Liver 1995; I S : 271-275 Printed in Denmark . All rights reserved

Copyrinht 0 Munksgaard 1995

LIVER ISSN 0106-9543

1 =Naphthyl isocyanate and 1 aaphthylamine as metabolites of 1 -naphthylisothiocvanate

J

Li Y, Yousef IM, Plaa GL. 1-Naphthyl isocyanate and 1-naphthylamine as metabolites of 1 -naphthylisothiocyanate. Liver 1995: 15: 271-275. 0 Munksgaard, 1995

Abstract: The importance of the bioactivation of 1 -naphthylisothiocyan- ate was studied. Forty minutes after 1-naphthylisothiocyanate adminis- tration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1 -naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1- naphthyl isocyanate, 1 -naphthylamine and the parent compound, 1- naphthylisothiocyanate. When rats were given cycloheximide, which at- tenuates 1 -naphthylisothiocyanate toxicity, 30 min before 1 -naphthylisothi- ocyanate (300 mg/kg), 1 -naphthyl isocyanate concentration was signifi- cantly lower than in rats receiving only 1 -naphthylisothiocyanate. The ap- pearance of 1 -naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepato- toxicity, enhanced 1-naphthyl isocyanate and 1 -naphthylamine forma-

l tion. It is suggested that l-naphthyl isocyanate, l-naphthylamine and the highly reactive sulfur released from 1 -naphthylisothiocyanate might be

I involved in the hepatotoxic effect of 1 -naphthylisothiocyanate.

Yao Li, lbrahim M. Yousef and Gabriel 1. Plaa Departernent de Phamacologie, Faculte de Medecine, Universite de Montreal, Montreal, Quebec, Canada

Key words: cholestasis - 1-naphthylarnine - 1-naphthyl isocyanate - 1- naphthylisothiocyanate

Dr. Gabriel L. Plaa, Departernent de Pharrnacologie, Faculte de Medehe, Universite de Montreal, C.P. 61 28, Succursale Centre-ville, Montreal, Quebec, Canada H3C 357.

Received 25 July 1994, accepted for publication 4 January 1995

For more than 30 years it has been known that acute administration of 1-naphthylisothiocyanate (ANIT) induces intrahepatic cholestatis in some laboratory animals (1). As hepatic lesions induced by ANIT resemble some of those seen in human biliary cirrhosis, ANIT has been employed to study human cirrhosis (2) and the mechanisms in- volved in drug-induced cholestasis (3). There is a marked species variation in the hepatotoxic effects of ANIT (4). This species difference may be trace- able to a critical enzyme system necessary for the biotransformation of ANIT to a toxic metabolite (5 ) . Although hepatic glutathione has also been implicated in ANIT cholestasis (6, 7 ) , evidence in- dicates that biotransformation of ANIT to toxic metabolites plays an important role in its acute cholestatic effect. Pretreatment of animals with phenobarbital (PB), a monooxygenase inducer, en- hances ANIT-induced cholestasis, while SKF 525- A and cycloheximide (CX), which are inhibitors of monooxygenase and protein synthesis, respectively, diminish the cholestatic response ( 5 , 8).

The results of distribution studies performed

with 14C-[isothiocyanate]- or 4-[3H-naphthyl]- labeled ANIT showed that the 3H/14C ratio of ANIT-derived material excreted into bile was dif- ferent from the ratio administered (9). This finding indicated that the isothiocyanate chain was modi- fied prior to biliary excretion. In rats pretreated with CX, however, the ratio did not change, indi- cating that biotransformation had not occurred. Traiger et al. (10) showed that ANIT may undergo toxic biotransformation via a cytochrome P-450- dependent S-oxidative pathway. Recently, four major compounds not present in control animals were found in the bile of ANIT-treated rats (11). One compound was ANIT, another was identified as 1-naphthylamine (1-N) and the two others were not identified. Their roles in the hepatotoxicity of ANIT, however, have not been assessed.

In order to test the hypothesis that biotrans- formation is involved in the hepatotoxicity of ANIT, ANIT and its metabolites were identified and quantified in the bile and liver of rats with or with- out pretreatment with an inhibitor or an enhancer of ANIT toxicity; the analyses were performed by

271

Li et al.

gas chromatography and mass spectrometry (GCI MS).

