Archives of
Arch. Toxicol. 41, 79-88 (1978)
TOXICOLOGY 9 Springer-Verlag t978
The Antihepatotoxic Activity of Dithiocarb as Compared with Six Other Thio Compounds in Mice C.-P. Siegers x, O. Strubelt x, and M. V/51pel2 1 Abteilung f/Jr Toxikologie, 2 Abteilung f'tir Pathologic der Medizinisehen Hochschule Liibeck, Ratzeburger Allee 160, D-2400 Lfibeck, Federal Republic of Germany Abstract. In mice, diethyldithiocarbamate (dithiocarb, 100 mg/kg i.p.) completely prevented the increments of serum enzyme activities (GOT, GPT, SDH) induced by oral administration of carbon tetrachloride (0.1 ml/kg), allyl alcohol (0.05 ml/kg), bromobenzene (0.25 rnl/kg), and thioacetamide (50 mg/kg). In this respect, cysteine (200 mg/kg i.p.) was active against CC14 and bromobenzene, cysteamine (100mg/kg i.p.) against CC14 and allyl alcohol, penicillamine (100 mg/kg i.p.) against allyl alcohol, thiazolidine carbonic acid (100 mg/kg i.p.) against bromobenzene, and thioctic acid (100 mg/kg i.p.) against allyl alcohol and thioacetamide. Dimercaprol (100 mg/kg i.p.) had a weak antidotal effect only against allyl alcohol poisoning. None of the tested antidotes inhibited serum enzyme elevations evoked by dimethyl nitrosamine (100 mg/kg p.o.). These findings prove the antihepatotoxic activity of diethyldithiocarbamate to be superior to that of all other thio compounds under observation. The lowest dose of dithiocarb active against carbon tetrachloride was 25 mg/kg i.p. The dose-response curves for serum-enzyme elevations induced by carbon tetrachloride (0.1--4 ml/kg p.o.) were shifted to the right under the influence of dithiocarb indicating a competitive antagonism. Dithiocarb (I00 mg/kg i.p.) depressed the p-hydroxylation of aniline in the 9000 x g liver homogenate supernatant of mice by about 55%. Thus, the antihepatotoxic activity of dithiocarb seems to be the consequence of a decreased oxidase activity.
Key words: Diethyldithiocarbamate -- Cysteine -- Cysteamine - D-Penicillamine -- Dimercaprol -- Thiazolidine carbonic acid - Thioctic acid - Carbon tetrachloride - Allyl alcohol -- Bromobenzene - Thioacetamide - Dimethyl nitrosamine - - Hepatitis, toxic - Aminotransferases - Mixed function oxidases.
Introduction
Diethyldithiocarbamate (dithiocarb) is a chelating agent which has been employed for many years in the therapy of thallium, nickel and copper poisoning (Sunderman, 1967). The antidotal effects of dithiocarb against CC14-induced hepatotoxicity were
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first described by Sakaguchi et al. (1966) and confirmed by other investigators (Lange and Jung, 1971; Lutz et al., 1973; Tonkelaar and van Loogten, 1974). Recently we found dithiocarb to protect mice against hepatotoxicity and death due to p a r a c e t a m o l poisoning (Strubelt et al., 1974). In the present experiments we investigated the antidotal effects of dithiocarb against liver injury induced by carbon tetrachloride, bromobenzene, allyl alcohol, thioacetamide, or dimethyl nitrosamine, respectively. Furthermore, the antihepatotoxic activity o f dithiocarb was c o m p a r e d with that o f six other thio compounds.
Materials and Methods Treatments of Animals. Male NMRI-mice weighing 20-30 g (Breeder: P. Bfiumler, Wolfratshausen) were fed with Altromin| pellets and tap water ad libitum. The hepatotoxines were given by gavage, whereas the antidotes were injected i.p. simultaneously. All doses refer to kg body weight. Substances. Allyl alcohol (Merck), cysteamine (Merck), cysteine (Fluka, Switzerland), dithiocarb (Merck), D-penicillamine (Knoll), thiazolidine carbonic acid (Medial S.A., Switzerland), thioacetamide (Merck) and thioctic acid (Homburg) were dissolved in saline. Bromobenzene (Merck-Schuchardt), carbon tetrachloride (Merck) and dimercaprol (Homburg) were diluted with olive oil. For all solutions a constant volume of l0 ml/kg body weight was instilled or injected. Determination of Serum Enzymes. Blood was obtained after decapitation 24 h following the treatments. Serum activities of glutamic oxaloaeetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and sorbitol dehydrogenase (SDH) were determined spectrophotometrically using the commercial kits of Boehringer, Mannheim. Aniline Hydroxylation. P-hydroxylation of aniline in a 9000 x g liver homogenate supernatant was determined with the method of Wilson and Frohman (1974) by measuring the formed p-aminophenol spectrophotometrically. Histological Examinations. For histological examinations, the livers were fLxedin 10% formaline and stained with hematoxylin-eosin. Statistics. Differences between controls and experimental groups were tested with the ranking method of Wilcoxon-Mann-Whitney taking P = 0.05 as a limit of statistical significance. The dose-response curves were calculated as regression lines with confidence limits for P = 0.05. For all statistical evaluations we used a programmed calculator (Hewlett-Packard 9820 A).
