Effect of thiocarbonyl compounds on α-naphthylisothiocyanate-induced hepatotoxicity and the urinary excretion of [35S]α-naphthylisothiocyanate in the rat

TOXICOLOGY AND APPLIED PHARMACOLOGY 72, 504-5 12 ( 1984)

Effect of Thiocarbonyl Compounds on wNaphthylisothiocyanate-Induced Hepatotoxicity and the Urinary Excretion of [35S]cY-Naphthylisothiocyanate in the Rat’v2 GEORGE

J. TRAIGER,~

Department

of Pharmacology

KAMLESH

University

Received

P. VYAS,~ AND ROBERT

and Toxicology and Department of Kansas, Lawrence, Kansas

June 24, 1983; accepted

September

of Medicinal

P. HANZLIK Chemistry,

66045

20, 1983

Effect of Thiocarbonyl Compounds on a-Naphthylisothiocyanate-Induced Hepatotoxicity and the Urinary Excretion of [35S]cY-Naphthylisothiocyanate in the Rat. TRAIGER, G. J., VYAS, K. P., AND HANZLIK, R. P. (1984). Toxicot. Appl. Pharmacol. 72,504-5 12. The effect ofdisulfiram (DSF), sodium diethyldithiocarbamate (DDTC), methyl diethyldithiocarbamate (Me-DDTC), and ethionamide on the hepatotoxic response of o-naphthylisothiocyanate (ANIT) was studied in the rat. The hyperbilirubinemic response of ANIT was significantly inhibited by ip or po DSF pretreatment. A more marked inhibition of toxicity occurred when DSF was given via ip injection. DDTC, Me-DDTC, and ethionamide significantly inhibited ANIT-induced hyperbilirubinemia. Me-DDTC is approximately three times more potent than DDTC as an inhibitor of toxicity. Approximately 16% of a dose of [35S]ANIT was excreted in the urine as inorganic sulfate 48 hr after dosing. Me-DDTC administered simultaneously with [35S]AMT significantly reduced urinary [‘*S]sulfate excretion in the first 24 hr. Ethionamide reduced urinary [35S]sulfateexcretion. Pretreatment with phenobarbital which stimulates toxicity in vivo increased urinary [35S]sulfate excretion 300% in the first 12 hr. Thus, this study shows that agents which sensitize or protect rats from the toxic effectsof AMT, correspondingly stimulate or inhibit the oxidative desulfumtion of ]35S]ANIT in viva.

The acute administration of a-naphthylisothiocyanate to mice and rats produces hyperbilirubinemia and cholestasis (Eliakim et al., 1959; Griffiths et al., 1961; Goldfarb et al., 1962; Becker and Plaa, 1965). These effects have led to the use of ANIT as an experimental ’ Supported in part by Biomedical Research Support Grant RR-5606 and ES-02335 from the National Institutes of Health. *Portions of this work were presented at the 1977 meeting of the Federation of American Societies for Experimental Biology (Fed. Proc. 36, 769). 3 Address correspondence to this author at The Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045. 4 Present address: Department of Drug Metabolism, Merck Sharp and Dohme Research Laboratories, West Point, Penn. 19486. 004 1-008X/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

tool for elucidating the mechanism(s) of chemically induced cholestasis. Several lines of evidence indicate that ANIT-induced hyperbilirubinemia and cholestasis are the result of its biotransformation by the hepatic mixed function oxidase system to toxic metabolite(s). Pretreatment with the microsomal enzyme inducer phenobarbital potentiates the hyperbilirubinemic response to ANIT (Roberts and Plaa, 1965). These workers demonstrated that the time course of the biphasic effects of SKF525A on ANIT’s response correlates with the time course of its biphasic effect on barbiturate metabolism. Disulfiram and its corresponding reduced thiol, diethyldithiocarbamate, have been shown to inhibit hepatic mixed function ox-

