arsphenamine. Since then, cholestasis has been attributed to chlorpropamide, chlor- promazine, methyltestosterone, norethandrolone, p-aminosalicylic acid, chlorthiazide, and sulfonamide derivatives as well as other agents (Popper and Schaffner, 195913; Hoffbauer, 1959; Schaffner et al., 1959; Rothfield et al., 1960; Reichel et al., 1960). All the ‘foregoing reports concern cases of hepatotoxicity in patients on drug therapy. Attempts to produce cholestasis in animals by most of these agents have generally been unsuccessful (Popper and Schaffner, 1959a). However, cholestasis has been pro- duced in the rat by oral administration of a-naphthylisothiocyanate (ANIT) accord- ing to several investigators (Eliakim et al., 1959; Goldfarb et aZ., 1962; Ungar et aZ., 1962; Steiner and Baglio, 1963; Becker and Plaa, 1964). The similarity of ANIT injury to chlorpropamide-induced toxicity in man has been pointed out by Goldfarb et al. (1962).

Cholestasis, with possible resulting jaundice, may arise from reduction of bile flow in intrahepatic or extrahepatic biliary passages. The interruption of flow in either case may result from mechanical obstruction, e.g., bile plugs, gallstones, strictures, edema pressure, or tumor growth (Popper and Schaffner, 1959a). Intrahepatic cholestasis may also result from the hepatocellular degeneration associated with viral hepatitis, toxic liver injury, or cirrhosis. Intrahepatic cholestasis may also occur without obvious cellular changes (Hoffbauer, 1959).

ANIT-induced cholestasis has been said to resemble intrahepatic cholestasis by some workers (Eliakim et al., 1959; Goldfarb et al., 1962; Ungar et al., 1962), and extra- hepatic cholestasis by others (Steiner and Baglio, 1963). One group of investigators denies that the compound is capable of producing cholestasis at all (McLean and Rees, 1958; Griffiths et al., 1961). Therefore, further investigation into the nature of ANIT-induced cholestasis, as to its intrahepatic or extrahepatic origin, is war- ranted.

1 These investigations were supported in part by U.S. Public Health Service grant A~-08083 and Special Fellowship AM-19,889, from the National Institute of Arthritis and Metabolic Diseases,

?. Special Fellow, National Institute of Arthritis and Metabolic Diseases.

680

ANIT-INDUCED CHOLESTASIS 681

The purpose of the present study is to compare the effects of the oral administration of ANIT with those of surgically induced extrahepatic cholestasis (bile duct ligation) on the basis of liver functions tests. A comparison with intrahepatic cholestasis can- not be made since there is no available method of producing experimental intrahepatic cholestasis in a laboratory animal.

METHODS

Surgical procedure. The animals used were adult, male, Swiss-Webster mice, housed in groups of 10 in stainless steel “shoe-box” cages and given food and water ad libitum. They were anesthetized for occlusion of the bile duct by the administration of ether or the intraperitoneal injection of 90 mg of sodium pentobarbital per kilogram. The viscera were exposed by a midline incision, and the common bile duct was isolated and ligated just above its junction with the duodenum. The incision was closed by sutures or wound clips.

Urinary excretion of bilirubin. Urinary bilirubin was detected qualitatively by the method of Free and Free (1953) using reagent tablets.3

Plasma concentration and metabolism of bilirubin. Two groups of 10 mice each were prepared, respectively, by (1) oral administration of ANIT, 150 mg/kg as a solution in corn oil; and (2) ligation of the common bile duct. A third group of 10 animals was subjected to a sham operation; these served as controls. At 24 hours after treatment, plasma was collected from all mice and assayed for conjugated and unconjugated bilirubin by the method of Weber and Schalm (1962).

This experiment was repeated with the modification that plasma was collected at 2 hours after treatment, rather than at 24 hours. In this case, ligation of the common bile duct was performed under light ether anesthesia.

Retention and metabolism of sulfobromophthalein. Five groups of 10 mice each were prepared by ligation of the bile duct, and five groups of 10 each were given ANIT, as a single oral dosage of 150 mg/kg. At 24 hours after the respective treat- ments, each animal received per kilogram an injection of 75 mg of sulfobromophthalein sodium (BSP) via the tail vein. Thirty minutes later, blood was collected from each animal and pooled by groups. The plasma was separated, and the 10 pools of plasma were analyzed for total BSP and for altered BSP (MBSP) by the method of Thomas and Plaa (1960). The ratio of MBSP to total BSP was calculated for each group.

