TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

Effects of Disulfiram

108,366-373

(1991)

on Hepatic P450llE1, Other Microsomal and Hepatotoxicity in Rats’

Enzymes,

JOHN F. BRADY, FANG XIAO, MONG-HENG WANG, YAN LI, SHU M. NING, JEANNE M. GAPAC,ANDCHUNGS.YANG' Laboratoryfbr

Cancer

Research, Department Rutgers University.

Received

November

of Chemical Piscataway,

Biology and Pharmacognosv, New Jersey 08855-0789

5, 1990; accepted

January

College

0fPharmac.v.

I I, 1991

Effects of Disulfiram on Hepatic P450IIE I, Other Microsomal Enzymes, and Hepatotoxicity in Rats. BRADY, J. F.. XIAO, F., WANG, M.-H., Lr, Y.. NING, S. M., GAPAC. J. M., AND YANG, C. S. (1991). Toxicol. Appl. Pharmacol. 108, 366-373. Disulfiram, widely used in avoidance therapy for alcohol abuse, has been shown to have protective effects against chemically induced toxicity and carcinogenesis. The purpose of this work was to elucidate the biochemical mechanisms of this protective action by examining its effects on cytochrome P4501IEl and other related microsomal enzyme activities. When a dose of disulfiram was given intragastrically to rats, a very rapid decrease of N-nitrosodimethylamine (NDMA) demethylase activity, possibly due to the inactivation of P450IIE 1, was seen. The loss of P450IIE 1 protein from the microsomal membrane was observed at 18 hr after receiving disulfiram, but not within the first 5 hr after the treatment. P450IIB1, on the other hand, was induced markedly between 15 and 72 hr after the disulfiram treatment. The treatment, however. caused only moderate changes in some other P450 isozymes. Carbon disulfide, a putative metabolite of disulfiram, produced similar effects on P4501IE 1. but with shorter duration. Carbon disullide. however, did not induce P450IIB 1. Diethyldithiocarbamate. a reductive product of disulfiram, was an inhibitor of P450IIEl activity in vitro, and upon preincubation with microsomes, it produced an NADPHdependent inactivation of NDMA demethylase activity. The results suggest that this or other metabolites of disulfiram are inhibitors of P4501IE 1 and are responsible for the inactivation of P450IIEl in viva. Hepatotoxicity of NDMA or CCIl in rats was blocked by pretreatment with disulfiram. The present work demonstrates that P450IIEl was inhibited and inactivated by disulfiram, and this mechanism can account for many of the reported inhibitory actions of disulfiram against chemically induced toxicity and carcinogenesis. 0 199 I Academic

Press. Inc.

dimethylhydrazine and the associated DNA methylation and colon tumorigenesis as reviewed by Fiala ( 198 1) and Wattenberg ( 1978) and protection against hepatotoxicity of CC&, CHC13, acetaminophen, N-nitrosodimethylamine (NDMA), and other compounds (Jorgensen et al., 1988; Masuda and Nakayama, 1982; Schmahl et al., 1976). In the case of NDMA, DSF inhibited carcinogenesis in the liver, but shifted the site of tumor formation to the nasal cavity (Bertram, 1989). The induction of glutathione Qtransferase by DSF (Spamins et al., 1982) may partially contribute

Disulfiram (DSF)3 has been used in avoidance therapy for alcohol abuse for decades. More recently, this compound has been investigated for its protective effect against chemically induced toxicity and carcinogenesis. Examples include inhibition of the metabolism of 1,2’ The work was supported by Grants ES03938 and CA37037 from the National Institutes of Health and Grant 88B18 from the American Institute for Cancer Research. ’ To whom correspondence should be addressed. 3 Abbreviations used: DSF, disulfiram: DDTC, diethyldithiocarbamate; NDMA, N-nitrosodimethylamine: P450, cytochrome P450. 0041-008x/91