504

ANIT

METABOLISM

idase activity both in vitro and in vivo (Stripp and Neal, 1975; Honjo and Netter, 1969; Zemaitis and Green, 1976b). Diethyldithiocarbamate protects against the hepatotoxicity of agents such as CC4 (Siegers et al., 1978) acetaminophen (Strubelt et al., 1974) bromobenzene, thioacetamide, and dimethylnitrosamine (Siegers and Strubelt, 1975; Abanobi et al., 1977). The hepatotoxicity of these agents is believed to be mediated by their toxic biotransformation by cytochrome P-450-dependent mixed function oxidases (Mitchell et al., 1973; Hunter et al., 1977; Jollow et al., 1974; Shank, 1975). The present investigation was undertaken to determine the effect of disulfiram and diethyldithiocarbamate on the toxicity and metabolism of ANIT. Since diethyldithiocarbamate is further metabolized to S-methyl diethyldithiocarbamate (Me-DDTC) (Gessner and Jakubowski, 1972) it was of interest to examine and compare the effect of this metabolite with the parent compound. et al., 1969; Hunter

METHODS Male Sprague-Dawley rats (200-270 g) obtained from ARS/Sprague-Dawley, Madison, Wisconsin were used throughout the study. They were maintained ad libitum on a diet of Purina chow and tap water in a temperaturecontrolled room with 12-hr periods of light and darkness. a-Naphthylisothiocyanate was obtained from Aldrich Chemical Co., Milwaukee, Wise. Sodium diethyldithiocarbamate and disulftram were purchased from Sigma Chemical Co., St. Louis, MO. Ethionamide was purchased from Ives Laboratories, New York, N.Y., as Tracator-SC tablets and was reextracted with hot acetone. Treatments. cu-Naphthylisothiocyanate (I 50 mg/k& 0.8 1 mmoljkg) was dissolved in corn oil and administered po or via ip injection in a final volume of 3 ml/kg body weight. Ethionamide was suspended in corn oil and administered by ip injection at 13.5 mg/kg (0.81 mmol/kg) in a final volume of 1.5 ml/kg. The following drug treatments were used in the time course studies: disulfiram, 240 mg/kg (0.8 1 mmol/kg) ip or po, dissolved in corn oil in a final volume of 3 ml/kg; sodium diethyldithiocarbamate - 3Hz0, 365 mg/kg (1.62 mmol/kg). ip dissolved in 0.9% NaCl in a final volume of IO ml/kg; Me-DDTC, ip. 66 mg/kg (0.4 mmol/kg) dissolved in corn oil in a final volume of 1.5 ml/kg.

