Protective action of dithiocarbamates on experimental liver damage produced by carbon tetrachloride

30,96-106(1974)

TOXICOLOGYANDAPPLIEDPHAdMACOLOGY

Protective Liver

Action Damage ENGELINAM.

of Dithiocarbamates Produced by Carbon DEN

TONKELAARAND M. J.

on Experimental Tetrachloride VAN

LOGTEN

Laboratory of Toxicology, National Institute of Public Health, Bilthoven, The Netherlandr Received July 13, 1973; accepted April 8,1974

Protective Action of Dithiocarbamateson Experimental Liver Damage Produced by Carbon Tetrachloride. DEN TONKELAAR, E. M. AND VAN LOGTEN, M. J. (1974). Toxicol. Appl. Pharmacol. 30,96-106. The protective action of dithiocarbamateson Ccl,-induced liver damagein rats was assessed with liver function tests(BSPretention testandSGPT). When given immediatelybefore CC14,all dithiocarbamatestestedshoweda protection againstCC& hepatotoxicity except for zineb and some“polymer” dithiocarbamates.For zineb it appearsplausiblethat this wascausedby a delayed absorption. No protection was found when one of the dithiocarbamates studiedwasgiven 1 hr after Ccl4 administration.From studieswith some metabolitesand relatedcompoundsit appearsthat only substances containing a carbon disulphidegroup have a protective action. Probably these compounds function as effective free radical scavengers.In addition, inhibition of microsomalliver enzyme activity and a lowering of body temperaturecould alsobe contributing factors. The hepatotoxic effect of carbon tetrachloride (CC&) is well known. A number of treatments and substances have been described that protect against this effect. In general,

these protective factors can be divided into several categories like agents which produce lowering of body temperature, inhibition of drug metabolizing enzymes, or have antioxidant properties.

Dithiocarbamates are widely used in industry and agriculture (Thorn and Ludwig, 1962). They are present in rubber products as antioxidants and accelerators and as residues of fungicides in crops. In addition, disulfiram, a dithiocarbamate, is used clinically as an antialcohol drug. Studies on the toxicity and alcohol intolerance of various dithiocarbamates have been summarized by van Logten (1972). According to Sakaguchi et al. (1966) dithiocarbamates also protect against the hepatotoxicity of Ccl,. Within the scope of studies in our laboratory concerning the mechanism of action of the dithiocarbamates, we were interested in the relation between molecular structure and protective action and the possible mechanism of this protective action. The hepatotoxic effects of CCI, and the protection given by administration of dithiocarbamates were measured using sulphobromophthalein (BSP) retention and serum glutamicpyruvic transaminase (SGPT) activity as liver function tests. Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