$3.00

Copyright g 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

366

EFFECTS

OF

DISULFIRAM

to these protective effects. In vivo, DSF is reduced to diethyldithiocarbamate (DDTC) which undergoes conversion to various products including CS2 (Fiala, 198 1; Stromme, 1965). Covalent trapping of reactive intermediates by DDTC has been demonstrated in the case of NDMA (Tacchi et al., 1984). However, the molecular basis for many of the observed effects of DSF was not known. A key hypothesis tested in this work is that DSF effects its protective function against chemically induced toxicity and carcinogenesis by inhibiting the activities of cytochrome P450IIE 1. P450IIE 1 is primarily responsible for the initial metabolism of many small organic compounds such as aniline, CCL,, CHC13, benzene, NDMA, azoxymethane, enflurane, diethyl ether, and acetaminophen (reviewed by Yang et al., 1990). This isozyme is present constitutively in the liver, kidney, brain (Hansson et al., 1990), and olfactory mucosa (Ding and Coon, 1990). P450IIEl is inducible, in the liver and perhaps other tissues, by various treatments (Koop et al., 1985; Yang et al., 1990) including fasting, diabetes, secondary ketones, alcohols, isoniazid, trichloroethylene, benzene, and pyrazole. Recent studies have shown that P450IIE l-dependent activities are decreased by treating animals with organic sulfur compounds such as diallyl sulfide (Brady et al., 1988), phenethyl isothiocyanate (Ishizaki et al., 1990), and dimethyl sulfide (Arndt et al., 1989). Pretreatment of rats with the first two compounds was found to lower the level of this isozyme in hepatic microsomes. DSF and its metabolites, DDTC and CS2, exhibit cancer protective properties that resemble those described for diallyl sulfide. The inhibition of microsomal aniline hydroxylase activity by DSF pretreatments (Zemaitis and Greene, 1976) suggested that P450IIEl could be inactivated by this compound or its metabolites. P450IIE 1 apparently metabolizes CS2 (Snyderwine et al., 1988), possibly resulting in enzyme inactivation due to the binding of the reactive product, atomic sulfur (Chengelis and Neal, 1987).

ON

367

P450IIEl

In the present study, the effects of treatment of rats with DSF or CS2 on hepatic P450IIE 1, other microsomal enzyme activities, and the toxicity of NDMA and CC& were examined. The in vitro inhibition of P450IIE l-mediated NDMA demethylase activity by DDTC was also examined. METHODS Materials. Crystalline disulfiram(bis[diethylthiocarbamoyl]disulfide). havingareportedmpof 69-7O”C,was obtainedfrom SigmaChemicalCo. (St. Louis,MO). DDTC (diethyldithiocarbamic acid, sodium salt, trihydrate; ACS reagent grade) was purchased from Aldrich Chemical Co. (Milwaukee, WI) and had a purity of >99%. CSZ (ACS reagent grade) was from Fisher Scientific (Fair Lawn, NJ). 7-Ethoxyresorufm, 7-pentoxyresorufin, and resorufin were from Pierce (Rockford. IL). Corn oil was a product of Mazola. CPC International, Inc. (Englewood Cliffs, NJ). Antibodies against P450IIBl which also recognize P4501IB2 were kindly provided by Drs. F. J. Gonzalez, S. S. Park, and H. V. Gelboin (Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD). Polyclonal antibodies against P450IIEl were prepared as described (Ma et al.. 1989; Yoo et al., 1987). Animal treatments and microsomal preparation. Male Sprague-Dawley rats (90-100 g) were purchased from Taconic Farms (Germantown, NY) and maintained as described (Brady et al.. 1988). DSF or CS2 was mixed with corn oil and administered intragastrically at the indicated doses and time points. Control animals received the same amount of vehicle (corn oil, usually I ml/kg body weight). The animals were killed by decapitation and liver microsomes were prepared (Brady et al.. 1988). Protein content was determined using the BCA method (Pierce) with bovine serum albumin (Fraction V from Sigma) as standard. Assaysfor monooxygenase activities. The conditions for the determination of microsomal P450 content by the reduced CO-difference spectra and NADPH-dependent reductase with cytochrome c as an electron acceptor have been described (Yoo ef al., 1987). Previous conditions were used for the assay of NDMA demethylase at a substrate concentration of 1 mM NDMA by measuring formaldehyde formation (Yoo ef al.. 1987). for the assays of pentoxyresorufin and ethoxyresorufin dealkylase activities by a fluorescence method (Brady et al., 1988). and for the quantitation of P450IIEl and P4501IBl by immunoblot analysis (Hong d al.. 1987).