Results Carbon Tetrachloride
The oral administration o f 0.1 ml/kg CC14 caused within 24 h strong increments of the activities o f G O T , G P T and S D H in the serum o f mice (Fig. 1). These enzyme elevations were completely prevented b y the simultaneous i.p. injection of 100 m g / k g dithiocarb. Cysteine (200 m g / k g i.p.) and cysteamine (100 m g / k g i.p.) only reduced the CCl4-induced SDH-elevations, whereas penicillamine (100 m g / k g
Antihepatotoxic Activity of Dithiocarb
81 20B0
U/L
Fig. 1. Serum enzyme activities (k _+s~; n = 12 each) 24 h after oral administration of 0.1 ml/kg carbon tetrachloride p.o. and simultaneous i.p. injection of seven thio compounds. C = controls (treatment with solvents); D = Dithiocarb; Cy = Cysteine; CA = Cysteamine; PA = Penicillamine; D M = Dimercaprol; TC = Thiazolidine Carbonic acid; TA = Thioctic acid
1500
1000
500
0
CCt~. CCI~+ b
C
+ c/
§ CA
+ PA
+ DM + TC
+ TA
h Influence of dithiocarb on the carbon tetrachloride-induced increments of serum enzyme activities. Values are means and their standard errors Table
CC14 p.o. (ml/kg)
Dithiocarb i.p. (mg/kg)
n
GOT (U/l)
-
--
0.1 0.1 0.1 0.1 0.1
-
16 8 8 8 8 8
133 + 856+ 957 + 570 + 109 + 153 +
10 25 50 100
9
176 315 149 14a 18a
GPT (U/l)
SDH (U/l)
75 + 19 1147+272 1353 + 608 813 + 313 54 + 5a 215 + 61a
14.2_+ 1.8 305 + 33 523 + 235 92 + 27a 12.7 + 2.6a 12.6 + 3.0 a
a Statistically significant difference to the corresponding value of the group treated with carbon tetrachloride only
i.p.), dimercaprol (100 mg/kg i.p.), and thiazolidine carbonic acid (100 mg/kg i.p.) failed to protect mice against CC14-induced liver injury. Thioctic acid even enhanced the hepatotoxic effects of CCI 4 (Fig. 1). The smallest dose of dithiocarb active against CC14-evoked hepatic damage was 25 mg/kg, causing a reduction of SDH-elevation (Table 1). The dose of 50 mg/kg dithiocarb inhibited the CC14-induced increments of G O T , G P T and S D H already completely. In a further series of experiments a constant dose of 100 mg/kg dithiocarb i.p. was tested against different doses of carbon tetrachloride ranging between 0.1 and 4 ml/kg (Fig. 2). C a r b o n tetrachloride caused a dose-dependent increase of serum enzyme activities. These dose-response curves were shifted to the right when 100 mg/kg dithiocarb were given simultaneously indicating a competitive antagonism between dithiocarb and carbon tetrachloride. Also after oral application dithiocarb (100 mg/kg) nearly completely inhibited the strong increments of G O T , G P T and S D H serum activities evoked by intraperitoneal administration of 0.1 mg/kg c a r b o n tetrachloride (not documented).
C.-P. Siegers et al.
82
u~ 8000
/
/o
7000,
o
GPT /
b000. 5000~000-
sp=.~_H
//
3000 Z000 1000
OS 1
01
2 3~-
O1
05
1
Z 3 ~"
0.1
0.':; 1
Fig. 2. Dose-response curves for orally applied carbon tetrachloride (O 9 under the influence of 100 mg/kg dithiocarb i.p. ( 0 - - - - 0 ) . The curves represent regression lines
with confidence limits for P = 0.95 Z 3
CCt~. w t / k B
I S00
I
U/L I 1000]
a
o
ALLYLA!LCOHOL *\\\\~ 6 0 T 6PT ~
~
c
SPH
A + D
A
+ cy
+ CA + PA + DM + TC
+ TA
ZOO0
Fig. 3. Serum enzyme activities (& + s~; n = 12 each) 24 h after oral administration of 0.05 ml/kg allyl alcohol (a), or 0.25 ml/kg bromobenzene (b), respectively, and simultaneous i,p. injection of seven thio compounds. C = controls (treatment with solvents); D = Dithiocarb; C y = Cysteine; C A = Cysteamine; P A = Penieillamine; D M = Dimercaprol; T C = Thiazolidine Carbonic acid; T A = Thioctic acid
BROr U/L I~00
~\\\x,x~GOT OPT SDH
1000
500
b
o
C
13
B + D
+ Cy 4- CA + PA + DM + TC + TA
Allyl Alcohol The oral administration of 0.05 ml/kg aUyl alcohol caused a nearly fivefold increase of GOT, GPT and SDH activities (Fig. 3, upper part). These increments were completely inhibited by the simultaneous treatment with 100 mg/kg dithiocarb, cysteamine, penicillamine or thioctic acid, respectively. Cysteine (200 mg/kg i.p.), dimercaprol and thiazolidine carbonic acid were ineffective against allyl alcohol-induced liver damage.
Antihepatotoxic Activity of Dithiocarb
83 2000 THIOACETAMIDE U/L
~',,",'-,',~GOT GPT SDH
1500
lO00
500
a
0L
C
T
T "," D
+ CY + CA + PA + DM + TC + T A
DMHA DMNA+D
+ CY + CA + PA + DM + TC + TA
ZO00~
Fig. 4. Serum enzyme activities (~c+ s~; n = 12 each) 24 h after oral administration of 50 mg/kg thioacetamide (T) or 100 mg/kg dimethyl nitrosamine (DMNA) respectively, and simultaneous i.p. injection of seven thio compounds. C = controls (treatment with solvents); D = Dithiocarb; Cy = Cysteine; CA = Cysteamine; PA = Penicillamine; D M = Dimercaprol; TC = Thiazolidine Carbonic acid; TA = Thioctic acid
O/t I soo~
1ooo-
s00 b
oC
B romobenzene
Serum enzyme elevations induced by 0.25 ml/kg bromobenzene p.o. were nearly completely inhibited by dithiocarb, cysteine or thiazolidine carbonic acid, respectively (Fig. 3). Cysteamine, penicillamine, dimercaprol and thioctic acid had no effect on bromobenzene-induced hepatotoxicity.
Thioacetamide
As shown in Figure 4, the increments of G O T , G P T and S D H produced by oral application of 50 mg/kg thioacetamide were completely abolished by the simultaneous administration o f dithiocarb and thioctie acid. Cysteine and cysteamine had no influence on the enzyme levels, whereas penicillamine, dimercaprol and thiazolidine carbonic acid even aggravated the hepatotoxic effects of thioacetamide.
Dimethyl Nitrosamine
None of the thio compounds antagonized the high elevations o f serum enzyme activities induced by oral application of 100 mg/kg dimethyl nitrosamine (Fig. 4).
84
C.-P. Siegers et al.
Fig. 5. Mouse liver 24 h after 0.1 ml/kg carbon tetrachloride p.o. (a) and simultaneous i.p. injection of 100 mg/kg dithiocarb (b). a centrilobular necroses, hydropic swelling and vacuolar degeneration of intact cells as indices of beginning nekrobiosis, b no cell necroses, centrilobular cells with eosinophilic cytoplasma and beginning vacuolar degeneration (H and E, • 100)
The small protective effects of dithiocarb, cysteine, cysteamine, and penicillamine were not statistically significant, whereas dimercaprol, thiazolidine carbonic acid and thioctic acid even enhanced the dimethyl nitrosamine-induced increments of serum enzyme activities.