In a second experiment of this type, the interval between BSP administration and sampling of the plasma was extended to 2 hours.

Urinary Excretion of Bilirubin RESULTS

At 4 hours after ligation of the common bile duct, mice had recovered sufficiently that a few drops of urine could be expressed from the bladder and tested for the presence of bilirubin. No bilirubin was found in the urine of any of 10 mice at this time. However, when the test was repeated at 24 hours after the operation, bilirubin

was found in the urine of all animals. On the other hand, bilirubin appeared in the urine as early as 35 minutes after the

administration of ANIT, 150 mg/kg, to mice. The test was positive in each of five

a “Ictotest” reagent tablets, Ames Laboratories, Elkhart, Indiana,

682 BERNARD A. BECKER AND GABRIEL L. PLAA

cases at this time. A similar result was obtained when the urine was tested at 24 hours after the dose.

Plasma Concentration and Metabolism of B&rub&

The results of these experiments are summarized in Table 1. ANIT at 150 mg/kg

TABLE 1 PLASMA BILIRUBIN CONCENTRATIONS~ IN MICE 2 HOURS AND 24 HOURS AFTER ORAL ADMINISTRATION

OF 150 MC OF ANIT PER KILOGRAM OR BILE DUCT LIGATION

2 Hours

ANIT Duct ANIT Bilirubin treatment ligation treatment fraction (mg %) bg %b) (me %I

Conjugated l.lb k 0.20 0.2 -t 0.03 3.0” I+ 0.10 Unconjugated 0.2 e 0.04 0.1 -c 0’.03 o.3b k 0.10

Total 1.3" IL 0.20 0.3 k 0.03 3.3 k 0.70

a Values stated as mean k standard error of 10 animals. b Significantly different from duct ligation, P < 0.05 (t test).

24 Hours

Duct ligation (mg %)

2.5 f 0.10

0.8 * 0.10 3.3 IL 0.40

Sham operated (w %I

0.1 -c 0.03 0.3 -c 0.04 0.4 -c 0.06

produced plasma bilirubin levels comparable to those produced by bile duct ligation. One difference between the chemically and surgically treated animals was the sig- nificant elevation of unconjugated bilirubin effected by the bile duct-ligated animals but not by the ANIT-treated animals. The 24hour findings prompted a comparison of the two techniques at only 2 hours after treatment. In this case, only the ANIT- treated mice showed an elevation of plasma bilirubin concentration. Thus, the action of ANIT differs from surgical occlusion in that it produces hyperbilirubinemia within a reasonably short interval, whereas surgical occlusion does not.

Retention and Metabolism of Sulfobromophthalein

These experiments are summarized in Table 2. When plasma was collected 30 min- utes after injection of BSP, there was no significant difference in the total BSP con- centration measured in both groups. However, the ratios of MBSP to total BSP were significantly greater by the Mann-Whitney U test (Siegel, 1956) for the ANIT- treated mice than for the corresponding bile duct-ligated mice. When the interval between BSP administration and sampling of the plasma was extended to 2 hours, the drug-treated animals were seen to remove BSP from the blood stream to a greater extent (t test) than the bile duct-ligated animals. Again the ratios of MBSP to total BSP were greater for the ANIT-treated animals.

DISCUSSION

One group (Steiner et al., 1963) has claimed that ANIT-induced ChGlestasis re-

sembles cholestasis due to surgical obstruction of the common bile duct, i.e., extra- hepatic cholestasis. The present study does not support this contention. Comparison of ANIT-treated and bile duct-ligated animals was made 2 and 24 hours after drug administration or surgical treatment. Plasma bilirubin was found to be significantly elevated at 2 hours in the case of the drug-treated mice, but the plasma bilirubin level of bile duct-ligated mice remained at the control level. Thus, a marked difference between the two treatments was seen in respect to onset of effects as reflected by

ANIT-INDUCED CHOLESTASIS 683

TABLE 2 PLASMA CONCENTRATIONS OF BSP AND ALTERED BSP (MBSP) AT 30 MINUTES AND 2 HOURS AFTER

INJECTION OF BSP INTO ANIT-TREATED AND BILE DUCT-LIGATED MICE

Pool numberc

Interval from BSP injection

(hours)