AND TOXICITY

505

Urinary excretion of [j5S]ANIT. [%]ANIT (0.7 mCi/ mmol, 0.8 1 mmol/kg) was dissolved in corn oil and administered po. Me-DDTC (0.4 mmol/kg, ip) or ethionamide (0.8 1 mmol/kg) was given via ip injection simultaneously with the po administration of [35S]ANIT. A group of rats was pretreated with sodium phenobarbital (60 mg/kg ip) daily for 3 days. [35S]ANIT was administered po 24 hr after the last phenobarbital injection. Control animals received injections of normal saline. The animals were kept in stainless-steel metabolism cages. They were provided with food and water ad libitum during the experiment. Urine samples were collected for a period of 48 hr, with care being taken to avoid contamination with feces. The urine samples were analyzed for total sulfur and inorganic sulfate according to the method of Folin (Hawk et al., 1954). Total plasma bilirubin concentration and plasma glutamic pyruvic transaminase (GPT) activities were measured 24 hr after dosing with ANIT. Blood was collected by aortic puncture and heparin-treated syringes. The plasma was separated and total bilirubin concentrations were determined by the method of Jendrassik and Grof (1938). as modified by Nosslin (1960) and Michaelsson (196 I). Plasma GPT activity was determined according to the method of Reitman and Frankel (1957). Synthesis of’&meth.vl diethyldithiocurbumate. Sodium diethyldithiocarbamate trihydrate (1 g, 0.0044 mol) and 5 ml of distilled water were placed in a 50-ml round bottom flask. Methyl iodide (1.25 g. 0.0088 mol) was added and the mixture stirred vigorously for 30 min. Anhydrous diethyl ether (20 ml) was added and the layers were separated. The organic layer was dried over anhydrous MgSO, and filtered; then the ether was removed. The crude methyl diethyldithiocarbamate was purified by vatuum distillation to give a 52.8% yield, bp 113-l 14°C (4 mm Hg). NMR (6 in CDCI,) 4.26-3.46 (b, 4, CH,): 2 56 (s. 3. CH,); 1.25 (t. 6. CHj). UV in ethanol solution. x, max at 251 nm, t 8500; hZ max at 277 nm. ( 11,410 (lit A, max 252 nm. c 9000; A2 max 276 nm. f 11,500). Synthesis o~[~‘SlcY-nuQhthy~j.sothi~~cyunute.[35S]AN1’i was prepared in 80% yield from [35S]ol-naphthylthiourea ([35S]ANTU) by the method of Cymerman-Craig er al (1963). [%]ANTIJ was synthesized essentially by the method of Frank and Smith (I 955). This procedure was modified to utilize the KSCN instead of the recommended NH,SCN. This modification allows synthesis ot [35S]ANTU starting with elemental sulfur-35 and KC‘N: K3%CN is formed in high yield simply by heating the two solids at 160°C for 90 min in a stoppered tube under nitrogen. Statistics. Student’s t test was used for statistical evaluation of the difference between two means. Multiple means were analyzed by a randomized one-way analysis of variance. When the analysis indicated that a significant difference existed, the means of each group were compared by Duncan’s Multiple Range test (Steel and Tonie. 1980). In all analyses the level of significance was p < 0.0.5

506

TRAIGER,

WAS,

RESULTS Eflects of Pretreatment of ANIT

on the Acute Toxicity

Pretreatment with disulfiram inhibited ANIT-induced hyperbilirubinemia. The time course for the effect of disulhram pretreatment is shown in Table 1. Oral pretreatment with disulfiram caused a significant inhibition of toxicity only when disulfiram was given 6 hr prior to ANIT. A more marked inhibition of toxicity was observed at all pretreatment times when rats were pretreated with ip doses of disulfiram. The ip injection of disulfiram caused a serofibrinous inflammation of the peritoneum as has been reported by Child and Crump (1952) and it may be that such in-

TABLE 1 EFFECTOFTIMEOFPRETREATMENT WITH DISULFIRAM ONANIT-INDUCEDHYPERBILIRUBINEMIA IN RATS' Plasma bilirubin mg/lOO ml Pretreatment time (hr)

ANIT

Disulfiram + ANIT

Disulfiram administered po, ANIT ip 0.25

1 lC 3

6

2.61 + 0.24 2.61 I? 0.24 3.29 k 0.33 2.61 * 0.24 2.61 + 0.24

2.53 2.49 3.45 2.18

r 0.23 r 0.34 r 0.14 -+ 0.21

0.62 1.19 0.82

+ 0.28 + 0.28 + 0.31 f 0.33

1.95 * 0.19*

Disulfiram administered ip, ANIT po 0.25

1 2 6

3.20 3.59 2.11 3.16

+ 0.11’

+ o.43b + 0.24'

1.51 -t 0.316

’ Disulfiram (0.8 1 mmol/kg) was given at various times prior to ANIT (0.8 1 mmol/kg). Blood samples were taken 24 hr aRer ANIT administration. Values shown are the mean + SE obtained from groups of four to five rats. * Significantly different from ANIT, p < 0.05. ’ Disulfiram was given at a dose of 3.24 mmol/kg in this experiment.

