Chem.-Biol. Interactions, 15 (1976) 277--287

277

© Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

MODIFICATIONS OF D R U G METABOLISM BY D I S U L F I R A M AND DIETHYLDITHIOCARBAMATE II. D-GLUCURONIC ACID PATHWAY

MARIOS MARSELOS, MATTI LANG and RIITTA TORRONEN Department of Physiology, University of Kuopio, SF-70101 Kuopio 10 (Finland)

(Received March 2nd, 1976) (Revision received July 15th, 1976) (Accepted August 12th, 1976)

SUMMARY Hepatic enzymes connected with the formation and metabolism of free Dglucuronic acid were affected in rats after treatment with disulfiram or diethyldithiocarbamate (300 mg/kg, intragastrically, per day, 4 X). The activities of UDPglucose dehydrogenase, UDPglucuronic acid pyrophosphatase, UDPglucuronosyltransferase and L-gulonate dehydrogenase were enhanced, while those of glucose-6-phosphate dehydrogenase, fl-glucuronidase and Dglucuronolactone dehydrogenase were inhibited. These changes were more pronounced with disulfiram than diethyldithiocarbamate. Treatment with phenobarbital (80 mg/kg, i.p., per day, 4 X) enhanced UDP glucuronosyltransferase, but brought about different effects on the other enzymes. Concurrent administration of phenobarbital with disulfiram or diethyldithiocarbamate led to potentiation or antagonism of the primary effects of each c o m p o u n d when given alone. The results suggest that activation of the Dglucuronic acid pathway may proceed in various ways, and that it is n o t necessarily followed by a simultaneous induction of the microsomal mixedfunction oxygenase activity.

INTRODUCTION

Administration of disulfiram causes an increased urinary excretion of free and conjugated D-glucuronic acid in rabbits [1], of L-ascorbic acid in rats [2] and of D-glucaric acid in guinea pigs [3]. These findings indicate a stimulation of the D-glucuronic acid pathway, a view supported also by the observed induction of the hepatic UDPglucuronosyltransferase activity [3]. In humans, disulfiram has been detected in the urine as the S-glucuronide of its reduced derivative diethyldithiocarbamate [4], and radioactive studies in

278 D-glucose

anaerobic ~ glycoiysis

glucose-6--phosphate ~ ~ pentose phosphate It shunt

glycogen ~

glucose- q -- phosphate U DP-glucose U DP-glucu~onic acid

D-glucuroniCl_phosphateaCid-

g~curonides

DIglUCuronic acid D-glucu~onolactone

L-gulonic ocid

I

116

I

L-gulonolactone i

D-glucQric acid

~

L-ascorbic acid

II7 i

V

L-×ylulose

Fig. 1. The D-glucuronic acid pathway and its relation to the metabolic routes of Dglucose. The numbers indicate enzymes determined in this study. (1) glucose-6-phosphate

dehydrogenase, (2) UDPglucose dehydrogenase, ( 3 ) UDPglucuronate pyrophosphatase, (4) UDPglucuronosyltransferase, (5) /3-glucuronidase, (6) n-glucuronolactone dehydrogenase, and (7) L-gulonate dehydrogenase. rats showed that this conjugate accounted for over 50% of the total metabolites excreted after administration of disulfiram or its reduced thiol [ 5]. These represent sporadic observations on various animal species and a systematic and comparative study of the effects of disulfiram and diethyldithiocarbamate on the D-glucuronic acid pathway is missing, Many experiments have pointed out that disulfiram could greatly impair the microsomal mixed-function oxygenase in the rat [6--8]. These results are in apparent contradiction with the generally accepted concept that the h y d r o x y l a t i o n and glucuronidation processes are in close functional relationship, as indicated by their similar response to induction or inhibition by foreign compounds [ 9--11]. In an attempt to study these two drug-metabolizing enzyme systems in detail, we used the same experimental animals after treatment with disulfiram or diethyldithiocarbamate. Results concerning the mixed-function oxygenase have been presented in another paper [12]. Herein, we report observations on enzymes connected with the hepatic D-glucuronic acid pathway (Fig. 1). MATERIALS AND METftODS

The origin of the animals, their treatment, and the preparation of the tissues have been described in a previous report [12]. The samples used for

