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Studies on the metabolic activation of benzidine by isolated rat hepatocytes
Toxicology, 23 (1982) 235--247 Elsevier/North-Holland Scientific Publishers Ltd.
STUDIES ON T H E METABOLIC ACT IV A T IO N OF BENZIDINE BY I S OLATED R A T H E P A T O C Y T E S
R.M.E. BROUNS, R.P. BOS, R. VAN DOORN and P.TH. HENDERSON Institute of Pharmacology and Toxicology, University of Nijmegen, Nijmegen (The Ne the rlands) (Received October 26th, 1981) (Accepted February 7th, 1982)
SUMMARY Isolated rat liver cells were shown t o metabolize the aromatic amine benzidine to reactive products which are mutagenic to S a l m o n e l l a t y p h i m u r i u m TA 1538 and which give rise t o DNA excision repair within the liver cells. Intact rat liver cells are shown to be m ore active in the form at i on of mutagenic metabolites than the 9000-g supernatant from these cells. Data are presented which are in favour of the role of N-acetylation in this respect. F u r t h e r m o r e , indications are presented t hat a sulfation reaction is involved in the generation of DNA m odi f yi ng metabolites, whereas f o r m a t i o n of mutagenic products is likely to proceed via deacetylation and/ or N,Oacyltransfer. Finally, data are given a b o u t the extrahepatocellular appearance of premutagenic metabolites which are more prone to metabolic activation by additional metabolic factors in the Salmonella assay than benzidine itself. The impact of these observations on the estimation of the genotoxic potential o f benzidine will be discussed.
INTRODUCTION The aromatic amine benzidine was epidemiologically shown to be a h u man carcinogen, producing urinary bladder tumors. In experimental animals it also produces tumors, f or example, in the liver of mice and hamsters, and in the liver, intestine and m a m m a r y gland of rats [1]. During the last 2 decades much information has become available a b o u t the mechanisms t h a t can be held responsible for the carcinogenic effects of arylamines and arylamides. An essential initial step in the bioactivation of Abbreviations: DMSO, dimethylsulphoxide, MEM, minimal essential medium; TdR, thymidine.
0300-483Xl8210000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.
23 5
aromatic amines and amides is claimed to be N-hydroxylation, mediated by monooxygenases, mainly localized in the endoplasmic reticulum of the liver [2--4]. For the generation of the ultimate reactive intermediates that bind covalently to cellular constituents and that are believed to represent the actual carcinogens, transferase catalyzed reactions appear to be implicated. In this respect sulfotransferase [ 4 - 6 ] , N,O-acyltransferase [ 7 - 9 ] , and glucuronyltransferase [10] catalyzed conjugation of the N-hydroxy moiety have been postulated. Only recently, in vitro studies showed that metabolic activation of benzidine can proceed via N-acetylation and subsequent N-hydroxylation leading to the formation of N-hydroxy-N,N'-diacetylbenzidine [11]. Furthermore, the role of both sulfotransferase and N,O-acyltransferase in the formation of metabolites leading to adduct formation and mutations have been indicated [11,12]. Recently we showed that the isolated rat hepatocyte, representing a morphological and biochemical entity, is able to metabolize the aromatic amines 2-aminofluorene and 2-acetylaminofluorene to genotoxic products, causing DNA-repair effects within the liver cell and mutations in S. typhimurium [ 13,14]. In the present study we investigated the role of hepatocellular metabolism of benzidine in the formation of reactive metabolites, using DNA-repair effects within the liver cell and bacterial mutagenicity as parameters for genotoxicity. MATERIALS AND METHODS
Chemicals [3H]Thymidine ([3H] TdR), specific activity 23 Ci/mmol was purchased from the Radiochemical Centre (Amersham, England). Collagenase type I (EC 3.4.24.3) was bought from Sigma Chemical Co. (St. Louis, USA). Eagle's minimal essential medium (MEM), including Hank's salts, and foetal calf serum were obtained from Biomed (Gibco). Benzidine, harmine-HC1, salicylamide and 4-aminoazobenzene (= p-phenylazoaniline) were from Aldrich Europe (Beerse, Belgium). All other reagents, obtained from different sources, were of the highest purity obtainable. Isolation of rat liver cells Parenchymal liver cells were isolated from adult male Wistar rats under sterile conditions by means of a collagenase perfusion method [15,16]. Twenty-four hours before the isolation procedure was started the animals were deprived of food. In the final cell preparation more than 95% of the cells were in a viable state as judged by the Trypan Blue exclusion method. Measurement o f hepatocellular DNA-repair Incubation of the hepatocytes, suspended in Eagle's MEM, supplemented with Hank's salts, 10% foetal calf serum, 0.01% streptomycin and 100 U/ml
236
penicillin G with the compounds to be investigated and subsequent measurem e n t of [3H] TdR incorporation into DNA via liquid scintillation counting was essentially as described previously [ 17].