Material and methods Chemicals and animals

ANIT and 2’-acetonaphthone were purchased from Aldrich Chemical Co. Inc. CX and 1-N were purchased from Sigma Chemical Co. PB (BDH Inc.) and l-naphthyl isocyanate (1 -NI) (Eastman Kodak Co.) were kindly provided by Dr. C. Lam- bert and Dr. Huy Ong, respectively.

Male Sprague-Dawley rats with an initial body weight of 200-225 g were purchased from Charles River (La Prairie, Qukbec). Animals were main- tained on Purina Chow and water ad libitum. After a 5-day acclimatization period, the rats were ran- domly divided into five groups (5-6 rats per groups) and placed in separate cages.

Treatment protocol

Treatment I (ANIT300) received 0.9% NaCl (2 ml/ kg i.p.) 30 rnin before ANIT (300 mg/kg P.o., dis- solved in corn oil to give a final concentration of 30 mg/ml). Group I1 (ANIT300-CX) received CX (2 mg/kg i.p. dissolved in 0.9%NaCl to give a final con- centration of 1 mg/ml) 30 min before ANIT (300 mg/kg) administration. Group I11 (ANIT100) re- ceived 0.9% NaCl (4 ml/kg i.p.) daily for 3 days be- fore ANIT (100 mg/kg P.o., dissolved in corn oil to give a final concentration of 10 mg/ml). Group IV (ANITIOO-PB) was pretreated daily for 3 days with PB (60 mg/kg i.p., dissolved in 0.9% NaCl to give a final concentration of 15 mg/ml); ANIT, 100 mg/kg p.0. was administered 24 h after the final drug pre- treatment. Group V (Control) received only 0.9% NaCl (2 mlkg i.p.) 30 rnin before a single dose of corn oil (10 ml/kg p.0.).

Surgical procedures and sample collection

Immediately after ANIT administration by gavage, animals were anesthetized with 25% urethane (1 g/ kg i.p.). Body temperature was maintained at 37°C with a temperature-controlled heating lamp. The trachea and common bile duct were cannulated with PE-240 and PE-10 tubing, respectively. Bile collec- tion was started 40 rnin after ANIT administration. Hepatic bile was collected in tared tubes at 30-min intervals over a 2.5-h period. Bile volumes were measured and the bile samples were stored at -20°C. The liver was perfused with cold 0.9% NaCl, removed, weighed and placed immediately in ice- cold saline. The liver was minced and homogenized with a Polytron homogenizer (speed setting 7,20 s) in cold saline (1 g: 1 ml) and stored at -20°C.

Extraction procedures

2’-Acetonaphthone (2.5 pg dissolved in 100 pl of 95% ethanol) was added as an internal standard (I. Std) to aliquots of 2 ml of liver homogenate and 0.5 ml of bile obtained from each sample. ANIT and its metabolites in liver and bile were extracted by shaking with 6 ml diethyl ether for 30 min. The extraction was repeated twice for liver homogen- ates and once for bile. The ether phases were com- bined and dried under a flow of nitrogen. The dry liver and bile extracts were dissolved in 2 ml and 0.3 ml of benzene, respectively.

GCIMS

ANIT and its metabolites were identified and quantified by GC/MS. One microliter of benzene extract was injected into an HP 5890 series I1 gas chromatograph (GC) coupled with an HP 5971A EI mode mass selective detector (MSD). Instru- ment conditions was as follows: HP-1 capillary column (cross-linked methyl silicone gum 0.33 pm film thickness, 12 MX0.2 mm); carrier gas, helium, column flow rate, 0.7 ml/min; injector block 220°C; GC oven temperature programmed from 100°C to 190°C at 6°C per min, and from 190°C to 280°C at 25°C per min; GCfMSD interface temperatul-e 280°C; ionizing beam 70eV. The mass spectra were corrected by background subtraction using HP G1034B software. ANIT and its metabolites were identified by comparing their GC elution times and their mass spectra with those of authentic samples. The calibration curve was constructed by adding known amounts of ANIT and internal standard to drug-free bile and liver homogenates. The amounts of ANIT and its metabolites in unknown samples were calculated from the calibration curves.