Histological Examinathgns The livers of controls (hepatotoxic treatment only) and dithiocarb-treated mice were also examined histologically. In all animals treated with carbon tetrachloride, aUyl alcohol, bromobenzene, thioacetamide and dimethyl nitrosamine, respectively, the well-known centrilobular liver cell necroses were observed. Treatment with dithiocarb completely protected the mice against the CC14-, allyl alcohol- and thioacetamide-induced liver damage. An example is given in Figure 5" the left part shows severe contrilobular necroses 24 h after 0.1 ml/kg CC14 p.o.; the right part documents the hepatoprotective effect of simultaneously injected dithiocarb (100 mg/kg i.p.) by the failure of any necroses. In the bromobenzene-poisoned mice the centrilobular liver necroses were only diminished by the treatment with dithiocarb. On the other hand, dithiocarb reduced
Antihepatotoxic Activity of Dithiocarb
85
Table 2. Influenceof dithiocarb (100-300 mg/kg i.p. 1 h before sacrifice) and
cobaltous chloride (60 mg/kg i.p. 24 h and 1 h before sacrifice) on the phydroxylation of aniline (0.044 mM) in the 9000 x g liver homogenate supernatant of mice. Values represent means and confidence limits for P = 0.95 Inhibitor
mg/kg
n
p-Aminophenol ~moles/g liver 9 30 min
Saline Dithiocarb
100 300 60
8 4 4 4 4
1.700 (1.600-1.800) 0.775 (0.751--0.799) 0.745 (0.711-0.779) 1.807 (1.726-1.888) 1.373 (1.281-1.464)
Saline Cobaltous chloride
the severity of liver necroses evoked by dimethyl nitrosamine although the increments of serum enzymes were not influenced.
A niline-Hydroxylase The parahydroxylation of aniline as a parameter of microsomai mixed-function oxidase activity was estimated in the mouse liver homogenate supernatant; the results are compiled in Table 2. Treatment with 100 mg/kg dithiocarb 1 h before sacrifice reduced the amount of p-aminophenol formed in the mouse liver homogenate by about 55% of controls; the dose of 300 mg/kg dithiocarb caused no further reduction. Cobaltous chloride, a well-known inhibitor of the synthesis of cytochrome P-450, produced a much weaker effect, namely a reduction by 24% of the activity of saline-treated controls.
Discussion
There are many reports on the protective and curative effects of thio compounds in experimental or clinical liver injury, especially for cysteine and cysteamine (Gutbrod et al., 1957; Eger, 1957; Kirnberger et al., 1958; Varga et al., 1963; Mitchell et al., 1973; Legros, 1976), but also concerning thioctic acid (Frimmer et al., 1968, 1975), dimercaprol (Janovics and Tako, 1950) or D-penicillamine (Lange and Jung, 1971). Dithiocarb till now was found effective against the hepatotoxic actions of carbon tetrachloride and paracetamol (references see introduction). Table 3 summarizes the present results on the antidotal effects of dithiocarb and six other thio compounds against five hepatotoxic agents. In these investigations dithiocarb afforded a nearly complete protection against liver damage induced by carbon tetrachloride, allyl alcohol, bromobenzene and thioacetamide, respectively. The other thio compounds were active against two of these hepatotoxines at the most. Thus the antihepatotoxic activity of dithiocarb proved superior to that of all other antidotes under observation.
86
C . - P . S i e g e r s et al.
Tabelle 3. C o m p a r i s o n
of the antihepatotoxic
Antidotes
Dithiocarb
activities of seven thio compounds
Hepatotoxines Carbon
Allyl
Bromo-
Thio-
Dimethyl
tetrachloride
alcohol
benzene
acetamide
nitrosamine
+
+
+
+
Cysteine
(+)
-
+
-
Cysteamine Penicillamine Dimercaprol
(+) -
+ + (+)
--
-
Thiazolidine carbonic acid
-
-
+
-
Thioctic acid
-
+
-
+
+
in m i c e
m
m
m
m
Total inhibition of serum enzyme elevations
(+) Partial inhibition of the increase of one or several enzymes
In the present experiments, cysteine, cysteamine and thioctic acid afforded protection against two of the five hepatotoxic agents. LDs0 values of these antidotes for oral administration in mice amount to 660, 450 or 502 mg/kg, respectively, whereas the LDs0 of dithiocarb is 1800 mg/kg (Strubelt et al., 1974; Homburg, 1975). Thus dithiocarb exceeds these other antidotes concerning the therapeutic index, too. What is the mechanism of the antihepatotoxic activity of dithiocarb? Hunter and Neal (1975) have demonstrated that dithiocarb inhibits in vivo and in vitro the mixed-function oxidase activity of the rat liver. This effect was explained by a reduction of the microsomal cytochrome P-450 content. In our own experiments, inhibition of the mixed-function oxidase system after dithiocarb was evident by a decrease of the aniline hydroxylase activity in the 9000 x g liver homogenate supernatant of the mouse liver. We suppose that this inhibition of the mixed-function oxidase is the main cause of the antihepatotoxic activity of dithiocarb. Namely, the hepatotoxic agents antagonized by dithiocarb are all activated by microsomal enzymes to toxic metabolites, as demonstrated by Fowler (1969), Bini et al. (1975), and Reynolds and Moslen (1974) for carbon tetrachloride, Mitchell et al. (1973) for paracetamol, Reid et al. (1971) and Jollow et al. (1974) for bromobenzene, and Ammon et al. (1967) and Schlicht (1971) for thioacetamide. Allyl alcohol seems to be an exception from this rule because it is said to be metabolized by the ADH system to acroleine (Rees and Tarlow, 1967). However, the microsomal ethanol oxidizing system (Lieber et al., 1970) might also be involved in the metabolic activation of allyl alcohol and be inhibited by dithiocarb. In mice dithiocarb had no statistically significant influence on serum enzyme elevations induced by 100 mg/kg dimethyl nitrosamine. The histological examinations, however, revealed some protective activity, and in rats dithiocarb also diminished serum enzyme elevations produced by dimethyl nitrosamine. The lack of efficacy in mice may be due to the high dose of dimethyl nitrosamine used in these experiments. Animal experiments as well as the clinical experience in the therapy of human thallium, nickel and copper poisoning have proved that dithiocarb is a relatively
Antihepatotoxic Activity of Dithiocarb
87
nontoxic substance (Sunderman, 1967). Furthermore, dithiocarb is an obligatory intermediate metabolite in the catabolism o f disulfiram ( S t r t m m e , 1965) which also b y itself is a largely innocuous drug. D i t h i o c a r b exerts its full antihepatotoxic activity also after oral application. Thus we presume that dithiocarb also might be of value in the prevention o f h u m a n toxic hepatitis if caused b y bioactivated substances. The ethanol-sensitizing effect o f dithiocarb (Sunderman and Sunderman, 1958) is no contraindication in this respect since toxic hepatitis obviously forbids the consumption o f ethanol. A n antidote for the treatment of h u m a n poisoning, however, should also be effective if applied therapeutically, i.e., after the poison. In this respect the value o f dithiocarb seems to be low regarding the mechanism p r o p o s e d above for its antihepatotoxic action. In severe intoxications, on the other hand, the absorption period often outcasts the beginning o f the clinical treatment and dithiocarb therefore might be effective when applied after the poison, too. In a n y case, dithiocarb treatment o f rats 4 or 8 h after intoxication with c a r b o n tetrachloride also resulted in a decrease o f CC14-hepatotoxicity.