RESULTS

E#ect of DSF treatment on hepatic monooxygenase activities. Rats were treated with a

368

n-.-v,

BKAU

I

600

I +p\:,’ t&q-:\

500 2 E 400

o----p 1 +

300 200

0 ii g 8 22 5. u”

100 0

C 0

12

24

36

Time

48

60

72

(h)

FIG. I. Hepatic monooxygenase activities after a single dose of DSF. DSF was given to rats intragastrically at a dose of 3.4 mmol/kg body wt. Each point represents the mean f SD of three separate microsomal preparations. Datum points above “c” were from vehicle-treated controls. The first time point was taken 1 hr after DSF administration. Microsomal P450 content (0) is expressed as nmol/mg protein. NADPH-dependent reductase activity (A) is given in nmol cytochrome c reduced/min/mg protein. NDMA demethylase activity (0) was measured as nmol HCHO/min/mg protein. Pentoxyresorufin (V) and ethoxyresorufin (0) dealkylase activities are expressed as pmol resorufin/min/mg protein. *p < 0.01 compared to control. ‘p < 0.05 compared to control.

single intragastric dose of DSF at different times prior to euthanization and the effects of the treatment on hepatic microsomal enzymes were analyzed (Fig. 1). The P450 content in the treated group was approximately 60-80% of that of controls throughout the period from 1 to 72 hr. Reductase activity increased gradually with treatment time, reaching 1.4-fold induction at 24 hr. NDMA demethylase activity, a rather specific assay for P450IIEl (Yang et ul., 1990; Yoo et al., 1990), was markedly decreased and this was observed 1 hr after DSF administration (the first time point). In contrast, pentoxyresorufin depentylase activity, primarily displayed by P450IIBl (Lubet et al., 1985), was induced

--

El

.

.

AL.

markedly between 15 and 72 hr. Ethoxyresorufin deethylase which probably reflects the activity of P450IAl and P450IA2 was 60% of that of controls between 2.5 and 15 hr and returned to control levels by 24 hr. A single dose of the vehicle (1 ml corn oil/kg) had no effect on these monooxygenase parameters during a similar time-course experiment. Dose-dependent responses of microsomal enzymes were studied with DSF administered 18 hr prior to killing (Fig. 2). Slight induction of NADPH-dependent reductase activity was produced by almost all the doses of DSF, whereas total cytochrome P450 content was not significantly affected. NDMA demethylase activity was 30% of that of controls at the lowest DSF dose used (0.1 mmol/kg) and 17% at the highest dose (3.4 mmol/kg). Pentoxyresorufin dealkylase activity was significantly induced only at doses around or above 1 mmol/kg. Ethoxyresorufin dealkylase activity appeared to be induced slightly at low DSF doses, but the data are scattered and the overall dose-response effect was not clear. 600

1.5 2

Disulfiram

dose

(mmol/kg)

FIG. 2. Dose response of DSF effects on hepatic monooxygenase activities. DSF was given to rats 18 hr before being killed. Each point represents the mean _+ SD of three separate microsomal preparations. The symbols and units correspond to those in Fig. 1.