96

DITHIOCARBAMATES

AND

ccl,

HEPATOTOXICITY

97

METHODS

Animals For all experiments female SPF-derived Wistar rats weighing about 150-200 g were used. They were obtained from the Central Institute for the Breeding of Laboratory Animals TNO, Zeist and received a commercial diet (Muracon, Trouw, Putten). Compounds Carbon tetrachloride, dissolved with olive oil, was given po at a dose of 1.2 ml/kg. The dithiocarbamates and other compounds tested were also given po in olive oil, except for metam-Na, NaDEDC, nabam, and phenergan, which were dissolved in water. For all compounds studied the names, chemical names, producer (for pesticides), and dose level in mg/kg (about l/10 of the oral LD50) are as follows: metam-Na (sodium-Nmethyldithiocarbamate, Van Hasselt, Amersfoort, SO), NaDEDC (sodium-N-diethyldithiocarbamate, 120), ziram (zinc-N-diethyldithiocarbamate, AAgrunol, Groningen, 140), nabam (sodium ethylenebisdithiocarbamate, AAgrunol, Groningen, 57), zineb (zinc ethylenebisdithiocarbamate, Van Hasselt, Amersfoort, 600), TMTM (tetramethylthiurammonosulphide, 75), thiram (tetramethylthiuramdisulphide, Van Hasselt, Amersfoort, 56), triaram (thiuramsulphide, exact composition not known, Vondelingenplaat Rozenburg, 198), tecoram (thiuramdisulphide, exact composition not known, AAgrunol, Groningen, 300), metiram (complex of zineb and polyethylene thiuramdisulphide, according to producer, BASF Ludwigshafen, Germany, 680), trivam (condensation product of ammoniamonomethyl dithiocarbamate and formaldehyde, according to producer, Vondelingenplaat, Rozenburg, 145), CS2 (carbon disulphide, 300), xanthogenate (sodium xanthogenate, 400), thiourea (60), ETU (ethylenethiourea, 30), captan [N-(trichloromethylthio)-4-cyclohexene-1,2-dicarboximide, Ligtermoet, Rotterdam, 12001, trapex (methylisothiocyanate, AAgrunol, Groningen, 13.5), eptam (ethylN-dipropylthiocarbamate, Ligtermoet, Rotterdam, 163), morestan [S,S-(6-methylchinoxalinediyl-2,3)dithiocarbonate, Bayer, Leverkusen, Germany, 3001, phenergan [lo-(2-dimethylaminopropyl)phenotriazine, 531, pip. butox. (piperonyl butoxide, ICI, Rotterdam, 1 ml), DPPD (N&‘-diphenyl-p-phenylenediamine, lOOO), gallate (npropylgallate, Chem. Fabriek Naarden, 380). Experimental Design The dithiocarbamates and other compounds were given once, immediately before administration of CC&, unless otherwise stated. All experiments were carried out in such a way that four or five groups of three animals each were compared, one of which served as a control group and one as a Ccl, group. The range of values for each combination and the range for the appropriate control and CC& values are given in the tables. For BSP retention and SGPT activity determinations, different animals were used. Analyses The SGPT activity was measured according to the method of Reitman and Frankel (1957) using Baker Diagnostic Reagents, the results are given in Karmen units. For the BSP test our own modification was used. When BSP is given according to body weight, elevated normal values are found for older and heavier animals (>300 g). Therefore

98

DEN

TONKELAAR

AND

VAN

LOGTEN

BSP was injected iv equivalent with the body weight raised to the power of 0.7 in such a way that for a rat of 150 g, 25 mg BSPjkg body weight was given. This method is particularly

valid when young and old rats have to be compared, because about the same

control values are obtained in that case. Blood was taken exactly 10 min after the injection of BSP. The concentration in serum was measured by diluting 0.3 ml serum with 2.7 ml saline. After addition of 0.05 ml of 10 % hydrochloric acid solution, the extinction was measured (EJ. Then 0.1 ml of a 10% sodium hydroxyde solution was added and the extinction measured again (I&). The difference between Es and EAis equivalent to the concentration of BSP in the serum. The results are expressed as pmol BSPiliter serum. RESULTS

In Table 1 the average + SD of all control values is given for the BSP test and the SGPT determinations. Values in the experiments are considered to be normal when they are lower than the average + 3SD, i.e., ~22 nmol/liter for BSP and < 19 U/liter for the SGPT. The values which are obtained 24 hr after Ccl, administration are also given in this table. TABLE MEAN

VALUES

FOR LIVER CC&-TREATED

AND

No. of animals

Mean + SD

Range

86 69 78 55

11.7f 3.3 116 + 62 10.6 f 2.6 117 f104

6.5- 26.6 25.9-305 5% 19.3 17.4-482

Control ccl, Control ccl,

BSP pmol/liter SGPT Karmen U/liter

1

FUNCTION TESTS OF CONTROL RATS AFTER 1 DAY”

’ Carbon tetrachloride was given po, I .2 ml/kg.