Acknowledgement. The authors thank Mrs. Hilke Herzberg for skilful technical assistance.
References Ammon, R., Berninger, H., Haas, H. J., Landsberg, I.: Thioacetamid-Sulfoxid, ein Stoffwechselprodukt des Thioacetamids. Arzneimittel-Forsch. 17, 521-523 (1967) Bini, A., Vecchi, G., Vivoli, G., Vannini, V., Cessi, C.: Detection of early metabolites in rat liver after administration of CCI4 and CBrCI 3. Pharmacol. Res. Commun. 7, 143--149 (1975) Eger, W.: Uber Cystein, Homocystein, Cystathionin und Cysteamin als nekrotrope Leberschutzstoffe in Verbindung mit Traubenzucker und Fruchtzucker. Medizinische 17, 618--623 (1957) Fowler, J. S. L.: Carbon tetraehloride metabolism in the rabbit. Brit. J. Pharmacol. 37, 733-737 (1969) Frimmer, M., Waldvogel, A., Weil, G.: Schutzwirkung yon Thiocts~iure gegen die Kalium-freisetzende Wirkung des Phalloidins an der isoliert perfundierten Rattenleber. Klin. Wschr. 46, 1281-1289 (1968) Frimmer, M., Petzinger, E., Homann, J.: Phalloidinantagonisten. 4. Mitteilung: Thiocts~iure, SH-Verbindungen, Rifampiein, Choleretika, Dexamethason, Ostradiol, unspezifische Hemmstoffe und unwirksame Verbindungen. Arzneimittel-Forsch. 25, 1881--1884 (1975) Gutbrod, H., Kornberger, E. J., Stille, G., Wolf, V.: Eigenschaften einer neuen antinekrotischen Kombination f/Jr die Behandlung akuter Lebersch~iden. Acta hepat. (Hamburg) 5, 1-12 (1957) Homburg, Chemiewerk; Thioctacid (Thiocts.~iureHomburg). Frankfurt 1975 Hunter, A. L., Neal, R. A.: Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thionosulfur-containing compounds. Biochem. Pharmacol. 24, 2199--2205 (1975) Janovics, T., Tako, J.: Die Anwendung yon 2,3-Dimercaptopropanol (BAL) bei diffusen Leberparenchymsch~idigungen. Schweiz. med. Wschr. 80, 933--936 (1950) Jollow, D. J., Mitchell, J. R., Zampaglione, N., Gillette, J. R.: Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenze oxide as the hepatotoxic metabolite. Pharmacology (Basel) 11, 151--169 (1974) Kirnberger, E. J., Braun, W., Stille, G., Wolf, V.: Beziehungen zwischen Leberschutz und Zuckerstoffwechsel. Arzneimittel-Forsch. 8, 72-76 (1958) Lange, P., Jung, F.: Die Verringerung der Lebertoxizit~it von Tetrachlorkohlenstoff dutch Diiithyldithiocarbamat und 3-(o-Tolyl)-4-(nitro)-sydnon. Acta biol. med. germ. 27, 425-434 (1971) Lange, J., Schumacher, K., Witscher, H. P.: Die Behandlung der chronisch-aggressiven Hepatitis mit D-Penicillamin. Dtsch. med. Wschr. 96, 139--145 (1971)
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Legros, J.: Paracetamol and the liver. Animal studies -- a theoretical basis for treatment. J. int. Med. Res. 4, (Suppl. 4), 46--54 (1976) Lieber, C. S., Rubin, E., De Carli, L. M.: Hepatic microsomal ethanol oxidizing system (MEOS): differentiation from alcohol dehydrogenase and NADPH oxidase. Biochem. biophys. Res. Commun. 40, 858-865 (1970) Lutz, L. M., Glende, E. A., Recknagel, R. O.: Protection by diethyldithiocarbamate against carbon tetrachloride lethality in rats and against carbon tetrachloride-induced lipid peroxidation in vitro. Biochem. Pharmacol. 22, 1729--1734 (1973) Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R., Brodie, B. B.: Acetaminopheninduced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. exp. Ther. 187, 185-194 (1973) Rees, K. R., Tarlow, M. J.: The hepatotoxic action of allyl formate. Biochem. J. 104, 757-761 (1967) Reid, W. D., Christie, B., Eichelbaum, M., Krishna, G.: 3-methyl-cholanthrene blocks hepatic necrosis induced by administration of bromobenzene or carbon tetrachloride. Exp. molec. Path. 15, 363-372 (1971) Reynolds, E. S., Moslen, M. T.: Chemical modulation of early carbon tetrachloride liver injury. Toxicol. appl. Pharmacol. 29, 377-388 (1974) Sakaguchi, T., Nishimura, H., Masuda, K., Tsuge, I., Onishi, K., Tatsumi, H.: The relationship between chemical structure and protective effect of dithiocarbamate derivates against experimental hepatic injury induced by carbon tetrachloride administration in rats. Biochem. Pharmacol. 15, 756--758 (1966) Schlicht, I.: Autoradiographische und radiochromatographische Untersuchungen der VerteiIung und des Stoffwechsels von Thioacetamid. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 268, 310-322 (1971) Str6mme, J. H.: Metabolism of disulfiram and diethyldithiocarbamate in rats with demonstration of an in vivo ethanol-induced inhibition of the glucuronic acid conjugation of the thiol. Biochem. Pharmacol. 14, 393--410 (1965) Strubelt, O., Siegers, C.-P., Sch/itt, Atsuko: The curative effects of cysteamine, cysteine and dithiocarb in experimental paracetamol poisoning. Arch. Toxicol. 33, 55-64 (1974) Sunderman, F. W." Diethyldithiocarbamate therapy of thallotoxicosis. Amer. J. reed. Sci. 253, 107-118 (1967) Sunderman, F. W., Sunderman, F. W., Jr.: Nickel poisoning. VIII. Dithiocarb: a new therapeutic agent for persons exposed to nickel carbonyl. Amer. J. med. Sci. 236, 26-31 (1958) Tonkelaar, E. M., van Loogten, M. J.: Protective action of dithiocarbamates on experimental liver damage produced by carbon tetrachloride. Toxicol. appl. Pharmacol. 30, 96-106 (1974) Varga, F., Mekes, J., Molnar, Z.: Die Leberschutzwirkung von Cystein, Homocysteinthiolacton und --N--Acetylhomocysteinthiolacton bei experimenteller Lebersch/idigung. Arzneimittel-Forsch. 13, 867-871 (1963) Wilson, J. T., Frohman, L. A.: Concomitant association between high plasma levels of growth hormone and low hepatic mixed-function oxidase activity in the young rat. J. Pharmacol. exp. Ther. 189, 255--270 (1974) Received Februar 10, 1978