ANIT-treateda Bile duct-ligatedb

Total Ratio of Total Ratio of BSP MBSP to BSP MBSP to

(ms %jd total BSPe (w k)” total BSPe

1 0.5 27.8 0.40 26.5 0.30 2 05 40.4 0.42 26.5 0.00 3 0.5 18.9 0.25 26.0 0.29

4 C.5 17.7 0.34 27.8 0.16

5 0.5 24.0 0.38 25.2 0.30

6 2.0 5.4 0.36 14.2 0.00 7 2.0 - 15.8 0.20

8 2.0 5.2 0.24 14.6 0.01

a A single oral dosage of 150 mg of ANIT per kilogram was given 24 hours prior to BSP injection. b The bile ducts of these animals were ligated 24 hours prior to BSP injection. c Pool of plasma from 10 animals. d Half-hour values of ANIT-treated do not differ from duct-ligated, P > 0.05 (t test). Two-hour

values of ANIT-treated differ significantly from duct-ligated, P < 0.05 (t test). 6 Ratios of ANIT-treated are significantly greater than those of duct-ligated, P < 0.05 (Mann-

Whitney U test).

the presence of excess bilirubin in the blood stream. The plasma total bilirubin levels at 24 hours, when cholestasis was established in both groups, were equal. However, bile duct-ligated animals showed an increase in both conjugated and unconjugated tbilirubin whereas the ANIT-treated animals showed an increase in the conjugated fraction only.

Furthermore, at 24 hours, drug-treated animals showed a significantly higher pro- portion of altered BSP in the plasma than did surgically prepared animals, possibly indicating a lesser degree of functional impairment of the parenchymal cells. When the period between BSP administration and plasma collection was lengthened from 30 minutes to 2 hours, the ANIT-treated animals had a significantly lower plasma BSP concentration than the bile duct-ligated animals. Thus, another difference be- tween the two types of cholestasis was demonstrated, namely, ANIT-treated mice appeared to be better able to remove BSP from the blood than bile duct-ligated mice. The lower plasma BSP levels of the drug-treated animals may indicate enhanced storage in hepatocytes, or the presence of some degree of biliary excretion. Direct evidence regarding relative degrees of BSP storage in the livers of drug-treated and surgically treated animals is not available. However, the possibility that bile flow is not completely halted by ANIT is supported by previous investigations in the bile duct-cannulated mouse (Becker and Plaa, 1964). Griffiths et al. (1961) reported that bile flow was reestablished in the ANIT-treated rat by 48 hours, whereas Gold- farb et al. (1962) reported return of normal bile flow in 4 days. Thus, the available evidence seems to indicate that ANIT-induced cholestasis is reversible and probably is not as complete as surgically induced cholestasis. Functional and morphologic damage to hepatocytes may also be less severe in ANIT-induced cholestasis.

Since ANIT-induced cholestasis does not resemble extrahepatic cholestasis, it must be assumed to be similar to intrahepatic cholestasis, the only remaining possibility.

684 BERNARD A. BECKER AND GABRIEL L. PLAA

Thus, ANIT may prove to be a valuable tool for production of experimental intra- hepatic cholestasis.

SUMMARY

Cholestasis was established in mice by the administration of a single oral dosage of 150 mg of a-naphthylisothiocyanate per kilogram and compared to extrahepatic cholestasis effected by sur- gical occlusion of the common bile duct. Plasma levels of conjugated and unconjugated bilirubin, and of intravenously injected sulfobromophthalein and its metabolic product, were measured as indicators of the functional integrity of the liver of the two preparations.

Equal degrees of hyperbilirubinemia and sulfobromophthalein retention were achieved by the two treatments. However, the plasma of a-naphthylisothiocyanate-treated mice contained increased levels of the metabolic products of bilirubin and sulfobromophthalein. The unconjugated form of bilirubin was elevated in animals with surgically cccluded bile ducts. The onset of hyperbili- rubinemia and bilirubinuria was more rapid following the drug treatment.

Therefore, a-naphthylisothiocyanate-induced cholestasis differs from extrahepatic cholestasis in the following ways: (1) the onset of stasis as judged by the appearance of hyperbilirubinemia is more rapid; (2) the increase in plasma bilirubin represents an increase solely in conjugated bili- rubin; (3) a higher proportion of intravenously injected sulfobromophthalein appears in the altered form; and (4) plasma levels of intravenously injected sulfobromophthalein decrease more rapidly.

ACKNOWLEDGMENT

The authors gratefully acknowledge the technical assistance of Mrs. Marcia Shaffer.

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