AND HANZLIK

flammation is involved in the reduction of toxicity when disulfiram is given by this route. The first step in the metabolism of disulfiram is its rapid reduction to diethyldithiocarbamic acid (DDTC) which mainly undergoes hydrolytic and conjugative metabolism to carbon disulfide, diethylamine, diethyldithiocarbamate-S-glucuronide, and S-methyl diethyldithiocarbamate (Me-DDTC) (Prickett and Johnston, 1953; Stromme, 1965; Kaslander, 1963; Eldjarn, 1950; Gessner and Jakubowski, 1972). Since DDTC, like disulfiram, also inhibits hepatic mixed function oxidase activity, it was of interest to examine its effect on the toxicity of ANIT. Also examined in this experiment is the disulfiram metabolite Me-DDTC. DDTC and Me-DDTC significantly blocked the hyperbilirubinemic and GPT responses due to ANIT (Figs. 1 and 2, respectively) when coadministered with or given 3 hr prior to the toxin. A 6-hr pretreatment with either agent yielded plasma bilirubin values which were not significantly different from those found in rats receiving ANIT alone. The dose-response relationship for the inhibition of ANIT’s hyperbilirubinemic response by DDTC and Me-DDTC is shown in Fig. 3. The data indicate that Me-DDTC is approximately three times more potent than DDTC as an inhibitor of ANIT-induced toxicity. Previous studies have suggested that an Soxidative pathway is required for the expression of the toxic effects of thiocarbonyl compounds (Neal and Halpert, 1982). The possibility that S-oxidation may be involved in the toxic biotransformation of ANIT was examined by pretreating rats with ethionamide, which is metabolized via an S-oxidative pathway (Johnston et al., 1967). As is evident from Table 2, ethionamide diminished ANIT-induced toxicity. Urinary [35S]Suifate Excretion [35S]ANIT was synthesized and an investigation of the fate of the sulfur atom was

ANIT

METABOLISM

GPT

I

I

Eflect of Pretreatments Excretion

ANIT DDTC’ANIT

0

q

36

1 PRETREATMENT

TIME

,“O”RsI

FIG. 1. Effect of times of pretreatment with sodium diethyldithiocarbamate (DDTC) on ANIT-induced hepatotoxicity. DDTC ( I .62 mmol/kg, ip) was administered along with or prior to ANIT (0.81 mmol/kg, po) at the times shown on the abscissa. On the ordinate is shown the mean plasma bilirubin concentration and the mean ghrtamic pyruvic transaminase (GPT) activity. Plasma bilirubin concentrations and GPT activities were determined 24 hr after dosing with ANIT. Each bar represents the mean ? SE obtained from a group of five to six rats. An asterisk indicates that the mean is significantly different from ANIT alone (p < 0.05).

carried out to determine if the toxin yields inorganic sulfate. The recovery of 35Sin urine (collected for a period of 48 hr) following a single po administration of [35S]ANIT is shown in Table 3. Approximately 29% of the dose of 35S was excreted in the urine during the 48-hr collection period. On analysis of total urinary radioactivity, 16% of the administered dose of 35S was found to be present as free sulfate. In order to check for the presence of SOz as a volatile metabolite in the expired air, rats were dosed with [35S]ANIT and placed in a sealed glass chamber for a period of 24 hr. The chamber was perfused with air which exited through sodium tetrachloromercurate traps. Analysis of the trapping solution for 35S by liquid scintillation spectrometry indicated the absence of 35S02 in the expired air.

507

AND TOXICITY

on Urinary

Sdfhte

A study was carried out to identify correlations between the effect of agents which alter the toxicity of ANIT and the metabolism of [35S]ANIT. The effect of Me-DDTC on the urinary excretion of [35S]sulfate after [35S]ANIT administration is shown in Fig. 4. Me-DDTC coadministered with [35S]ANIT significantly reduced the urinary excretion of [35S]sulfate. Ethionamide also reduced urinary [35S]sulfate excretion in the first 24 hr (Fig. 5). Phenobarbital pretreatment, which stimulates both the metabolism and toxicity of ANIT in viva (Roberts and Plaa, 1965; Capizzo and Roberts, 197 1b), resulted in a threefold increase of urinary [35S]sulfate excretion at 12 hr (Fig. 5). Thus agents which either inhibit or augment the hyperbilirubinemic response of ANIT correspondingly inhibit or stimulate