Statistical methods

Results are expressed as mean2S.E.M. Analyses of variance and differences were carried out using the Mann-Whitney U test; p<0.05 was considered to be significant.

Results

Since the concentrations of ANIT in different tissues were shown to be elevated 0.5 h after ANIT administration (9), bile and liver samples were ob- tained 40 rnin and 190 rnin after ANIT administra- tion, respectively. The in vivo biotransformation of ANIT was examined by measuring ANIT and its metabolites in the fourth bile collection period (1 30-1 60 rnin after ANIT) and in the liver.

Gas chromatographic profiles of bile (Fig. 1)

272

Metabolites of 1-naphthylisothiocyanate

ceiving ANIT (100 mg/kg) alone. The amount of ANIT in the bile and liver in the ANITlOO-PB group was lower than in the ANIT100 group. The liver 1-NI concentration (4.0220.28 pg/g) in rats treated with PB and ANIT (1 00 mg/kg) was nearly the same as the corresponding value in the ANIT300 group (4.0820.39 pg/g), and was sig- nificantly higher than the amounts obtained in rats treated with CX and ANIT (300 mg/kg).

2 0 0 0 0 -

Abundance 500000 i

127

4 . 5

200000 p 5 11 l 3 n

6 1 I\

jw1---'I------- 0 , , , , l , , , l l , l l , l r l l l , , , , , , , , l ,

rnin -> 7 . 0 0 8 . 0 0 9.00 1 0 . 0 0 1 1 . 0 0 1 2 . R e t e n t i o n Time

Fig. 1. Gas chromatogram of bile extracts. Bile was obtained 130-160 rnin after 1-naphthylisothiocyanate (ANIT) adminis- tration. (a) Bile from rat receiving only ANIT (300 mg/kg); (b) bile from control group receiving only corn oil. Three main compounds not found in normal bile were detectable in ANIT- treated animals. The peaks were identified as: (1) natural bile component; (2) 1 -naphthyl isocyanate; (3) natural bile compon- ent; (4) 1-naphthylamine; ( 5 ) natural bile component; (6) 2'- acetonaphthone (internal standard); (7) ANIT.

and liver showed that ANIT was completely separ- ated under the conditions used; 1-NI and 1-N, however, overlapped other natural components in bile and liver. 1-NI and 1-N in the samples were identified by mass spectrometry.

The ions selected for 1-NI, 1-N, and ANIT were 169, 143, and 185 a.m.u., respectively. To correlate the extent of biotransformation with the toxic ef- fect of ANIT, the concentration of 1-NI, 1-N and ANIT in bile and liver were quantified by gener- ation of extracted ion chromatograms for m/z 169, 143 and 185. Peaks corresponding to the three compounds were found in bile (Fig. 2) and liver of the rats receiving ANIT with or without CX or PB pretreatment, but not in control untreated rats. The GC elution time (Fig. 2a, b, c) and the mass spectra (Fig. 2d, e, f) of the three peaks correspond well with those of the standards (Fig. 3).

When rats were treated with ANIT alone (Fig. 4 and 5) , the amounts of ANIT and its metabolites in the bile and liver were significantly lower in rats receiving 100 mdkg of ANIT than in rats receiving 300 mg/kg of ANIT. In rats given CX (2 mg/kg) 30 rnin before ANIT (300 mg/kg), the excretion rate of 1-NI in bile (Fig. 4) and its concentration in liver (Fig. 5 ) were significantly lower than ob- served in rats receiving only ANIT (300 mg/kg). The excretion rate of 1-N and ANIT in bile and the concentration in liver of the ANIT300-CX group were also lower than the respective values in the ANIT300 group.