EFFECTS OF DISULFIRAM

Efict of CS, treatment. Treatment with CS2 led to a marked reduction in NDMA demethylase activity at 2.5 and 5 hr; but in contrast to the response to DSF, this activity began to recover by 15 hr, and remained slightly depressed up to 72 hr (Fig. 3). The treatment also caused a rapid decrease in ethoxyresorufin dealkylase activity which recovered by 15 hr. No induction of pentoxyresorufin dealkylase activity, cytochrome P450 content, or NADPH-dependent reductase activity occurred. When CS2 was administered 18 hr prior to microsomal P450 content euthanization, seemed to decrease and the reductase activity seemed to be elevated but the effect was not clear (Fig. 4). NDMA demethylase activity was decreased and dropped to 35% of that of control with CS2 at 3.4 mmol/kg. Pentoxyresorufin dealkylase activity was unchanged. Ethoxyresorufin dealkylase activity increased slightly as the dose of CS2 was raised, but the dose response was not clear. ?

‘S tj.0 6 3

1.5

O\O

3.0 . i 2.0

0 50

I\

I /

QE

+ 8---b-p+

z e ii 2 5

1.0. yw

_

e.

'9 *p

0.0 c

0

0 12

24

36 48 Time (h)

60

72

FIG. 3. Hepatic monooxygenase activities after a single dose of CS2. CS2 was given to rats intragastrically at a dose of 3.4 mmol/kg. Each point represents the mean 2 SD of three separate microsomal preparations. The first time point was taken 10 min after CS2 administration. The symbols

1.5 ‘; ‘5 i; Fi Ln 1.0 c 0 E 5

700

0.5 -

6 r

100

?=0.01

JO

CS2

Dose

(mmol/kg)

FIG. 4. Dose response of CS2 effects on hepatic monooxygenase activities. CS2 was given to rats 18 hr before being killed. Each point represents the mean f SD of three separate microsomal preparations. The symbols and units correspond to those in Fig. I.

600

z

z 9

369

ON P45011El

and units correspond

to those in fig.

1.

Efect of DSF pretreatment on microsomal contents of P450IIEl and P45OIIB. Microsomes prepared from animals pretreated with DSF for 18 hr were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis (Fig. 5). A decline in P45OIIEl content to 20% of that of controls (Panel A) and an elevation of P450IIB1&2 to more than IO-fold (Panel B) were observed, in agreement with the decrease in the rate of NDMA demethylase and the elevation in pentoxyresorufin dealkylase activities, respectively (Fig. 1). When samples from rats pretreated with DSF for 1, 2.5, and 5 hr were analyzed. a change in the content of P450IIEl protein was not observed (data not shown). In vitro inhibition of NDMA demethylase activity by DDTC. When 0.05, 0.1, and 0.5 mM of DDTC were added to a microsomal NDMA demethylase assay mixture, 5 1, 76, and 86% inhibitions were observed, respectively. The possibility that DDTC was metab-

--

370

BKAUY

1

2

3

6

7

4

5

6

7

8

9

-_. -tl

10

0

1

2

3

4 5

8

910

111213141516171819

FIG. 5. Immunoblot analysis of hepatic microsomes. Microsomes were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by immunoblot analysis with anti-P450IIE 1 IgG (Panel A) or antiP450IIBl (IIBZ) IgG (Panel B). In Panel A. individual microsomal preparations were obtained from controls (lanes 3-6. 5 rg protein each) or from animals treated with DSF 18 hr prior to being killed (lanes 7-10. 5 pg each). As references for P450IIE 1, microsomes from acetone-treated rats were applied to lanes 1 and 2 at 2.66 and 1.33 pg protein, respectively. In Panel B. lanes l-4 contained purified P450IIBl applied at 0.04, 0.08, 0.12. and 0. I6 /*g protein per lane, respectively. Lanes 5-8 contained microsomes from phenobarbital-treated rats at 2.7. 4.0. 5.3, and 6.6 Kg protein, respectively. as references for the induction of P450IIB I. Lanes 9- 12 each contained 5 peg protein from individual control microsomes showing primarily P450IIB2. Lanes 13-16 each contained 5 pg microsomal protein from rats treated with DSF for 18 hr. The lower band is due to P4501IB I. Lanes 17 and I8 contained the same microsomes as applied to lane 9 but using 10 and 15 pg protein, respectively. Lane 19 contained 10 fig of the microsomes used for lane 10.