PRETREATMENT

TIME

CHOJRSI

FIG. 2. Effect of time of pretreatment with methyl diethyldithiocarbamate (ME-DDTC) on ANIT-induced hepatotoxicity. Me-DDTC (0.40 mmol/kg, ip) was administered along with or prior to ANIT (0.81 mmol/kg. po) at the times shown on the abscissa. On the ordinate are shown the mean plasma bihrubin concentration and the mean ghrtamic pyruvic transaminase (GPT) activity. Plasma bilirubin concentrations and GPT activities were determined 24 hr after dosing with ANIT. Each bar represents the mean -t SE obtained from a group of five to six rats. An asterisk indicates that the mean is significantlv different from ANIT alone (p < 0.05).

TRAIGER,

508

02

04

OS QE

LO 12 Dose

o

Me-DDTC

l

DDTC

14

l6

I6 24

22

24

VYAS. AND HANZLIK

26

’

hMOLES/KGl

FIG. 3. Dose-response relationship for the inhibition of ANIT-induced hyperbilirubinemia by diethyldithiocarbamate (DDTC) or methyl diethyldithiocarbamate (MeDDTC). Abscissa: dose of DDTC or Me-DDTC administered via ip injection with a simultaneous challenge dose of ANIT (0.81 mmol/kg, PO). Ordinate: percentage of hyperbilirubinemic response taking the mean plasma bilirubin concentration of animals receiving only ANIT as 100%. Plasma bilirubin concentrations were determined 24 hr after dosing. Each point represents the mean -C SE for a group of four to five animals.

their oxidative desulfuration (De Matteis, 1974; Hunter and Neal, 1975; Dalvi et al., 1974; Neal and Halpert, 1982). The present finding that 35S-labeled inorganic sulfate is a major metabolite of rats treated with [35S]ANIT is consistent with the involvement of desulfuration in its metabolism. The oxidative desulfuration of ANIT by the hepatic mixed function oxidase system could be expected to result in the release of atomic sulfur and the formation of cu-napthylisocyanate. Elemental sulfur can react nonenzymatically with membrane nucleophiles (e.g., sullhydryl group of cysteine) or free tissue thiols (e.g., sulphydryl group of reduced glutathione) resulting in the formation of a persulfide. Persulfides are unstable and can react with thiol, giving hydrogen sulfide and the corresponding disulfide (Sorbo, 1957; Roy and Trudinger, 1970). R - SH + [S*] - R - S - S* - H R-SH+R-S-S*-HH$*

+ R - S- S- R

The overall reaction is thus the metabolism of [35S]ANIT as measured by [35S]sulfate excretion in the urine. DISCUSSION ANIT and several other thiocarbonyl compounds, including carbon disulfide, phenylthiourea, and parathion, have previously been shown to decrease the concentration of cytochrome P-450 when incubated in the presence of NADPH whereas their oxygen containing analogs were inactive (De Matteis, 1974). A similar effect on benzphetamine metabolism has been reported for carbon disulfide and thioacetamide (Hunter and Neal, 1975). Based on the results obtained with carbon disulfide and parathion, it has been postulated that the inhibitory as well as toxic effects of these thiono-sulfur compounds may be the result of the liberation of atomic sulfur during

S* + 2 RSH -

H$*

+ RSSR.

Sulfide can be oxidized to sulfate by mitochondrial enzymes present in liver and kid-

TABLE 2 EFFECT OF ETHIONAMIDE HEPATOTOXICITY

ON ANIT-INDUCED IN THE RAT’

Treatment

Total plasma bilirubin mg/lOO ml

GPT activity (units/ml)

Control ANIT Ethionamide + ANIT

0.20 + 0.02 2.47 k 0.07 1.24 + 0.24’

16? 2 104 f 20 44+ 3b

’ Ethionamide (0.8 1 mmol/kg, ip) was administered simultaneously with ANIT (0.81 mmol/kg, po). Each value represents the mean -+ SE obtained from five animals. ‘p < 0.05 when compared with ANIT alone.