Pretreatment of rats with PB (60 mg/kg per day) for 3 days before ANIT (100 mg/kg) administra- tion, produced excretion rates on 1-NI and 1-N in bile (Fig. 4) and their concentrations in liver (Fig. 5) that were higher than those observed in rats re-

AbundanceIon 169. LL 20000

0 min ->6.'50 7.'25

Abundance

10000 4

Ion 185. l o o o o ~ Ion 143 .50000 o";

0 7 . 0 0 7 . 7 5 1 0 . 7 0 1 1 . 3 9

R e t e n t i o n Time

(169 M+' 4

114 141 180197 233

0 I " ' i

4199 3 e' i 1 4 3 M+" 0

$'

Abundance 1 "-Po

1 0 0 0 0 0 0

0 min ->

1 0 0 0 0 0 0

0 min ->

R e t e n t i o n T i m e

Fig. 3. Gas chromatogram obtained from a synthetic mixture of standards. Peaks were identified by retention time. The peaks correspond to the following: 6.90, I-naphthyl isocyanate; 7.47, I-naphthylamine; 8.83, 2'acetonaphthone (internal standard); 11.19, 1-naphthylisothiocyanate.

273

Li et al.

0 I -NI

I-N

ANIT

ANIT300 ANIT300-CX ANIT100 ANIT100-PB

Fig. 4. Excretion rates of 1 apht,&cyanate (1-NI), I-naph-

bile. ANIT was administered orally. In the ANIT300 group, rats received only ANIT (300 mg/kg). In the ANIT300-CX group, rats were pretreated with cycloheximide (2 mgikg) i.p. 30 min be- fore ANIT (300 mgikg). In the ANIT100 group, rats received only ANIT (100 mgikg). In the ANITIOO-PB group, rats were pretreated with phenobarbital (60 mg/kg) i.p. daily for 3 days prior to ANIT (100 mg/kg). 1-NI, 1-N and ANIT were deter- mined at the fourth bile collection period (130-160 rnin) after ANIT administration. Results (mean?S.E.M.) are expressed as ngimin per g liver. a indicates p<0.05 vs. ANIT300 group.

thylamine (1-N) and 1-nap f thylisothiocyanate (ANIT) in rat

Discussion

ANIT and two of its metabolites, namely 1-NI and 1 -N, were identified by GUMS in the liver and bile of rats. The quantitative aspects of this study are consistent with the hypothesis that biotransforma- tion of ANIT is involved in the hepatotoxic effect of ANIT and in its potentiation or inhibition by various chemicals.

When rats were pretreated with CX, an inhibitor of protein synthesis and an attenuator of ANIT hepatotoxicity (8), the amounts of ANIT, 1-NI and 1-N in bile and liver were lower than those found in rats receiving ANIT alone (Fig. 4 and 5) . The concentrations of ANIT in the liver and bile were less than those observed in the ANIT300 group, which, in turn, produced fewer metabolites. This observation is consistent with previous work that showed that treatment with CX reduces ANIT ab- sorption in the gut (9). However, the effect of CX on the absorption of ANIT, might not be the only reason for the protection afforded from ANIT toxicity. Plaa and his coworkers showed that pre- treatment with CX prevented the alteration in 3H/ 14C ratio in bile normally observed in nonpre- treated rats even 16 h after ANIT administration (9, 12). It is thought that CX inhibition of protein synthesis (1 3-1 5 ) might modify the biotransforma- tion of ANIT by preventing the formation of

metabolites that may be toxic. The present study shows that while the hepatic ANIT concentration in the ANIT300-CX group was significantly higher than in the ANIT100 group (Fig. 5) , the hepatic 1-NI levels in both groups were in about the same range (ANIT300-CX 3.150.21 pg/g liver; ANIT100 2.81 20.32 pg/g liver). These findings suggest that decreased biotliansformation of ANIT occurs following CX pretreatment.

The ANITlOO-PB group showed that PB, an in- ducer of monooxygenase and a potentiator of ANIT toxicity enhanced the desulfuration of ANIT, and that concentrations of ANIT metabolites were increased following PB treatment. On the other hand, the excretion rate of ANIT into bile and the concentration of ANIT in liver were lower than in those that received ANIT alone. This finding sup- ports the suggestion of Capizzo & Roberts (16) that enhanced absorption is not an explanation for the mechanism by which PB potentiates ANIT-induced hyperbilirubinemia.4ur data are consistent with the hypothesis that PB enhances ANIT toxicity, possibly by increased biotransformation.