In the first experiment, the protective action of metam-Na was studied. The results of BSP retention and SGPT activity after 1,2, and 4 days are given in Table 2. When the animals were pretreated with the dithiocarbamate, protection was found 1,2, and 4 days TABLE 2 LIVER

FUNCTION

RESULTS

AFTER ORAL ADMINISTRATION AND 1.2 ml CCl,/kg

CC& BSP SGPT

after after after after after after

1 day 2 days 4 days 1 day 2 days 4 days

38-l 621317-l 287-

136 93 46 74 96 22

Control 6-8 lo-16 10-15 12-13 8-14 8-12

OF

80 mg

Metam-Na + ccl, 8-14 15-20

7-12 IO-22 IO-21 9-13

METAM-Na

Protective score” 313 313 313 213 213 313

dlProtective score for metam-Na + CC& is the number of values lower than the mean control value + 3SD/total number. For BSP this is x22 pmol/liter and for SGPT 49 Karmen U/liter. b Range of values for BSP in pmol/liter and for SGPT in Karmen U/liter.

DITHIOCARBAMATES

AND

ccl,

99

HEPATOTOXICITY

after Ccl, administration. In this experiment metam-Na was given immediately before Ccl,. In the next experiment the effect of metam-Na given some time before or after CCL, administration was studied. The results of the BSP test are shown in Table 3. From the results shown it is obvious that metam-Na shows protection when given within 16 hr before CCl,. With a longer time interval this dithiocarbamate partially lost its protective action. In sharp contrast are the results when metam-Na is administered after Ccl,. Given 15 min after CC14, metam-Na gave only partial protection, and given later no protection was found at all. TABLE

3

BSP LIVER FUNCTION RESULTS WHEN METAM-Na IS GIVEN VARIOUS TIMES BEFORE OR AFTER Ccl., ADMINISTRATION

AT

Time

of administration”

-

BSP assessed 24 hr ccl,

0 hr 0 hr Ohr 0 hr 0 hr 0 hr 0 hr 0 hr Ohr 0 hr 0 hr 0 hr Control

cc14

metam-Na -48 -24 -16 -8

hr

hr hr hr hr

-4

-2 hr

-1 hr -0 hr +15

min

t30 min +l hr +2 hr (6 animals) (10 animals)

Protective

after CCL

score*

38-21gc lo- 49 ll- 26 17- 26 12- 15 14- 28 16- 19 lo- 24

W-5

12-200

315

9-115 66-118 42-104

216 016

416 516 213 313 213 313

516

o/3

6- 12 52-l 69

’ Minus (-) sign denotes metam-Na was given before sign denotes it was given after Ccl4 (1.2 ml/kg, PO). h Number of values ~22 pmol/liter,/total number. c Range of BSP values in ,umol/liter.

CC&;

plus (+)

Subsequently, a number of dithiocarbamates and related compounds were tested and compared to study whether there is a relation between structure and action. In all cases the dithiocarbamates were given just before CC&, and liver function tests were measured after 1 day. The dithiocarbamates are divided into metal dithiocarbamates, bisdithiocarbamates, thiuram(di)sulphides, and “polymers.” In these experiments the dithiocarbamates exerted no effect themselves on the liver function tests. Tables 4 and 5 show that nearly all dithiocarbamates tested exhibit a protective action. One exception, besides the “polymer” dithiocarbamates, is zineb, which was expected to react like other bisdithiocarbamates. However, this compound has a very low solubility, and possibly this results in a slow uptake from the gastrointestinal tract and the liver. Therefore the protective action of this agent was studied by giving it at

100

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VAN

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TABLE 4 SULPHOBROMOPHTHALEIN RETENTION 24 hr AFTER ADMINISTRATION OF DITHIOCARBAMATES (DTC), FOLLOWED BY CCq”