279 the determination of enzymes in these experiments were the same with those used in studies on the mixed-function oxygenase system. The activity of dehydrogenases was determined from the hepatic cytosolic fraction (about 0.5 mg protein), by monitoring the reduction of NADP or NAD at 340 nm with a Perkin--Elmer double-beam spectrophotometer (model 402). Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was determined b y slightly modifying the method of Glock and McLean [13]. The reaction was carried out at 25°C in a cuvette containing tris buffer (70 mM, pH 7.6), 2.5 mM glucose-6-phosphate (Merck AG, Darmstadt, W. Germany), 20 mM MgC12 and 0.3 mM NADP (Boehringer and Sohne, Mannheim, W. Germany). UDPGlucose dehydrogenase (EC 1.1.1.22) was measured according to Strominger et al. [14] at 25°C, in a final volume of 1 ml containing glycine buffer (50 raM, pH 8.7), 0.15 mM uridine-5-diphosphoglucose (Sigma) and 0.8 mM NAD (Boehringer). D-Glucuronolactone dehydrogenase (EC 1.1.1.70) was determined at 38°C, in a final volume of 1 ml containing 28 mM D-glucuronolactone (Fluka AG, Buchs, Switzerland), 1,6 mM NAD and 80 mM phosphate buffer (pH 7.8) [15]. L-Gulonate dehydrogenase (3-hydroxyacid dehydrogenase, EC 1.1.1.45) measurement was carried out at 38°C in 1 ml containing 50 mM glycine buffer (pH 8.7), 10 mM sodium L-gulonate, 2 mM L-cysteine (Merck) and 0.8 mM NAD [16]. L-Gulonate was synthesized from 0.2 M L-gulono-l,4lactone (Nutritional Biochem. Co., Cleveland, Ohio) incubated overnight with an equimolar amount of sodium hydroxide [ 17]. UDPGlucuronosyltransferase (EC 2.4.1.17) was determined from the microsomal fraction (about 0.5 mg protein) at 38°C, as described by H~/nninen and Puukka [18]. The incubation mixture was 1 ml and contained 0.35 mM p-nitrophenol (Merck) as aglycone, 4.5 mM uridine diphosphate glucuronic acid (ammonium salt, 98%, Sigma) and 10 mM K2-EDTA (Merck}. ~-Glucuronidase (EC 3.2.1.31) was measured according to the method of Bernfeld et al. [19] slightly modified. The microsomal fraction {about 0.1 mg protein) was incubated in 1 ml containing 80 mM sodium acetate buffer (pH 4.5) and 1 mM phenolphthalein glucuronide (Sigma). After incubation for 30 rain at 38°C the reaction was stopped with 0.5 ml 3% trichloroacetic acid, the tubes were centrifuged to remove precipitated protein ( 2 0 0 0 g , 10 min), and 1 ml of 1 M Na2CO3 was added for the development of colour. Absorbance was measured at 540 nm with a Perkin--Elmer spectrophotometer (model 139), and liberated phenolphthalein was estimated with the aid of a standard curve. UDPGlucuronic acid pyrophosphatase (probably EC 3.6.1.9) was determined from the microsomal fraction (about 0.1 mg protein} with a new method developed in our laboratory (Puhakainen, unpublished work). The products of the reaction, a-D-glucuronic acid 1-phosphate and UMP, were hydrolyzed by alkaline phosphatase and the liberated phosphate was quanti-

280 tated as described by Harper [20]. Incubation was carried out in a final volume of 150 pl containing 67 mM tris buffer (pH 8.9), 2 mM UDPglucuronic acid (Merck}, 25 mM MgC12 and 0.002 I.U. of alkaline phosphatase (Boehringer). The reaction was started by adding microsomes suspended in 2.5% albumin and immersing the tubes in a 38°C water-bath. After 15 min the reaction was terminated with I ml trichloroacetic acid (10% solution in 2% L-ascorbic acid). Denatured protein was removed with centrifugation ( 5000 g, 5 min), and 0.25 ml of 1% a m m o n i u m - h e p t a m o l y b d a t e {Merck} was added to the supernatants. About 10 rain later, 0.5 ml of arsenite-citrate solution (2% in 2.8 N acetic acid) was added, the samples were mixed and the developed colour was measured after 15 min at 700 nm in a Perkin--Elmer spectrophotometer {model 139). Potassium phosphate standard curve (0.0-0.2 mM) was used for the final calculation of the results. Protein determination and statistical analysis of the results were carried out as described before [12]. RESULTS