The metabolism of ANIT in vivo has not been complete elucidated. A number of thiono-sulfur- contai 'ng co ounds, including ANIT and car- bon sulfi e (CS,), exhibit toxic properties in

characteristics: 9 0 t h possess a C=S fragment; mammas. fY ANiTpnd CS2 share some commdn

20

h L ? .- I 15 z v

E .- 0 10 CI E 2 5 s CI

0 ANIT300 ANIT300-CX ANIT100

0 I-N1

0 1.N

ANIT

b

, , , . ... ...... ...... ...... ...... . .... . . . . . . ......

ANIT100-PB

Group Fig. 5. Concentrations of 1-naphthyl isocyanate (1 -NI), l-naph- thylamine (1-N) and I-naphthylisothiocyanate (ANIT) in rat liver. ANIT was administered orally. In the ANIT300 group, rats received only ANIT (300 mg/kg). In the ANIT300-CX groups, rats were pretreated with cycloheximide (2 mg/kg) i.p. 30 min before ANIT (300 mgikg). In the ANIT100 group, rats received only ANIT (100 mgikg). In the ANIT100-PB group, rats were pretreated with phenobarbital (60 mgikg) i.p. daily for 3 days prior to ANIT (100 mgikg). Livers were excised 190 min after ANIT administration. Results (mean5S.E.M.) are ex- pressed as pg/g liver. a indicates p<0.05 vs. ANIT300 group; b indicates p<0.05 vs. ANITlOO group.

274

Metabolites of 1-naphthylisothiocyanate

5. ROBERTS R J, PLAA G L. Potentiation and inhibition of a - naphthylisothiocyanate-induced hyperbilirubinemia and cholestasis. J Pharmacol Exp Ther 1965: 150: 499-506.

6 . DAHM L J, ROTH R A. Protection against a-naphthylisothi- ocyanate-induced liver injury by decreased hepatic non-pro- tein sulfhydryl content. Biochem Pharmacol1991: 42: 1181- 1188.

7 . DAHM L J, BAILIE M B, ROTH R A. Relationship between a-naphthylisothiocyanate-induced liver injury and elev- ations in hepatic non-protein sulfhydryl content. Biochem Pharmacol 1991: 42: 1189-1 194.

8 . INDACOCHEA-REDMOND N, WITSCHI H, PLAA G L. Effect of inhibitors of protein and ribonucleic acid synthesis on the hyperbilirubinemia and cholestatis produced by a-naphthy- lisothiocyanate. J Pharmacol Exp Ther 1973: 184: 780-786.

9. LOCK S, WITSCHI H, SKELTON F S , HANASONO G, PLAA G L. Effect of cycloheximide on the distribution of a-naphthyliso- thiocyanate in rats. Exp Mol Path011974 21: 237-245.

10. ~ I G E R G J, VYAS K P, HANZLIK R l? Effect of inhibitors of a-naphthylisothiocyanate-induced hepatotoxicity on the in vitro metabolism of a-naphthylisothiocyanate. Chem Biol Interact 1985: 52: 335-345.

11. CONNOLLY A K, PRICE S C, STEVENSON D, CONNELLY J C, HINTON R H. Factors influencing the toxicity of alpha- naphthylisothiocyanate towards bile duct lining cells. In: Guillouzo A, ed. Liver cells and drugs, Vol. 164, Colloque INSERM/John Libbey Eurotext Ltd. 1988: 191-196.

12. SKELTON F S, WITSCHI H, PLAA G L. The effects of cyclo- heximide, actinomycin D, and ethionine on the biliary ex- cretion of labelled alpha-naphthylisothiocyanate in rats. Exp Mol Pathol 1975: 23: 171-180.

13. VERBIN R S , FARBER E. Effect of cycloheximide on the cell cycle of the crypts of the small intestine of the rat. J Cell Bid 1967: 35: 649-658.

14. VERBIN R S, SULLIVAN R J, FARBER E. The effects of cyclo- heximide on the cell cycle of the regenerating rat liver. Lab Invest 1969: 21: 179-182.

15. WITSCHI H P The effects of diethylnitrosamine on ribo- nucleic acid and protein synthesis in the liver and lung of the Syrian golden hamster. Biochem J 1973: 136: 789-794.