AL.

min incubation. In this experiment, the microsomal NADPH-cytochrome c reductase activity was only slightly lowered (data not shown), suggesting that the metabolism-dependent decrease of NDMA demethylase activity is mainly due to an inactivation of P450IIEl by metabolites of DDTC. The results indicate that DDTC itself is an inhibitor of NDMA demethylase, and it can also cause a metabolism-dependent inactivation of P450IIE 1. Prevention of hepatotoxicity by DSF. Previous studies have shown the potentiation of the hepatotoxicity of NDMA (Lorr et al., 1984) and CC& (Hewitt et al., 1980) by treatments that induce P450IIEl. Depression of this isozyme by DSF was expected to lower the toxic responses to these compounds. Pretreatment of rats with DSF (3.4 mmol/kg) virtually eliminated the CC& and NDMA-induced elevations in serum transaminase activities, suggesting that pretreatment with DSF effectively alleviated the hepatotoxicity (Table 1). a.0

6.0

v

4.0

2.0

0.0

olized to reactive intermediates that inactivate P450IIEl was investigated using a two-stage incubation protocol in which microsomes were incubated with DDTC (0.1 mM) in the presence or absence of an NADPH-generating system. The mixture was then diluted 10 times to determine the NDMA demethylase activity. In the absence of an NADPH-generating system, the microsomal NDMA demethylase activity was decreased only slightly after a lomin incubation (Fig. 6). In the presence of an NADPH-generating system, demethylase activity was decreased more than 50% after a lo-

0

4

Time

6

8

10

(min)

FIG. 6. Time-dependent inhibition of NDMA demethylase activity by DDTC. First-stage incubations consisting of acetone-induced microsomes ( 1.1 mg). 50 mM TrisHCI buffer (pH 7.4) 10 mM MgC12, and 150 mM KCI, with (0) and without (0, 0) NADPH-generating system. in the presence (0.0) or absence (0) of 0. I mM DDTC. were conducted at 37°C in a total volume of 0.6 ml. At 0.5. 1, and 10 min. an aliquot was diluted IO-fold by the addition to a second-stage assay mixture containing the same buffer, NADPH-generating system, and 1.0 mM NDMA in a total volume of 0.5 ml. The reactions were continued for IO min. The activity (v) is expressed as nmol HCHO/min/mg protein. Each point represents the average of two incubations.

EFFECTS TABLE

OF

DISULRRAM

I

EFFECTOF DSFON SERUM TRANSAMINASE ACTIVITIES IN RATSTREATEDWITH NDMA ORCCL~O Treatment

I 2 3 4 5 6

Transaminase

NDMA

Ccl,

DSF

+ + -

-

+

+ +

+ +

GPT 25& 9 35* 4 860 + 365 36i9h 2250 f 755 38% II*

activity GOT 171 + 29 154* 15 2540 k 850 136t 42h 7525 f 1943 160? 44h

” DSF (3.4 mmol/kg body wt) was administered intragastrically 18 hr prior to intraperitoneal injection of aqueous solutions of NDMA (75 mg/kg) or Ccl, (1 ml/ kg). Blood sample was obtained 24 hr after the injection of NDMA or Ccl, for the determination of serum glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) activities (according to Sigma ProcedureNo. 505).Thetransaminaseactivities.inSigmaFrankel Units/ml (multiplication by 0.48 gives International Units in U/ml), are the mean i SD from five animals in each group. h Significantly different from the corresponding values on rows 3 and 5, p < 0.01.