Arch. Toxicol. 41, 79-88 (1978)
TOXICOLOGY 9 Springer-Verlag t978
The Antihepatotoxic Activity of Dithiocarb as Compared with Six Other Thio Compounds in Mice C.-P. Siegers x, O. Strubelt x, and M. V/51pel2 1 Abteilung f/Jr Toxikologie, 2 Abteilung f'tir Pathologic der Medizinisehen Hochschule Liibeck, Ratzeburger Allee 160, D-2400 Lfibeck, Federal Republic of Germany Abstract. In mice, diethyldithiocarbamate (dithiocarb, 100 mg/kg i.p.) completely prevented the increments of serum enzyme activities (GOT, GPT, SDH) induced by oral administration of carbon tetrachloride (0.1 ml/kg), allyl alcohol (0.05 ml/kg), bromobenzene (0.25 rnl/kg), and thioacetamide (50 mg/kg). In this respect, cysteine (200 mg/kg i.p.) was active against CC14 and bromobenzene, cysteamine (100mg/kg i.p.) against CC14 and allyl alcohol, penicillamine (100 mg/kg i.p.) against allyl alcohol, thiazolidine carbonic acid (100 mg/kg i.p.) against bromobenzene, and thioctic acid (100 mg/kg i.p.) against allyl alcohol and thioacetamide. Dimercaprol (100 mg/kg i.p.) had a weak antidotal effect only against allyl alcohol poisoning. None of the tested antidotes inhibited serum enzyme elevations evoked by dimethyl nitrosamine (100 mg/kg p.o.). These findings prove the antihepatotoxic activity of diethyldithiocarbamate to be superior to that of all other thio compounds under observation. The lowest dose of dithiocarb active against carbon tetrachloride was 25 mg/kg i.p. The dose-response curves for serum-enzyme elevations induced by carbon tetrachloride (0.1--4 ml/kg p.o.) were shifted to the right under the influence of dithiocarb indicating a competitive antagonism. Dithiocarb (I00 mg/kg i.p.) depressed the p-hydroxylation of aniline in the 9000 x g liver homogenate supernatant of mice by about 55%. Thus, the antihepatotoxic activity of dithiocarb seems to be the consequence of a decreased oxidase activity.
Key words: Diethyldithiocarbamate -- Cysteine -- Cysteamine - D-Penicillamine -- Dimercaprol -- Thiazolidine carbonic acid - Thioctic acid - Carbon tetrachloride - Allyl alcohol -- Bromobenzene - Thioacetamide - Dimethyl nitrosamine - - Hepatitis, toxic - Aminotransferases - Mixed function oxidases.
Introduction
Diethyldithiocarbamate (dithiocarb) is a chelating agent which has been employed for many years in the therapy of thallium, nickel and copper poisoning (Sunderman, 1967). The antidotal effects of dithiocarb against CC14-induced hepatotoxicity were
0340-5761/78/0041/0079/$ 02.00
80
c.-P. Siegers et al.
first described by Sakaguchi et al. (1966) and confirmed by other investigators (Lange and Jung, 1971; Lutz et al., 1973; Tonkelaar and van Loogten, 1974). Recently we found dithiocarb to protect mice against hepatotoxicity and death due to p a r a c e t a m o l poisoning (Strubelt et al., 1974). In the present experiments we investigated the antidotal effects of dithiocarb against liver injury induced by carbon tetrachloride, bromobenzene, allyl alcohol, thioacetamide, or dimethyl nitrosamine, respectively. Furthermore, the antihepatotoxic activity o f dithiocarb was c o m p a r e d with that o f six other thio compounds.
Materials and Methods Treatments of Animals. Male NMRI-mice weighing 20-30 g (Breeder: P. Bfiumler, Wolfratshausen) were fed with Altromin| pellets and tap water ad libitum. The hepatotoxines were given by gavage, whereas the antidotes were injected i.p. simultaneously. All doses refer to kg body weight. Substances. Allyl alcohol (Merck), cysteamine (Merck), cysteine (Fluka, Switzerland), dithiocarb (Merck), D-penicillamine (Knoll), thiazolidine carbonic acid (Medial S.A., Switzerland), thioacetamide (Merck) and thioctic acid (Homburg) were dissolved in saline. Bromobenzene (Merck-Schuchardt), carbon tetrachloride (Merck) and dimercaprol (Homburg) were diluted with olive oil. For all solutions a constant volume of l0 ml/kg body weight was instilled or injected. Determination of Serum Enzymes. Blood was obtained after decapitation 24 h following the treatments. Serum activities of glutamic oxaloaeetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and sorbitol dehydrogenase (SDH) were determined spectrophotometrically using the commercial kits of Boehringer, Mannheim. Aniline Hydroxylation. P-hydroxylation of aniline in a 9000 x g liver homogenate supernatant was determined with the method of Wilson and Frohman (1974) by measuring the formed p-aminophenol spectrophotometrically. Histological Examinations. For histological examinations, the livers were fLxedin 10% formaline and stained with hematoxylin-eosin. Statistics. Differences between controls and experimental groups were tested with the ranking method of Wilcoxon-Mann-Whitney taking P = 0.05 as a limit of statistical significance. The dose-response curves were calculated as regression lines with confidence limits for P = 0.05. For all statistical evaluations we used a programmed calculator (Hewlett-Packard 9820 A).