Measurement o f mutagenicity Experiments on mutagenicity were basically performed according to the m e t h o d described by Ames et al. [18]. Salmonella typhimurium TA 1538 was used as bacterial tester strain. Thereto, an overnight nutrient broth culture was centrifuged and the bacterial pellet was resuspended in saline at a concentration of approximately 5 X l 0 s bacteria/ml. The experimental procedure was as follows: a suspension of liver cells in HEPES buffer (Seglen), 2.5 X 106 viable cells/ml, was added to 3 ml of molten topagar, together with the compounds under test and the bacteria, and, when indicated, supplemented with a postmitochondrial fraction from rat liver. In the case of pre-incubation (see below), cell free supernatant was added to the topagar together with the test compounds, bacteria and different subcellular liver fractions as an additional activating system. Detailed experimental conditions are given in the legends. The topagars supplemented with the different components were poured onto minimal agar petri dishes. After 48 h at 37°C the number of histidine prototrophic colonies was counted on each dish.
Pre-incubation o f liver cells Suspensions of freshly isolated hepatocytes, 2.5 X 106 viable cells/ml, were incubated in 50-ml erlenmeyer flasks in a shaking water bath (Infors WTR1) at 37°C together with the c o m p o u n d under test, added as a concentrated solution in dimethylsulphoxide (DMSO). The final a m o u n t of DMSO did not exceed 10 t~l/ml incubation volume. After various time intervals 2-ml aliquots were taken and centrifuged immediately. Next 0.5-ml aliquots of the supernatant were added to the molten topagar together with the other components. Detailed descriptions are given in the legends. RESULTS
Mutagenic effects after activation o f benzidine by hepatocytes and hepatocellular 9000-g supernatant When intact rat liver cells were used as metabolizing system, it was found that benzidine could be activated to metabolites exerting mutagenic effects in S. typhimurium TA 1538. The mutagenicity, as a function of the benzidine concentration, is presented in Fig. 1. When, instead of intact cells, the 9000-g supernatant of the same cells, prepared by ultrasonic treatment as described previously [ 19], was given as a metabolizing component, fortified with an external NADPH regenerating system, mutagenicity was considerably lower.
237
revertants TA1538/prate 200 160
120
/o/e/e
80
/"
40
'
2 0' 0
'
4 60
benzidine
'
cone (,uM)
Fig. 1. Mutagenic activation of benzidine by intact rat liver cells, o - ~ - - o , or 900O-g supernatant from these cells after ultrasonic treatment, ----.---. To 3 ml of molten topagar were added: 0.5 ml cell suspension (2.5 x 104 viable cells/ml) or 0.5 ml 9000-g supernatant, equivalent to 2.5 x 10' cells, fortified with a NADPH regenerating system, consisting of 8 mM MgCl2, 4 mM NADP, 5 mM glucose 6-phosphate and 2.8 U/ml glucose-6-phosphate dehydrogenase, 0.1 ml of bacteria (TA 1538) suspended in saline and 41 ul of different freshly prepared solutions of benzidine in DMSO. Values (revertants/ plate), representing one typical experiment out of at least four, are means of determinations in triplicate.