DISCUSSION The rapid and prolonged suppression of P450IIEl activity by DSF observed in the present work provides an explanation for many of the previously reported protective effects of DSF and its metabolites against toxicity and carcinogenesis.A number of studies by Fiala and by Wattenberg and their associateshave shown the inhibition of 1,2-dimethylhydrazine metabolism and carcinogenic responses by DSF and its products. Since P450IIE 1 is vital to the activation of 1,2-dimethylhydrazine (Sohn e?al.. 1987, 1990) the suppressionof this isozyme could largely account for the observed protective properties. The inhibition and inactivation of hepatic P4501IE 1by DSF would also decreasethe metabolism of NDMA in the liver and increase the exposure of nonhepatic tissuesto this carcinogen and thus increase the tumorigenesis in the nonhepatic tissuessuch as in the nasal

ON

P450IIEl

371

cavity (Bet-tram, 1989). Since P450IIEl is responsible for only a small portion of the NDMA demethylase activity in the nasal mucosa, the possible inactivation of the enzyme by DSF would have a minimal effect on the activation of this carcinogen in the nasal cavity. In female Wistar rats, DSF prevents the hepatotoxicity of acetaminophen (Jorgensen et al., 1988), another P450IIE 1 substrate. The inhibition of the metabolism and toxicity of NDMA in rats by severaldithiocarbamates has been reported recently (Frank et al., 1990). DDTC and CSI have been shown to protect mice against liver and kidney injury caused by several chemicals(Masuda and Nakayama, 1982, 1983), most of which are substratesof P450IIEl. In addition to the inhibition of P4501IE1, other mechanisms may also be involved in the protective action of DSF, especially against compounds which are not metabolized by P450IIE 1. In the present study, DSF pretreatment gave more pronounced and persistent inhibition of NDMA demethylase activity than CS2. It has been suggestedthat DSF, a lipophilic agent, is sequestered and is continuously released, forming DDTC and CS2 (Eckes and Buch, 1985). However, the quantity of CS2 formed appears to be too low to account for all the effect exerted by DSF on P450IIE I. This conclusion is consistent with that of Miller et al. (1983) based on the study concerning aniline metabolism. The induction of P450IIBl by DSF is clearly not related to the formation of CS2 (Fig. 3). DDTC is rapidly formed from DSF in vivo (Stromme, 1965). Since DSF is relatively insoluble, DDTC was used in the present experiments in vitro. The results suggest that DDTC itself is an inhibitor of NDMA demethylase and it can also produce a metabolism-dependent inactivation of P450IIE 1 upon incubation in the presenceof NADPH. DDTC is structurally similar to substratesof the havin-containing monooxygenase, as described by Taylor and Ziegler (1986). The dithioperacid of DDTC, if it is formed, would be a strong oxidant that could bind to nucleophilic

372

BRADY

groups at the active site of P450IIE 1. However, the enzymology of the metabolism of DSF and the mechanism of P450IIEl inactivation remain to be investigated. DSF pretreatment led to a pronounced decrease in NDMA demethylase activity and induction of pentoxyresorufin dealkylase activity. The changes in these activities were a reflection of the levels of P450IIEl and P450IIB1, respectively, as determined in immunoblot analysis (Fig. 5). At early time points, however, the inactivated P450IIE 1 probably still remained in microsomes. An alternative interpretation, especially for the first time point, is that some of the inhibition was due to the residual DSF present in the microsomes. The effects on P450 isozymes are rather selective, and the total microsomal P450 content was changed only moderately. DSF treatment also caused a 30% increase in 7c+hydroxylation of testosterone, suggesting an elevation in P450IIA1, and a 25% decrease in 6/Y-hydroxylation of testosterone, possibly related to changes in P450IIIA 1, IIIA2, or other enzymes (data not shown). Overall, the results indicate that DSF pretreatment did not lead to a general suppression of all P450 activities, but was somewhat selective. The extent of selectivity of the effect toward P450IIE 1 or other enzyme systems (Eneanya et al., 198 1) appears to be affected by the dose of DSF administered. In conclusion, DSF, DDTC, CS2, and other organic sulfur compounds such as diallyl sulfide (Brady et al., 1988) and phenethyl isothiocyanate (Ishizaki et al., 1990) can inactivate P450IIE1, and may act as competitive and mechanism-based inhibitors. While the mechanism and consequences of the induction of P450IIEI have received considerable attention, the inhibition of this enzyme by organic sulfur compounds is a novel observation that may have potential practical applications in the prevention of chemical toxicity and carcinogenesis. ACKNOWLEDGMENTS The authors thank Dr. Jun-Yan Hong and Professor H. Newmark for helpful discussions, Dr. J.-S. H. Yoo and