ccl‘+

Control

DTC + CCL,

Protective scoreb

Metal DTC metam-Na metam-Na metam-Na NaDEDC ziram

38-113’ 26-170 164-244 82-163 44- 72

6-8 13-16 14-16 9-20 lo-1 1

8121668-

14 15 18 10 13

313 313 313 313 313

Bis-DTC nabam zineb zineb

44- 72 70-117 31- 66

10-l 1 9-12 7-27

ll- 12 43- 47 51,101*

313 O/3 o/2

Thiuram-S TMTM thiram triaram triaram triaram tecoram

109-166 109-l 66 31- 66 131-151 38-113 70-l 17

8- 9 8- 9 7-27 6-10 6- 8 9-12

10, lo* 162192114-

18 37 13 28 19

212 313 l/3 313 l/3 313

Polymers metiram metiram trivam trivam trivam

70-232 64-134 106-141 41- 67 131-151

11-13 7- 8 12-14 9-10 6-10

244738916-

56 70 49 16 97

O/3 O/3 o/3 313 113

’ Carbontetrachloridedosewas1.2ml/kg, po. bNumberof values~22 mol/liter/total number. ’ Rangeof BSPvaluesin pmol/liter. * Only two animals. various times before Ccl,. The results on the BSP test measured 24 hr after CCL, are

given in Table 6. It is thus possible that the nonprotective action of zineb shown in Tables 4 and 5 may be explained by a delayed uptake, and that the compound may protect as well as the other dithiocarbamates. Tables 7 and 8 show the results obtained using other related compounds and metabolites. Only CS2, xanthogenate, and captan showed a clear protective action. In order to elucidate the mechanism of action of the protection offered by the dithiocarbamates, a number of other substances were tested such as phenergan (a well-known protective agent), piperonyl butoxide (known for inhibition of the microsomal liver enzymes), and diphenyl-p-phenylenediamine (DPPD) and propylgallate (known as antioxidants). Some of these compounds were also given 4 hr before CCL. As can be

DITHIOCARBAMATES

AND

ccl,

TABLE

101

HEPATOTOXICITY

5

SER.UM GLUTAMIC-PYRUVIC TRANSAMINASE ACTIVITY 24 ADMINISTRATION OF DITHIOCARBAMATES (DTC), FOLLOWED

Metal DTC metam-Na metam-Na metam-Na metam-Na NaDEDC ziram Bis-DTC nabam zineb zineb Thiuram-S TMTM thiram triaram triaram triaram triaram tecoram Polymers metiram trivam trivam trivam

hr

AFTER BY Ccl,”

Protective scoreb

cc14

Control

17-174’ 30-241 20- 36 96-241 72-193 44- 98

12-13 11-12 11,15* 9-10 10-13 13-16

lo1513ll139-

22 20 15 14 22 11

213 213 3/3 313 213 313

44- 98 48- 96 30-241

13-16 8-11 10-13

12- 14 lo- 11 32-176

313 313 O/3

289, 482d 289, 482d 17-174 30-241 37-145 29- 37 48- 96

10-l 1 lo-11 12-13 lo-13 8- 9 8-12 8-11

17161287912-

21 51 16 19 14 18 24

l/3 l/3 313 213 313 313 213

96-241 62-212 20- 36 37-145

9-10 6-9 11-15 8- 9

43-289 14- 69 17- 96 17- 58

O/3 l/3 l/3 113

DTC + Ccl4

a Carbon tetrachloride dose was 1.2 ml/kg, po. bNumber of values x19 Kannen U/liter/total number. c Range of SGPT values in Karmen U/liter. d Only two animals. TABLE 6 SULPHOBROMOPHTHALEIN

RETENTION WHEN ZINEB IS GIVEN VARIOUS TIMES BEFORE CCL ADMINISTRATION

Time of administration ccl*

zineb

BSP24hr after Ccl,

Oh Ohr Oh Ohr Ohr

-24 hr -6hr -4hr -2 hr -0hr

lO-17b 14-25 lo-22 17-55 14-42

Control (5 animals) CCI, (5 animals)

9-17 733-266

a Number of values ~22 qol/liter/total * Range of BSP values in mol/liter.

number.