ANIT

METABOLISM

TABLE 3 EXCRETION OF 35S IN URINE OF RATS DOSED WITH [35S]ANlT” Urine sample

inorganic sulfate (% dose)

Urine sample (total % dose)

O-24 hr 24-48 hr O-48 hr

8.20 + 0.25 8.09 + 1.32 16.29 f 1.55

15.10 f 0.45 14.14 f 1.54 29.24 + 1.98

’ [‘SIANIT (0.81 mmoI/kg, 0.7 mCi/mmoI) was administered orally. Urine samples were collected at the indicated time. Each value represents the mean +- SE obtained from a group of four animals.

ney (Siegel, 1975; Bartholomew et al., 1980). Thus the present demonstration that pretreatments which enhance or decrease oxidative desulfuration of [35S]ANIT in vivo respectively sensitize or protect animals from ANIT-induced liver injury supports the hypothesis that the toxic effects of this thione-containing compound are initiated at least in part by Soxidative bioactivation.

509

AND TOXICITY

The desulfuration of ANIT could occur via oxidative attack on the sulfur atom or via a hydrolytic mechanism which would lead to extrusion of sulfide rather than atomic sulfur. The available evidence indirectly suggests that an oxidative process is involved. The present results as well as those of Roberts and Plaa ( 1965) indicate that pretreatments with agents which inhibit or induce hepatic mixed function oxidase activity respectively inhibit or stimulate ANIT induced toxicity. An oxidative mechanism is further suggested by requirement of oxygen and NADPH for the in vitro binding and metabolism of ANIT (El-Hawaii and Plaa, 1977; Vyas, 1979). Although there is no direct evidence that the toxic biotransformation of ANIT involves esterase-mediated hydrolysis, such a mechanism cannot be excluded. Disulfiram and to a lesser extent DDTC, while known to inhibit oxidative drug metabolism, have also been shown to impair hepatic microsomal esterase activity (Zemaitis and Greene, 1976a).

2 B m

g 24 s.E E t

-

I’“sl-ANIT

Time

After[‘%]

-ANIT

Administration

(Hr)

FIG. 4. Effect of Me-DDTC (0.40 mmoI/kg, ip) on urinary excretion of 35S-labeled inorganic sulfate from rats dosed with [?S]ANIT (0.81 mmoI/kg, po). Each point represents the cumulative [35S]sulfate content (mean t SE) in urine, expressed as a percentage of original dose from four rats. The asterisk represents values significantly different from animals receiving [‘?3]ANIT alone (p < 0.05).

510

TRAIGER,

WAS,

24-

ANIr-=S

-

ANIT-35S

+ Phenabarbttal

-

ANIT-SSS

+ Ethionamide

20: 0” 2 b ‘, I P 9 ‘0 z

16-

‘2-

Time After

ANlT-35S

Admtniatratiar

(Hr)

FIG. 5. Effect of ethionamide (0.81 mmol/kg, ip) or phenobarbital pretreatment (60 mgJkg, ip, daily for 3 days) on urinary 35S-labeled inorganic sulfate excretion from rats dosed with [35S]ANIT (0.8 I mmol/kg, po). Each point represents the cumulative [“S]sulfate content (mean + SE) in urine, expressed as a percentage of original dose from four rats. The asterisk represents values significantly different from animals receiving [‘?S]ANIT alone

(p < 0.05).