ET AL.

Mr. M. J. Lee for assistancein the testosterone metabolism study, and Ms. Dorothy Wong for excellent secretarial assistance in the preparation of this manuscript.

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DING. X.. AND COON, M. J. (1990). Induction of cytochrome P450 isozyme 3a (P45011E I) in rabbit olfactory mucosa by ethanol and acetone. Drug Metab. Dispos. l&742-745. ECKES, L.. AND BUCH. H. P. (1985). Influence of disulhram. diethyldithiocarbamate and carbon disulfide on the metabolic formation of trifluoroacetic acid from halothane in the rat. .4rzneim-Forsch. 35, 1447-145 1. ENEANYA, D. J., BIANCHINE, J. R., DURAN, D. O., AND ANDERSEN. B. D. (1981). The actions and metabolic fate of disulfiram. Annu. Rev. Pharmacol. To-xicol. 21, 575-596.

FIALA, E. S. (198 1). Inhibition of carcinogen metabolism and action by disulfiram, pyrazole, and related compounds, In Inhibition of Tumor Induction and Development (M. S. Zadeck and M. Lipkin. Eds.). pp. 2369. Plenum, New York. FRANK, N.. BERTRAM, B., SCHERF.H. R.. AND WIESSLER. M. (1990). Influence of dithiocarbamates on the metabolism and toxicity of N-nitrosodimethylamine in rats. Carcinogenesis 11. 199-203. HANSSON,T.. TINDBERG. N., INGELMAN-SUNDBERG,M., AND KOHLER, C. (1990). Regional distribution of ethanol-inducible cytochrome P450IIEl in the rat central nervous system. Neuroscience 34, 45 l-463. HEWITT, W. R.. MIYAJIMA, H.. COTE, M. G.. AND PLAA, G. L. (1980). Modification of haloalkane-induced hepatotoxicity by exogenous ketones and metabolic ketosis. Fed. Proc. 39, 3 118-3 123.

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MASUDA, Y., AND NAKAYAMA, N. (1983). Protective action of diethyldithiocarbamate and carbon disulfide against acute toxicities induced by l.l-dichloroethylene in mice. Toxicol. Appl. Pharmarol. 71, 42-53. MILLER, G. E., ZEMATITIS. M. A., AND GREENE, F. E. ( 1983). Mechanisms of diethyldithiocarbamate-induced loss of cytochrome P450 from rat liver. Biochem. Pharmacol. 32, 2433-2442.

SCHMAHL. D.. KRUGER, F. W., HABS, M., AND DIEHL. B. ( 1976). Influence of disulfiram on the organotropy of the carcinogenic effect of dimethylnitrosamine and diethylnitrosamine in rats. 2. Krebsforsch 85,27 l-276. SNYDERWINE,E. G., KROLL. R.. AND RUBIN. R. J. (1988). The possible role of the ethanol-inducible isozyme of

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Effects of disulfiram on hepatic P450IIE1, other microsomal enzymes, and hepatotoxicity in rats.

Disulfiram, widely used in avoidance therapy for alcohol abuse, has been shown to have protective effects against chemically induced toxicity and carc...
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