Protective score” 414 415 314 215 l/4

AT

102

DENTONKELAAR

AND VAN LOGTEN

TABLE

7

SULPHOBROMOPHTHALEIN RETENTION 24hr AFTER ADMINISTRATION OF COMPOUNDSRELATED TODITHIOCARBAMATES,FOLLOWED BY CCL,”

cc14 cs2 xanthogenate xanthogenate xanthogenate thiourea ETU captan trapex trapex eptam morestan

77-l 63’ 55-l 66 64-133 179-244 106-141 25- 42 55-166 40- 67 106141 164-244 66-157

Compound + CCL

Control 9-20 12-18 7- 8 G-10 12-14 7-10 12-18 6-10 12-14 14-16 10-13

Protective scoreb

8- 10 ll- 15 14- 22 16- 22 130,200d 66- 73 ll- 15 12- 96 71-151 162-184 170-288

313 313 213 2/3

o/2 O/3 313 213 O/3 O/3 O/3

a Carbon tetrachloride dose was 1.2 ml/kg, po. b Number of values ~22 pmol/liter/total number. c Range of BSP values in ,umol/liter. d Only two animals. TABLE

8

SERUM GLUTAMIC-PYRUVICTRANSAMINASEACTIVITY 24 hr AFTER ADMINISTRATION OF COMPOUNDS RELATED TO DITHIOCARBAMATES, FOLLOWED BY Ccl,”

cs2 xanthogenate xanthogenate thiourea thiourea thiourea ETU ETU captan captan trapex trapex trapex eptam morestan

CCI.$

Control

72-193’ 32-173 33-206 39- 58 62-212 20- 36 68-104 54-137 33-206 30-241 39- 58 2@- 36 62-212 41-347 32-173

10-12 13-18 9-10 7- 9 6-9 11-15 17,21d 12-16 9-10 lo-12 7- 9 11-15 6-9 7- 8 13-18

0 Carbon tetrachloride dose was 1.2 ml/kg, po. b Number of values ~19 U/liter/total number. c Range of SGPT values in Karmen U/liter. d Only two animals.

Compound + cc14 16, 18d 18- 31 13- 14 8- 29 61-116 36, 4gd 15- 28 29- 47 lo- 12 12- 20 12- 13 13- 40 51-116 434482 81-193

Protective scoreb

212 l/3 313

213 O/3

012 213 O/3

313 213 313 113

o/3 o/3 o/3

DITHIOCARBAMATES

AND

ccl,

103

HEPATOTOXICITY

seen in Table 9, phenergan, pip. butox., and DPPD did not protect when they were given in the same way as the dithiocarbamates, that is, just before Ccl,. DPPD and propylgallate also provided no protection administered 4 hr before Ccl,. TABLE 9 LIVER

FUNCTION RESULTS 24 hr AFTER ADMINISTRATION OF ANTIOXIDANTS AND OTHER PROTECTIVE AGENTS, FOLLOWED BY Ccl,”

cc14

Control

Compound + cc14

55-l 66’ 26-170 55-l 66 49-144 109-166 49-144 49-144

12-18 13-16 12-18 8-14 7- 9 8-14 8-14

32- 59 20- 58 13- 47 47- 98 18- 91 23, 29d 46- 74

O/3 l/3 l/3 O/3 l/3

30-241” 33-206 33-206 91-333 289,482” 91-333 91-333

11-12 8-10 8-10 lo-19 lo-11 lo-19 IO-19

26 69

O/3 l/3 O/3 O/3 O/3 O/3 O/3

Protective score*

BSP phenergan phenergan pip. butox. pip. butox. DPPD DPPD 4 hr before gallate 4 hr before SGPT phenergan phenergan pip. butox. pip. butox. DPPD DPPD 4 hr before gallate 4 hr before

16- 63

22- 47 30482 68-116 50- 65 482-482

a Carbon tetrachloride dose was 1.2 ml/kg, po. * Number of values ~22 pmol/liter for BSP and 49 Karmen U/liter for SGPT/total c Range of BSP values in rmol/liter; range of SGPT values in Karmen U/liter. d Only two animals.

012

O/3

number.

In Table 10 a summary is given of the protective scores found for BSP and SGPT in all experiments. In this table an attempt is made to divide the compounds that were tested into strong, moderate, and weak protectors, and compounds that did not protect at all. Finally, some compounds were also studied for protection 3 days after CCL administration. At that time some recovery is already found from the Ccl, hepatotoxicity. The results are not given in detail, but it was found that for each compound the protective score after 3 days was as good or better than on day 1.