Results Carbon Tetrachloride
The oral administration o f 0.1 ml/kg CC14 caused within 24 h strong increments of the activities o f G O T , G P T and S D H in the serum o f mice (Fig. 1). These enzyme elevations were completely prevented b y the simultaneous i.p. injection of 100 m g / k g dithiocarb. Cysteine (200 m g / k g i.p.) and cysteamine (100 m g / k g i.p.) only reduced the CCl4-induced SDH-elevations, whereas penicillamine (100 m g / k g
Antihepatotoxic Activity of Dithiocarb
81 20B0
U/L
Fig. 1. Serum enzyme activities (k _+s~; n = 12 each) 24 h after oral administration of 0.1 ml/kg carbon tetrachloride p.o. and simultaneous i.p. injection of seven thio compounds. C = controls (treatment with solvents); D = Dithiocarb; Cy = Cysteine; CA = Cysteamine; PA = Penicillamine; D M = Dimercaprol; TC = Thiazolidine Carbonic acid; TA = Thioctic acid
1500
1000
500
0
CCt~. CCI~+ b
C
+ c/
§ CA
+ PA
+ DM + TC
+ TA
h Influence of dithiocarb on the carbon tetrachloride-induced increments of serum enzyme activities. Values are means and their standard errors Table
CC14 p.o. (ml/kg)
Dithiocarb i.p. (mg/kg)
n
GOT (U/l)
-
--
0.1 0.1 0.1 0.1 0.1
-
16 8 8 8 8 8
133 + 856+ 957 + 570 + 109 + 153 +
10 25 50 100
9
176 315 149 14a 18a
GPT (U/l)
SDH (U/l)
75 + 19 1147+272 1353 + 608 813 + 313 54 + 5a 215 + 61a
14.2_+ 1.8 305 + 33 523 + 235 92 + 27a 12.7 + 2.6a 12.6 + 3.0 a
a Statistically significant difference to the corresponding value of the group treated with carbon tetrachloride only
i.p.), dimercaprol (100 mg/kg i.p.), and thiazolidine carbonic acid (100 mg/kg i.p.) failed to protect mice against CC14-induced liver injury. Thioctic acid even enhanced the hepatotoxic effects of CCI 4 (Fig. 1). The smallest dose of dithiocarb active against CC14-evoked hepatic damage was 25 mg/kg, causing a reduction of SDH-elevation (Table 1). The dose of 50 mg/kg dithiocarb inhibited the CC14-induced increments of G O T , G P T and S D H already completely. In a further series of experiments a constant dose of 100 mg/kg dithiocarb i.p. was tested against different doses of carbon tetrachloride ranging between 0.1 and 4 ml/kg (Fig. 2). C a r b o n tetrachloride caused a dose-dependent increase of serum enzyme activities. These dose-response curves were shifted to the right when 100 mg/kg dithiocarb were given simultaneously indicating a competitive antagonism between dithiocarb and carbon tetrachloride. Also after oral application dithiocarb (100 mg/kg) nearly completely inhibited the strong increments of G O T , G P T and S D H serum activities evoked by intraperitoneal administration of 0.1 mg/kg c a r b o n tetrachloride (not documented).
C.-P. Siegers et al.
82
u~ 8000
/
/o
7000,
o
GPT /
b000. 5000~000-
sp=.~_H
//
3000 Z000 1000
OS 1
01
2 3~-
O1
05
1
Z 3 ~"
0.1
0.':; 1
Fig. 2. Dose-response curves for orally applied carbon tetrachloride (O 9 under the influence of 100 mg/kg dithiocarb i.p. ( 0 - - - - 0 ) . The curves represent regression lines
with confidence limits for P = 0.95 Z 3
CCt~. w t / k B
I S00
I
U/L I 1000]
a
o
ALLYLA!LCOHOL *\\\\~ 6 0 T 6PT ~
~
c
SPH
A + D
A
+ cy
+ CA + PA + DM + TC
+ TA
ZOO0
Fig. 3. Serum enzyme activities (& + s~; n = 12 each) 24 h after oral administration of 0.05 ml/kg allyl alcohol (a), or 0.25 ml/kg bromobenzene (b), respectively, and simultaneous i,p. injection of seven thio compounds. C = controls (treatment with solvents); D = Dithiocarb; C y = Cysteine; C A = Cysteamine; P A = Penieillamine; D M = Dimercaprol; T C = Thiazolidine Carbonic acid; T A = Thioctic acid
BROr U/L I~00
~\\\x,x~GOT OPT SDH
1000
500
b
o
C
13
B + D
+ Cy 4- CA + PA + DM + TC + TA
Allyl Alcohol The oral administration of 0.05 ml/kg aUyl alcohol caused a nearly fivefold increase of GOT, GPT and SDH activities (Fig. 3, upper part). These increments were completely inhibited by the simultaneous treatment with 100 mg/kg dithiocarb, cysteamine, penicillamine or thioctic acid, respectively. Cysteine (200 mg/kg i.p.), dimercaprol and thiazolidine carbonic acid were ineffective against allyl alcohol-induced liver damage.
Antihepatotoxic Activity of Dithiocarb
83 2000 THIOACETAMIDE U/L
~',,",'-,',~GOT GPT SDH
1500
lO00
500
a
0L
C
T
T "," D
+ CY + CA + PA + DM + TC + T A
DMHA DMNA+D
+ CY + CA + PA + DM + TC + TA
ZO00~
Fig. 4. Serum enzyme activities (~c+ s~; n = 12 each) 24 h after oral administration of 50 mg/kg thioacetamide (T) or 100 mg/kg dimethyl nitrosamine (DMNA) respectively, and simultaneous i.p. injection of seven thio compounds. C = controls (treatment with solvents); D = Dithiocarb; Cy = Cysteine; CA = Cysteamine; PA = Penicillamine; D M = Dimercaprol; TC = Thiazolidine Carbonic acid; TA = Thioctic acid
O/t I soo~
1ooo-
s00 b
oC
B romobenzene
Serum enzyme elevations induced by 0.25 ml/kg bromobenzene p.o. were nearly completely inhibited by dithiocarb, cysteine or thiazolidine carbonic acid, respectively (Fig. 3). Cysteamine, penicillamine, dimercaprol and thioctic acid had no effect on bromobenzene-induced hepatotoxicity.
Thioacetamide
As shown in Figure 4, the increments of G O T , G P T and S D H produced by oral application of 50 mg/kg thioacetamide were completely abolished by the simultaneous administration o f dithiocarb and thioctie acid. Cysteine and cysteamine had no influence on the enzyme levels, whereas penicillamine, dimercaprol and thiazolidine carbonic acid even aggravated the hepatotoxic effects of thioacetamide.
Dimethyl Nitrosamine
None of the thio compounds antagonized the high elevations o f serum enzyme activities induced by oral application of 100 mg/kg dimethyl nitrosamine (Fig. 4).
84
C.-P. Siegers et al.
Fig. 5. Mouse liver 24 h after 0.1 ml/kg carbon tetrachloride p.o. (a) and simultaneous i.p. injection of 100 mg/kg dithiocarb (b). a centrilobular necroses, hydropic swelling and vacuolar degeneration of intact cells as indices of beginning nekrobiosis, b no cell necroses, centrilobular cells with eosinophilic cytoplasma and beginning vacuolar degeneration (H and E, • 100)
The small protective effects of dithiocarb, cysteine, cysteamine, and penicillamine were not statistically significant, whereas dimercaprol, thiazolidine carbonic acid and thioctic acid even enhanced the dimethyl nitrosamine-induced increments of serum enzyme activities.