Primary effects of disulfiram, diethyldithiocarbamate and phenobarbital Both disulfiram and diethyldithiocarbamate produced an increase in the relative liver weight. The absolute liver weight (g) was 12.2 -+ 0.1 (S.E.M.) for untreated controls, and 12.0 ± 0.2 and 11.7 +- 0.1 for disulfiram- and diethyldithiocarbamate-treated animals. The respective values as a percentage of the body weight were 3.6 +- 0.06, 4.0 + 0.08 (P < 0.001) and 3.7 + 0.05 (P < 0.01). Changes caused by the tested compounds on the overall hepatic c o n t e n t of protein, phospholipids and glycogen, as well as the microsomal protein and phospholipid contents, are described in detail in the first part of this work [121. Administration of disulfiram and diethyldithiocarbamate produced changes on enzyme activities of the D-glucuronic acid pathway, which were mostly of the same quality. The former c o m p o u n d , however, caused always more pronounced effects than its reduced derivative. On the other hand, the action of phenobarbital on the determined enzymes were both qualitatively and quantitatively different than those of the other compounds (Table I (A) and (B)). The activity of glucose-6-phosphate dehydrogenase was slightly decreased by disulfiram, and this effect was even more pronounced by diethyldithioearbamate. On the contrary, phenobarbital enhanced the activity of this enzyme. The priming enzyme of the D-glucuronic acid pathway, UDPglucose dehydrogenase, was induced both by disulfiram and diethyldithiocarbamate, whereas it was not affected by phenobarbital. Again enzymes leading to the formation of free D-glucuronic acid were influenced in a different way according to the treatment. Thus, while disulfiram and its reduced thiol significantly induced the activity of D-glucuronate pyrophosphatase, this enzyme was inhibited by phenobarbital. A 2.5-fold increase of UDP-

I

ACTIVITIES

OF ENZYMES

with disulfiram

(Pb)

THE

solely

+ 2.4

45.1

f 0.4 + 1.7

2.9 20.9

20.6 3.3 43.6 0.57 70.5 2.4 16.9

Control * 2.0 + 0.6 + 4.0 + 0.03 * 3.3 +_0.3 t 0.7,

(x)

or in combination

f 3.3

68.3

+ 0.01

r 0.6

4.1

0.44

+_ 1.3

(x)

21.6

Control

or diethyldithiocarbamate.

Glucose-6-phosphate dehydrogenase UDPGlucose dehydrogenase UDPGlucuronate pyrophosphatase UDPGlucuronosyltransferase &Glucuronidase D-Glucuronolactone dehydrogenase L-Gulonate dehydrogenase

(B) Treatment with phenobarbital All data are expressed as above.

WITH

D-GLUCURONIC

ACID

f 5.2

75.3

+ 1.3

* 0.5

33.1 3.9 26.5 0.85 58.8 7.0 16.5

PB (t) f * + + + + f

4.4 0.06 1.2 0.08 2.0 0.6 0.7

with disulfiram

28.2

traces

11.2

(xx)

(xxx)

(xxx)

(xxx)

(xxx)

(x)

f 7.3

73.3

19.9

0.44

26.0

t 0.7

+ 0.07

+ 1.7

t 0.02

+ 0.1

6.9

0.71

t 0.8

7.6

(@§)

(xxx)

(xxx,@$)

(xXx,$@,

(xx)

(xx,@,

(xxx)

Diethyldithiocarbamate

(xx) (x) (x) (xxx)

(x)

30.3 5.1 31.0 1.5 9.2 0.6 23.8

t 1.8 _+0.3 + 2.2 t 0.04 + 1.1 f 0.1 f 1.6

Pb + DS (5) (x) (x,j.) (x) (xxx,tl_t) (xxx,j.$j-) (xx,tt$) (xx#)

LIVER statistical

f f + f + + *

0.9 0.2 2.3 0.1 1.3 0.5 1.0

(xx) (t) (t) (xxx,tt) (xxx,ttt,~§§) (xx,t,§@, (@)

Pb + DEDTC 29.2 4.4 33.8 1.5 30.3 4.6 17.1

signif-

(Pb + DEDTC).

to which

(Pb + DS) and diethyldithiocarbamate

(5)

f 0.02

f 0.6

9.4

1.10

* 2.8

14.7

Disulfiram

IN RAT

with regard

PATHWAY

are quoted, The symbols in parentheses refer to the group xx,ti,@, P < 0.025; xxx,?-/-t&j@, P < 0.001).