DISCUSSION The results of these experiments indicate that certain dithiocarbamates have a strong protective effect on the hepatotoxicity of Ccl,. The results of Sakaguchi et al. (1966) are confirmed. The protective action of metal dithiocarbamates and ethylenebisdithiocarbamates is, in general, better than that of the thiuram(di)sulphides. The “polymer”

104

DENTONKELAARAND TABLE

VANLOGTEN 10

SUMMARYOFDATAONPROTECTIO~

Total protective score for BSP

Total protective score for SGPT

metam-Na NaDEDC ziram nabam zineb

919

TMTM

212

10112 213 313 313 316 113 113 11112 213 Of3 319 212 416

thiram triaram tecoram metiram trivam cs2 xanthogenate thiourea ETU

313 313 313 015 313

519 213

O/6

419 313 719

012 O/3 313

218 216 516

captan trapex eptam morestan phenergan pip. butox.

116

‘76

DPPD

l/3

o/3 013 013

DPPD (4 hr before) gallate (4 hr before)

216 O/3 013 O/3

012 o/3

419 o/3 o/3 116

Total 19/21 516 616 616 3/l 1 315 416

16121 416 019 7118 515

11115 2110 219 819 6115

016 016 119 l/12 116 015 016

Protective action ++ ++ ++ ++ kb + + + + iz ++ + rk f ++ k -

-

-

a SeeTables 4-9 for details. b This compound gets a ++ score when given 4-24 hr before Ccl,.

dithiocarbamates, trivam and metiram, showed little or no protective action, possibly due to a slower uptake in the body or a slower metabolism. This rationale is also plausible for zineb. Metam-Na gives maximal protection when given between 0 and 16 hr before Ccl,. However, when given after CCL,, little or no protection was afforded. Studies with related compounds were carried out to provide information on which part of the molecule may be responsible for the protective effect. From the results it is obvious that only carbon disulphide, xanthogenate, and captan produce good protection. These compounds comprise the CS2 group. The protective action of captan looks remarkable because no CS, group is initially present. However, from metabolism studies (Menzie, 1969) it follows that CS, originates from this fungicide, after degradation of thiophosgene. Thus, the conclusion can be drawn that a CS2 group may be necessary for the protective action. Moreover, trapex, thiourea, and ethylenethiourea give only weak protection, and eptam and morestan give no protection at all. These compounds do not possess a CS2 group. Recknagel(l967) and Slater (1966) have described the toxic action of CCI, by postulating the formation of a *Ccl, radical as the first reaction step. This starts a chain of