Histological Examinathgns The livers of controls (hepatotoxic treatment only) and dithiocarb-treated mice were also examined histologically. In all animals treated with carbon tetrachloride, aUyl alcohol, bromobenzene, thioacetamide and dimethyl nitrosamine, respectively, the well-known centrilobular liver cell necroses were observed. Treatment with dithiocarb completely protected the mice against the CC14-, allyl alcohol- and thioacetamide-induced liver damage. An example is given in Figure 5" the left part shows severe contrilobular necroses 24 h after 0.1 ml/kg CC14 p.o.; the right part documents the hepatoprotective effect of simultaneously injected dithiocarb (100 mg/kg i.p.) by the failure of any necroses. In the bromobenzene-poisoned mice the centrilobular liver necroses were only diminished by the treatment with dithiocarb. On the other hand, dithiocarb reduced
Antihepatotoxic Activity of Dithiocarb
85
Table 2. Influenceof dithiocarb (100-300 mg/kg i.p. 1 h before sacrifice) and
cobaltous chloride (60 mg/kg i.p. 24 h and 1 h before sacrifice) on the phydroxylation of aniline (0.044 mM) in the 9000 x g liver homogenate supernatant of mice. Values represent means and confidence limits for P = 0.95 Inhibitor
mg/kg
n
p-Aminophenol ~moles/g liver 9 30 min
Saline Dithiocarb
100 300 60
8 4 4 4 4
1.700 (1.600-1.800) 0.775 (0.751--0.799) 0.745 (0.711-0.779) 1.807 (1.726-1.888) 1.373 (1.281-1.464)
Saline Cobaltous chloride
the severity of liver necroses evoked by dimethyl nitrosamine although the increments of serum enzymes were not influenced.
A niline-Hydroxylase The parahydroxylation of aniline as a parameter of microsomai mixed-function oxidase activity was estimated in the mouse liver homogenate supernatant; the results are compiled in Table 2. Treatment with 100 mg/kg dithiocarb 1 h before sacrifice reduced the amount of p-aminophenol formed in the mouse liver homogenate by about 55% of controls; the dose of 300 mg/kg dithiocarb caused no further reduction. Cobaltous chloride, a well-known inhibitor of the synthesis of cytochrome P-450, produced a much weaker effect, namely a reduction by 24% of the activity of saline-treated controls.
Discussion
There are many reports on the protective and curative effects of thio compounds in experimental or clinical liver injury, especially for cysteine and cysteamine (Gutbrod et al., 1957; Eger, 1957; Kirnberger et al., 1958; Varga et al., 1963; Mitchell et al., 1973; Legros, 1976), but also concerning thioctic acid (Frimmer et al., 1968, 1975), dimercaprol (Janovics and Tako, 1950) or D-penicillamine (Lange and Jung, 1971). Dithiocarb till now was found effective against the hepatotoxic actions of carbon tetrachloride and paracetamol (references see introduction). Table 3 summarizes the present results on the antidotal effects of dithiocarb and six other thio compounds against five hepatotoxic agents. In these investigations dithiocarb afforded a nearly complete protection against liver damage induced by carbon tetrachloride, allyl alcohol, bromobenzene and thioacetamide, respectively. The other thio compounds were active against two of these hepatotoxines at the most. Thus the antihepatotoxic activity of dithiocarb proved superior to that of all other antidotes under observation.
86
C . - P . S i e g e r s et al.
Tabelle 3. C o m p a r i s o n
of the antihepatotoxic
Antidotes
Dithiocarb
activities of seven thio compounds
Hepatotoxines Carbon
Allyl
Bromo-
Thio-
Dimethyl
tetrachloride
alcohol
benzene
acetamide
nitrosamine
+
+
+
+
Cysteine
(+)
-
+
-
Cysteamine Penicillamine Dimercaprol
(+) -
+ + (+)
--
-
Thiazolidine carbonic acid
-
-
+
-
Thioctic acid
-
+
-
+
+
in m i c e
m
m
m
m
Total inhibition of serum enzyme elevations
(+) Partial inhibition of the increase of one or several enzymes
In the present experiments, cysteine, cysteamine and thioctic acid afforded protection against two of the five hepatotoxic agents. LDs0 values of these antidotes for oral administration in mice amount to 660, 450 or 502 mg/kg, respectively, whereas the LDs0 of dithiocarb is 1800 mg/kg (Strubelt et al., 1974; Homburg, 1975). Thus dithiocarb exceeds these other antidotes concerning the therapeutic index, too. What is the mechanism of the antihepatotoxic activity of dithiocarb? Hunter and Neal (1975) have demonstrated that dithiocarb inhibits in vivo and in vitro the mixed-function oxidase activity of the rat liver. This effect was explained by a reduction of the microsomal cytochrome P-450 content. In our own experiments, inhibition of the mixed-function oxidase system after dithiocarb was evident by a decrease of the aniline hydroxylase activity in the 9000 x g liver homogenate supernatant of the mouse liver. We suppose that this inhibition of the mixed-function oxidase is the main cause of the antihepatotoxic activity of dithiocarb. Namely, the hepatotoxic agents antagonized by dithiocarb are all activated by microsomal enzymes to toxic metabolites, as demonstrated by Fowler (1969), Bini et al. (1975), and Reynolds and Moslen (1974) for carbon tetrachloride, Mitchell et al. (1973) for paracetamol, Reid et al. (1971) and Jollow et al. (1974) for bromobenzene, and Ammon et al. (1967) and Schlicht (1971) for thioacetamide. Allyl alcohol seems to be an exception from this rule because it is said to be metabolized by the ADH system to acroleine (Rees and Tarlow, 1967). However, the microsomal ethanol oxidizing system (Lieber et al., 1970) might also be involved in the metabolic activation of allyl alcohol and be inhibited by dithiocarb. In mice dithiocarb had no statistically significant influence on serum enzyme elevations induced by 100 mg/kg dimethyl nitrosamine. The histological examinations, however, revealed some protective activity, and in rats dithiocarb also diminished serum enzyme elevations produced by dimethyl nitrosamine. The lack of efficacy in mice may be due to the high dose of dimethyl nitrosamine used in these experiments. Animal experiments as well as the clinical experience in the therapy of human thallium, nickel and copper poisoning have proved that dithiocarb is a relatively
Antihepatotoxic Activity of Dithiocarb
87
nontoxic substance (Sunderman, 1967). Furthermore, dithiocarb is an obligatory intermediate metabolite in the catabolism o f disulfiram ( S t r t m m e , 1965) which also b y itself is a largely innocuous drug. D i t h i o c a r b exerts its full antihepatotoxic activity also after oral application. Thus we presume that dithiocarb also might be of value in the prevention o f h u m a n toxic hepatitis if caused b y bioactivated substances. The ethanol-sensitizing effect o f dithiocarb (Sunderman and Sunderman, 1958) is no contraindication in this respect since toxic hepatitis obviously forbids the consumption o f ethanol. A n antidote for the treatment of h u m a n poisoning, however, should also be effective if applied therapeutically, i.e., after the poison. In this respect the value o f dithiocarb seems to be low regarding the mechanism p r o p o s e d above for its antihepatotoxic action. In severe intoxications, on the other hand, the absorption period often outcasts the beginning o f the clinical treatment and dithiocarb therefore might be effective when applied after the poison, too. In a n y case, dithiocarb treatment o f rats 4 or 8 h after intoxication with c a r b o n tetrachloride also resulted in a decrease o f CC14-hepatotoxicity.