CONNECTED

Glucose-6-phosphate dehydrogenase (nmole NADPH/min/mg protein) UDPGlucose dehydrogenase (nmole NADH/min/mg protein) UDPGlucuronic acid pyrophosphatase (nmole Pi/min/mg protein) UDPGlucuronosyltransferase (nmole p-nitrophenol/min/mg protein) fi-Glucuronidase (nmole phenolphthalein/min/mg protein) D-Glucuronolactone dehydrogenase (nmole NADH/min/mg protein) L-Gulonate dehydrogenase (nmole NADH/min/mg protein)

(A) Treatment

Mean values (t S.E.M.) from five animals icance has been assessed (x,t,$, P < 0.05;

SPECIFIC

TABLE

282 glucuronosyltransferase activity could be detected after treatment with disulfiram, while diethyldithiocarbamate and phenobarbital enhanced the enzyme only by a factor of 1.5. Disulfiram was more effective than the other compounds also in decreasing the activity of ~-glucuronidase. In contrast to the dramatic inhibition of D-glucuronolactone dehydrogenase by disulfiram and diethyldithiocarbamate, phenobarbital markedly induced the activity of this enzyme. L-Gulonate dehydrogenase was affected only by disulfiram, which brought about a significant enhancement of its activity. These differences in enzyme activities remained the same when expressed in terms of a m o u n t in total liver. Effects of the concurrent administration of the tested compounds. Combination of tested compounds affected the liver weight of the animals. The liver weight (g) of untreated controls was 10.9 ± 0.3 (S.E.M.), and 13.1 _+ 0.3, 14.0 +_ 1.0 and 12.6 +- 0.5 for animals treated with phenobarbital, phenobarbital plus disulfiram, and phenobarbital plus diethyldithiocarbamate, respectively. These values were higher and differed statistically from the controls (P ~ 0.01), when expressed as a percentage of the body weight. Combination of phenobarbital with disulfiram or diethyldithiocarbamate had a different effect on enzyme activities than when the compounds were given alone {Table I (A) and (B)). The final result was often found to depend on the primary effect of each c o m p o u n d per se. As a rule, qualitatively similar alterations produced by each c o m p o u n d alone were potentiated under combined treatment. On the other hand, primary effects of opposite nature resulted in a balance between stimulation and inhibition. Thus, although disulfiram and diethyldithiocarbamate given separately decreased the activity of glucose-6-phosphate dehydrogenase, this effect was virtually abolished in the presence of phenobarbital. The enhancement of UDPglucuronate pyrophosphatase caused by disulfiram and diethyldithiocarbamate was completely prevented by phenobarbital, and inhibition of this enzyme still occurred. Phenobarbital partially overcame the inhibition of D-glucuronolactone dehydrogenase produced by disulfiram and diethyldithiocarbamate, leading to an intermediate level of activity in the case of the latter compound. On the contrary, the inhibition and induction caused by phenobarbital on resp. ~-glucuronidase and UDPglueuronosyltransferase was potentiated by concurrent t r e a t m e n t with either of the other compounds tested. Although phenobarbital alone did n o t affect UDPglucose and L-gulonate dehydrogenases, its presence markedly hindered or even abolished the actions of the other compounds on these enzymes. Due to the enlargement of the liver after phenobarbital treatment, UDPglucuronate pyrophosphatase activity did not show any difference among the experimental groups when expressed on a total liver basis. DISCUSSION It has been found that soon after disulfiram administration a great part of it is reduced to diethyldithiocarbamate [21], which is a known p o t e n t metal-