DITHIOCARBAMATES

AND cc&

105

HEPATOTOXICITY

autoxidations land results in lipid peroxidation. The formation of the radical is catalyzed by microsomal liver enzymes:Recknagel(l967) describes a number of factors that offer protection against the hepatotoxicity of Ccl,. In general these protective factors can be divided into three different categories : (1) decrease of the activity of the microsomal liver enzymes; (2) lowering of body temperature; (3) antioxidant action. Dithiocarbamates can belong to all three classes of protective agents. They give a prolongation of hexobarbital sleeping time and are said to impair drug-metabolizing enzymes (Stripp et al., 1969). The prolongation of hexobarbital sleeping time was confirmed in our laboratory (unpublished results). Scholler et al. (1970) explained the protective action of disulfiram against chloroform toxicity by assuming an enzyme-inhibiting effect causing a suppression of metabolism. It is, however, not probable that the protective action of dithiocarbamates is only based on inhibition of microsomal liver enzymes. In general, drug-induced inhibition of these enzymes is maximal after 1 or 2 hr. Dithiocarbamates protect when given simultaneously with CCL, but when given 15 or 30 min later the effect is drastically reduced. The lack of effect of piperonyl butoxide, a well-known enzyme inhibitor, also points in this direction. Dithiocarbamates also lower body temperature (van Logten, 1972). This cannot be the main protection mechanism, since the protective effect of dithiocarbamates can also be demonstrated 2, 3, or 4 days after Ccl, administration. This is in contrast with the findings of Larson and Plaa (1963, 1965). They showed only a delay in the Ccl,induced hepatotoxicity under the influence of a lower body temperature. The dithiocarbamate-induced lower temperature lasts only 2430 hr. Dithiocarbamates are known as antioxidants (Tollenaar, 1951); however, their protective action is much stronger than that of phenergan, DPPD, and propylgallate. One possible explanation for their protection against Ccl, hepatoxicity can stem from the fact that dithiocarbamates function as very effective free radical scavengers (Slater, 1966). Since it is known that CC& exerts some of its effects on the liver after only 5 min (Rao and Recknagel, 1968) the dithiocarbamates must catch the first radicals formed. This may explain why the protection is drastically decreased when metam-Na is given some time after Ccl,. From the results described in this paper the conclusion can be drawn that dithiocarbamates are among the most potent protective agents against Ccl, hepatotoxicity, perhaps as a combination of the lowering of body temperature, partial inhibition of drug-metabolizing enzymes, and a primary antioxidant property. The protection was based on liver function tests, but experiments to study the influence of this combination on fat accumulation in the liver and other biochemical and histological parameters are currently in progress. REFERENCES LARSON, R. E. AND PLAA, G. L. (1963). Spinal cord transection and Ccl,-toxicity.

Experienta

19, 604-606. LARSON, R. E. AND FLAA, G. L. (1965). A correlation of the effects of cervical cordotomy,

hypothermia

and catecholamines on carbon tetrachloride-induced

hepatic necrosis.

J. Pharmacol.Exp. Ther. 147, 103-111. MENZIE, C. M. (1969). Metabolism of pesticides. Special Scientific Report. Wildlife No. 127,

Washington, D.C.

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RAO, K. S. AND RECKNAGEL, R. 0. (1968).Early onset of lipoperoxidation in rat-liver after carbon tetrachloride administration. Exp. Molec. Pathol. 9, 271-278. RECKNAGEL, R. 0. (1967).Carbon tetrachloride hepatotoxicity. Pharmacol. Rev. 19, 145-208. REITMAN, S. AND FRANKEL, S. (1957).A calorimetric method for the determinationof serum glutamic oxalacetic and glutamic pyruvic transaminases. Amer. J. Clin. Pathol. 28, 56-63. SAKAGUCHI, T., NISHIMURA, H., MAWDA, K., ISUGE, J., ONISHI, K. AND TATSUMI, H.(1966).

The relationshipbetweenchemicalstructureand protective effect of dithiocarbamatederivatives againstexperimentalhepatic injury inducedby carbon tetrachloride administrationin rats. Biochem. Pharmacol. 15, 756-758. SCHOLLER, K. L., MUELLER, E. AND VON PLEHWE, U. (1970).Versttikung und Unterdriickung der Toxizitgt von Chloroform fiir dieLeberdurch Pharmaka.Arzneim.-Forsch. 20,289-292. SLATER, T. F. (1966).Necrogenicaction of carbon tetrachloride in rat : A speculativemechanism basedon activation. Nature (London) 209, 36-40. STRIPP, B., GREENE, F. E. AND GILETTE, J. R. (1969).Disultiam impairmentofdrugmetabolism by rat liver microsomes.J. Pharmacol. Exp. Ther. 170, 347-354. THORN, G. D. AND LUDWIG, R. A. (1962). Dithiocarbamates and Related Compounds. Elsevier, Amsterdam. TOLLENAAR, F. D. (1951).The inhibition of oxidative defectsin cold-storedbutter by some tetraalkylthiuramdisulphides.Ned. Melk Zuiveltijdschr. 5, 46-50. VAN LOGTEN, M. J. (1972).De dithiocarbamaat-alcoholreactie bij de rat. Thesis,University of Utrecht.