Acknowledgement. The authors thank Mrs. Hilke Herzberg for skilful technical assistance.
References Ammon, R., Berninger, H., Haas, H. J., Landsberg, I.: Thioacetamid-Sulfoxid, ein Stoffwechselprodukt des Thioacetamids. Arzneimittel-Forsch. 17, 521-523 (1967) Bini, A., Vecchi, G., Vivoli, G., Vannini, V., Cessi, C.: Detection of early metabolites in rat liver after administration of CCI4 and CBrCI 3. Pharmacol. Res. Commun. 7, 143--149 (1975) Eger, W.: Uber Cystein, Homocystein, Cystathionin und Cysteamin als nekrotrope Leberschutzstoffe in Verbindung mit Traubenzucker und Fruchtzucker. Medizinische 17, 618--623 (1957) Fowler, J. S. L.: Carbon tetraehloride metabolism in the rabbit. Brit. J. Pharmacol. 37, 733-737 (1969) Frimmer, M., Waldvogel, A., Weil, G.: Schutzwirkung yon Thiocts~iure gegen die Kalium-freisetzende Wirkung des Phalloidins an der isoliert perfundierten Rattenleber. Klin. Wschr. 46, 1281-1289 (1968) Frimmer, M., Petzinger, E., Homann, J.: Phalloidinantagonisten. 4. Mitteilung: Thiocts~iure, SH-Verbindungen, Rifampiein, Choleretika, Dexamethason, Ostradiol, unspezifische Hemmstoffe und unwirksame Verbindungen. Arzneimittel-Forsch. 25, 1881--1884 (1975) Gutbrod, H., Kornberger, E. J., Stille, G., Wolf, V.: Eigenschaften einer neuen antinekrotischen Kombination f/Jr die Behandlung akuter Lebersch~iden. Acta hepat. (Hamburg) 5, 1-12 (1957) Homburg, Chemiewerk; Thioctacid (Thiocts.~iureHomburg). Frankfurt 1975 Hunter, A. L., Neal, R. A.: Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thionosulfur-containing compounds. Biochem. Pharmacol. 24, 2199--2205 (1975) Janovics, T., Tako, J.: Die Anwendung yon 2,3-Dimercaptopropanol (BAL) bei diffusen Leberparenchymsch~idigungen. Schweiz. med. Wschr. 80, 933--936 (1950) Jollow, D. J., Mitchell, J. R., Zampaglione, N., Gillette, J. R.: Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenze oxide as the hepatotoxic metabolite. Pharmacology (Basel) 11, 151--169 (1974) Kirnberger, E. J., Braun, W., Stille, G., Wolf, V.: Beziehungen zwischen Leberschutz und Zuckerstoffwechsel. Arzneimittel-Forsch. 8, 72-76 (1958) Lange, P., Jung, F.: Die Verringerung der Lebertoxizit~it von Tetrachlorkohlenstoff dutch Diiithyldithiocarbamat und 3-(o-Tolyl)-4-(nitro)-sydnon. Acta biol. med. germ. 27, 425-434 (1971) Lange, J., Schumacher, K., Witscher, H. P.: Die Behandlung der chronisch-aggressiven Hepatitis mit D-Penicillamin. Dtsch. med. Wschr. 96, 139--145 (1971)
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Legros, J.: Paracetamol and the liver. Animal studies -- a theoretical basis for treatment. J. int. Med. Res. 4, (Suppl. 4), 46--54 (1976) Lieber, C. S., Rubin, E., De Carli, L. M.: Hepatic microsomal ethanol oxidizing system (MEOS): differentiation from alcohol dehydrogenase and NADPH oxidase. Biochem. biophys. Res. Commun. 40, 858-865 (1970) Lutz, L. M., Glende, E. A., Recknagel, R. O.: Protection by diethyldithiocarbamate against carbon tetrachloride lethality in rats and against carbon tetrachloride-induced lipid peroxidation in vitro. Biochem. Pharmacol. 22, 1729--1734 (1973) Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R., Brodie, B. B.: Acetaminopheninduced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. exp. Ther. 187, 185-194 (1973) Rees, K. R., Tarlow, M. J.: The hepatotoxic action of allyl formate. Biochem. J. 104, 757-761 (1967) Reid, W. D., Christie, B., Eichelbaum, M., Krishna, G.: 3-methyl-cholanthrene blocks hepatic necrosis induced by administration of bromobenzene or carbon tetrachloride. Exp. molec. Path. 15, 363-372 (1971) Reynolds, E. S., Moslen, M. T.: Chemical modulation of early carbon tetrachloride liver injury. Toxicol. appl. Pharmacol. 29, 377-388 (1974) Sakaguchi, T., Nishimura, H., Masuda, K., Tsuge, I., Onishi, K., Tatsumi, H.: The relationship between chemical structure and protective effect of dithiocarbamate derivates against experimental hepatic injury induced by carbon tetrachloride administration in rats. Biochem. Pharmacol. 15, 756--758 (1966) Schlicht, I.: Autoradiographische und radiochromatographische Untersuchungen der VerteiIung und des Stoffwechsels von Thioacetamid. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 268, 310-322 (1971) Str6mme, J. H.: Metabolism of disulfiram and diethyldithiocarbamate in rats with demonstration of an in vivo ethanol-induced inhibition of the glucuronic acid conjugation of the thiol. Biochem. Pharmacol. 14, 393--410 (1965) Strubelt, O., Siegers, C.-P., Sch/itt, Atsuko: The curative effects of cysteamine, cysteine and dithiocarb in experimental paracetamol poisoning. Arch. Toxicol. 33, 55-64 (1974) Sunderman, F. W." Diethyldithiocarbamate therapy of thallotoxicosis. Amer. J. reed. Sci. 253, 107-118 (1967) Sunderman, F. W., Sunderman, F. W., Jr.: Nickel poisoning. VIII. Dithiocarb: a new therapeutic agent for persons exposed to nickel carbonyl. Amer. J. med. Sci. 236, 26-31 (1958) Tonkelaar, E. M., van Loogten, M. J.: Protective action of dithiocarbamates on experimental liver damage produced by carbon tetrachloride. Toxicol. appl. Pharmacol. 30, 96-106 (1974) Varga, F., Mekes, J., Molnar, Z.: Die Leberschutzwirkung von Cystein, Homocysteinthiolacton und --N--Acetylhomocysteinthiolacton bei experimenteller Lebersch/idigung. Arzneimittel-Forsch. 13, 867-871 (1963) Wilson, J. T., Frohman, L. A.: Concomitant association between high plasma levels of growth hormone and low hepatic mixed-function oxidase activity in the young rat. J. Pharmacol. exp. Ther. 189, 255--270 (1974) Received Februar 10, 1978