Coadministration of ethionamide, DDTC, or Me-DDTC with ANIT protected from the hepatotoxicity due to ANIT. Inhibition of ANIT’s hyperbilirubinemic response was three to four times greater with Me-DDTC than with DDTC. One interpretation for the above finding could be that biotransformation of MeDDTC is more closely related to the biotransformation of ANIT than is that of DDTC. The greater potency of Me-DDTC over DDTC correlates very well with the findings of Gessner and Jakubowski (1972). These authors reported that DDTC undergoes S-methylation to Me-DDTC by rat liver and kidney microsomal fraction. Furthermore, when [35S]MeDDTC and [35S]DDTC were fed to rats, over 60% of the radioactivity from Me-DDTC was excreted as inorganic sulfate in urine during the 48-hr period following dosing, compared to only 16% from DDTC-treated animals. This finding indicates that Me-DDTC is a better substrate than DDTC for a pathway to sulfate formation. DDTC mainly undergoes hydrolytic and conjugative metabolism to carbon disulfide, diethylamine, and DDTC-Sglucuronide (Strbmme, 1965). The authors sug-

AND HANZLIK

gested that the formation of inorganic sulfate from DDTC occurs via the intermediate formation of Me-DDTC. Although there is no direct evidence that DDTC and Me-DDTC are metabolized to their corresponding S-oxides, this pathway is plausible since it would account for the eventual formation of inorganic sulfate. Thus the inhibition of in vivo toxicity of ANIT by these thione compounds further suggests that ANIT may also undergo toxic biotransformation via an S-oxidase pathway. Inhibition of hepatic microsomal drug oxidase activity in vivo after oral treatment with disulfiram or DDTC has been sugested in part to involve their metabolism to carbon disulfide, and it is the latter which represents the effective inhibitor (Zemaitis and Greene, 1979). Me-DDTC has been shown to be a major metabolite of DDTC in the dog (Cobby et al., 1978) and of disulfiram in man (Cobby et al., 1977). The results obtained with the methyl ester in the present investigation indicate that the formation of this metabolite of disulfiram and DDTC may also contribute to the decrease of oxidase activity seen after dosing with these agents. Unpublished studies in our laboratory are consistent with such an effect. Me-DDTC in vitro significantly inhibited NADPH-dependent metabolism of aminopyrine and ANIT by hepatic microsomes from phenobarbital-treated rats (Vyas, 1979). Approximately 16% of the dose of [35S]ANIT was recovered in the urine as sulfate during the 48-hr collection period. This value is in agreement with previous estimates for the excretion of 14C02 in the expired air after dosing rats with [ 14C]ANIT labeled in the isothiocyanate group (Capizzo and Roberts, 197 1a). About 18% of the administered dose was recovered from the expired air collected for 48 hr. To our knowledge, the only other identified metabolite of ANIT is cu-naphthylamine, which is excreted in urine and bile (Mennicke et al., 1978). The formation of (Ynaphthylamine as well as COz and sulfate derived from ANIT’s isothiocyanate group is consistent with the oxidative desulfuration of

ANIT

METABOLISM

ANIT yielding the corresponding oxygen analog, cw-naphthylisocyanate, and atomic sulfur. Further metabolism of the sulfur in vivo as mentioned above results in sulfate formation. In contrast to ANIT, a-naphthylisocyanate is very reactive toward water and is readily hydrolyzed to a-naphthylamine and C02. The delayed effect of orally administered disulfiram on ANIT’s toxicity correlates with the time course of its impairment of oxidative drug metabolism by hepatic microsomes (Honjo and Netter, 1969; Stripp et al., 1969). The pronounced inhibition of toxicity seen when rats were pretreated with disulfiram ip 0.25 to 2 hr prior to ANIT is not in accord with the temporal aspects of this effect. However ip administration of disulfiram resulted in marked peritoneal inflammation and edema. Therefore, the possibility exists that the presence of inflammation per se or the accompanying stress may contribute to the reduction of ANIT’s toxic response. This suggestion is supported by the observation of Indacochea-Redmond et al. (19731, who noted that 12 hourly ip injections of saline to ANITtreated rats significantly reduced toxicity. A difference in the effect of disulfiram on the hepatotoxicity of vinyl chloride after po or ip dosing has been reported by Jaeger et al. (1977). Disulfiram given by the ip route produced a twofold greater augmentation of vinyl chloride-induced hepatotoxicity relative to that seen when disulfiram was administered orally. The authors proposed the difference may represent the contributory effect of peritonitis.

AND TOXICITY

511

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