16. CAPIZZO E ROBERTS R J. Effect of phenobarbital, chlor- promazine actinomycin D and chronic a-naphthylisothi- ocyanate administration on a-naphthylisothiocyanate-in- duced hyperbilirubinemia. J Pharmacol Exp Ther 1971: 179: 455464.

7. DALVI R R. Mechanism of the neurotoxic and hepatotoxic effects of carbon disulfide. Drug Metabol Drug Interact 1988: 6: 275-284.

8. EI-HAWARI A M, PLAA G L. Impairment of hepatic mixed- function oxidase activity by a- and P-naphthylisothiocyan- ate: relationship to hepatotoxicity. Toxicol Appl Pharmacol 1979: 48: 445458.

9. CHENGELIS C l? Paradoxical effect of cobaltous chloride on carbon disulfide induced hepatotoxicity in rats. Res Com- mun Chem Pathol Pharmacol 1988: 61: 83-96.

20. CHENGELIS C l? Changes in hepatic glutathione concen- trations during carbon disulfide induced hepatotoxicity in the rat. Res Commun Chem Pathol Pharmacol1988: 61: 97- 109.

21. NEAL R A, HALPERT J. Toxicology of thiono-sulfur com- pounds. Ann Rev Pharmacol Toxicol 1982: 22: 321-339.

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

23. TRAIGER G J, VYAS K P, HANZLIK R P Effect of thiocarbonyl compounds on a-naphthylisothiocyanate-induced hepato- toxicity and the urinary excretion of [35S] a-naphthylisothi- ocyanate in the rat. Toxicol Appl Pharmacol 1984: 72: 504- 512.

%

hepatotoxicity appears to be mediated by biotrans- formation for both agents (5 , 17); hepatotoxicity is accompanied by a decrease in hepatic cyto- chrome P-450 with both agents (18, 19); and both agents result in an increase in hepatic GSH (7, 20). Therefore, ANIT may be metabolized similarly to CS2. The metabolic pathways and the toxic nature of CS2 have been extensively studied. It is specu- lated that CS2 is biotransformed to COS and then C02 in two sequential steps, leading to the release and covalent binding of one or both of the sulfur atoms to cell macromolecules (17, 21). The data presented in the present study suggest that ANIT was metabolized by monooxygenase to 1 -NI, poss- ibly by the release and covalent binding of a reac- tive species of sulfur. Furthermore, 1-NI might be converted to 1-N and C02. The overall reaction could be the following:

N = C = S N = C = O

(ANIT)

This reaction scheme is supported by the 14C- ANIT disposition studies carried out by Capiz- zo & Roberts (4, 16, 22). They showed that about 7-8% of the total amount of radioactivity adminis- tered is eliminated as 14C02 within the first 12 h. More recently, a study carried out by Traiger et al. (23) demonstrated that 35S-labeled inorganic sul- fate was a major metabolite of rats treated with 35S-ANIT. In the present study, 1-NI and I-N were found in ANIT-treated animals.

Since the change in 1-NI concentration could be correlated to the release of sulfur, we suggest that the latter may bind covalently to hepatocyte macromolecules in a manner similar to that seen with CS2 (17, 21) to induce hepatotoxicity.

Acknowledgements

We thank Dr. Gilles Caille for use of his GUMS equipment, Dr. Wang Tao for expert technical assistance with GC/MS, and Dr. Orval A. Mamer for his expert advice on GC/MS. This work was supported in part by the Medical Research of Canada.

References 1 . GOLDFARB S, SINGER E J, POPPER H. Experimental cholan-

gitis due to alpha-naphthylisothiocyanate (ANIT). A m J Pathol 1962: 40: 685-695.

2. POPPER H, RUBIN E, SCHAFFNER E The problem of primary biliary cirrhosis. A m J Med 1962: 33: 807-810.

3. PLAA G L, PRIESTLY B G. Intrahepatic cholestasis induced by drugs and chemicals. Pharmacol Rev 1976: 28: 207-273.

4. CAPIZZO F, ROBERTS R J. a-Naphthylisothiocyanate (ANIT)-induced hepatotoxicity and disposition in various species. Toxicol Appl Pharmacol 1971: 19: 176-187.

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