283 chelating agent. Moreover, both disulfiram and diethyldithiocarbamate are capable of binding to protein --SH groups [22]. These properties are probably responsible for the unspecific inhibitory effects of the c o m p o u n d s on several enzymes possessing --SH groups in their active site, or requiring metal ions as cofactors. However, although most of the enzymes determined in our study are sensitive to --SH binding agents [17], induction rather than inhibition of their activity was observed. Perhaps regulatory changes in the metabolism of D-glucose and its increased utilization in the D-glucuronic acid pathway is the principal factor contributing to the overall increase of enzyme activities. In the case of UDPglucuronosyltransferase, induction might be due also to the fact that diethyldithiocarbamate is an exogenous substrate for the enzyme [4]. Disulfiram and diethyldithiocarbamate can interfere in many ways with the intermediary metabolism of carbohydrates. Direct influence is pointed out by the inhibition of enzymes such as glyceraldehyde-3-phosphate dehydrogenase [23], glucose-6-phosphate dehydrogenase [24] and hexokinase [25]. In addition, indirect implications may ensue due to the inhibition of dopamine-~-hydroxylase [26], and the following changes of cate: cholamine concentration in the central nervous system [27] and the periphery [28]. These changes could be expected to have an impact on the Dglucuronic acid pathway, which is closely associated with the metabolism of D-hexoses [29]. The increased flow through the D-glucuronic acid pathway, reflected by enhanced urinary excretion of D-glucuronic acid [1] and Lascorbic acid [2], may be due to the inhibition by disulfiram of the anaerobic glycolysis and the pentose phosphate shunt [30], which represent alternative routes in the metabolism of D-glucose. Enhancement of enzyme activities due to increased substrate availability is a recognized regulatory mechanism [31], and in the case of the D-glucuronic acid pathway this has been also proven after experimental loading with carbohydrates [ 32,33 ]. Treatment with phenobarbital might enhance the activity of glucose-6phosphate dehydrogenase, as has been shown with mice [34]. The fact that many xenobiotics activate concomitantly the pentose phosphate and the glucuronate pathways [34] led to the hypothesis that an initial alteration in the redox state of the liver cell could be the c o m m o n underlying mechanism for these changes [35]. Inhibition by disulfiram of glucose-6-phosphate dehydrogenase was first detected in insect muscle [24]. Our results show that inhibition also occurs in hepatic tissue. Thus, enhancement of the pentose phosphate shunt is not a prerequisite for the activation of the Dglucuronic acid pathway. This finding, however, does not necessarily argue against a possible regulatory role of the hepatic redox state in the observed alterations. Although phenobarbital, disulfiram and diethyldithiocarbamate activated the pathway, they clearly affected many of the measured enzymes in a different way. Combined administration of these c o m p o u n d s resulted in potentiation or abolition of their primary effects. This variety of actions implies that stimulation of the D-glucuronic acid pathway takes place in

284 various ways, depending on the nature of the administered foreign compound. The end result may represent primary effects of the c o m p o u n d and its metabolites, secondary adaptation to changes in the intermediary metabolism, or a combination of these. Formation of free D-glucuronic acid takes place via either the pyrophosphatase-phosphatase, or the UDPglucuronosyltransferase-~-glucuronidase route {Fig. 1). The observed inhibition of fi-glucuronidase suggests that the former route prevails after treatment with disulfiram and diethyldithiocarbamate. Further metabolism of free D-glucuronic acid seems to be directed mainly towards the formation of L-ascorbic acid [2], and also of L-xylulose judging from the increased L-gulonate dehydrogenase activity observed. The marked inhibition of D-glucuronolactone dehydrogenase does not favour metabolism towards D-glucaric acid, a view compatible with the reported decrease of its urinary excretion in humans given disulfiram [36]. Notten and Henderson [3], however, have noticed even a slight increase of D-glucaric acid excretion in guinea pigs after prolonged administration of disulfiram, despite the inhibition of D-glucuronolactone dehydrogenase. This further indicates an enhanced flow through the pathway and a great availability of D-glucuronic acid. Changes in the glucuronidation enzymes caused by disulfiram and diethyldithiocarbamate are in contrast to those found in the mixed-function oxygenase system [6--8,12]. Impairment of the microsomal h y d r o x y l a t i o n and a parallel activation of the UDPglucuronosyltransferase have been reported with carbon tetrachloride [37,38] and n-hexane [39]. A possible mechanism is the action of these compounds on the microsomal membrane lipids [40], by analogy to the in vitro effect of surfactants and chaotropic agents [41, 42]. This does not seem, however, to apply to disulfiram and diethyldithiocarbamate. In this study most of the key-enzymes of the D-glucuronic acid pathway had enhanced activities, including those of cytosolic origin. Moreover, the increased glucuronidation capacity in vivo after treatment with disulfiram [1] suggests that a real stimulation of the pathway takes place, and n o t a simple activation of the membrane-bound UDPglucuronosyltransferase. To date, increased urinary excretion of L-ascorbic acid is conventionally considered as an in vivo measure of induced microsomal drug-metabolism in the rat [43--47]. Nonetheless, from our experiments it is evident that this is not always the case, since stimulation of the glucuronidation enzymes was followed by a parallel inhibition of the hydroxylation system [12]. The clearly differentiated action of disulfiram and its reduced thiol on the two enzyme systems presents a useful tool for in vitro and in vivo studies on their functional correlation and on the mechanism involved in their induction. ACKNOWLEDGEMENTS We would like to thank Professor O. H~inninen for his interest in the work and his advice and criticism during the preparation of the manuscript. We are

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