Effect of modified hepatocellular N-acetylation and sulfation The involvement of N-acetyltransferase and sulfotransferase catalyzed reactions in the metabolic activation of benzidine was demonstrated by Morton et al. [11,12]. It was now studied whether liver cell mediated mutagenicity of benzidine could be affected by modifying these transferase catalyzed reactions. For that purpose harmine was used to inhibit N-acetylation [20], whereas the sulfation reaction was antagonized by salicylamide [21]. The influence of these modifying agents on the activation of benzidine by isolated rat liver cells to mutagenic species is presented in Fig. 2. Mutagenicity was considerably reduced by harmine (500 t~M), whereas inhibition of the sulfation reaction by salicylamide (1 mM) had no influence. Stimulation of the sulfate conjugation even resulted in a marked decrease of mutagenicity. It should be remarked that none of these treatments had any effect on bacterial survival, nor did they reduce hepatocellular viability. It cannot fully be excluded by these experiments that the strong reduction in mutagenicity by harmine is partly due to an interference by this c o m p o u n d with the N-oxidation of benzidine. However, we did not find any inhibitory effect of 0.5 mM harmine on microsomal monooxygenase activity, as measured in vitro with aniline or aminopyrine. On the contrary, it was reported [20] that the N-acetylating capacity in vitro is inhibited by about 90% in the presence of 0.5 mM harmine.
238
rever tants TA153B/prate 160 O
O
120
8O
z~o
r '
:25o
4~o benzidine
'
660
conc (/uM)
Fig. 2. Mutagenic activation of benzidine by intact rat liver cells in the presence of harmine (500 uM), e---o-.; salieylamide (1 mM), o---o---o ; Control, o--o--o. To 3 ml of molten topagar were added: 0.5 ml cell suspension (2.5 x 106 viable cells/ml), 0.1 ml bacteria (TA 1538) suspended in saline and the compounds under test, added as concentrated solutions in ethanol (salicylamide), DMSO (4-aminoazobenzene) or saline (harmineHC1) to reach the final concentrations in the topagar as indicated. Values (revertants/ plate), representing one typical experiment out of at least four, are means of determinations in triplicate.
Influence o f pre-incubation and additional metabolic activation on mutagenic potential of benzidine Besides the observation t h a t upon incubation of benzidine with rat liver cells metabolic products are formed that exert direct mutagenic effects in S. typhimurium, evidence was obtained that liver cell mediated metabolism of benzidine gives rise to the formation of products that are not mutagenic per se but need an additional activation to become mutagenic. The experimental approach was based on a pre-incubation of benzidine with liver cells, whereafter the extracellular medium was subjected .to an additional metabolic activation according to the standard Ames-assay with rat liver 9000-g supernatant. In Fig. 3 the effect of pre-incubation with rat liver cells on the mutagenic activation by rat liver 9000-g supernatant is visualized. Due to pre-incubation with intact cells a 4-fold increase in the a m o u n t of revertant colonies was found. Furthermore, the observed difference in mutagenicity was found to be dependent upon the pre-incubation time, as illustrated in Fig. 4. In order to obtain information a b o u t the nature of the premutagenic products released by the hepatocytes, it was investigated which metabolic factors were required for the additional mutagenic activation. As can be seen from Table I, a combined action of both microsomes and cytosol was essential in this respect, whereas NADPH was also required. These findings suggest the involvement of a microsomal oxidative step, followed by either a sulfotransferase or N,O-acyltransferase catalyzed reaction [11,12] in the
239
revertants 1200
TA 1 5 3 8 / p l . a t e
lOOO
BOO
6OO 4OO
200
400 6~)0 benzidine conc. ()JM)
Fig. 3. Mutagenic activation of benzidine by hepatic 9000-g supernatant after preincubation with, o--c--~, or without, ,--a--o, intact rat liver cells. Benzidine (final conc. on the axis) was pre-incubated either with or without rat liver cells for 60 min as indicated in "Materials and Methods". After removal of the cells by centrifugation, 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar, together with 0.5 ml 9000-g supernatant from liver of phenobarbital pretreated rats (75 mg/kg, i.p. for 5 days), corresponding to 12.5 mg of liver tissue, fortified with a NADPH regenerating system (see legend to Fig. 1), and 0.1 ml of bacteria (TA 1538) suspended in saline. Values (revertants/plate), representing one typical experiment out of at least four, are means oi determinations in triplicate. g e n e r a t i o n o f t h e m u t a g e n i c species. F r o m T a b l e II it can be seen t h a t s a l i c y l a m i d e , a s u b s t r a t e t h a t is k n o w n t o a n t a g o n i z e t h e s u l f a t i o n r e a c t i o n [ 21] had n o i n h i b i t o r y e f f e c t on t h e m u t a g e n i c i t y data, whereas s u p p l e m e n t a t i o n w i t h a P A P S g e n e r a t i n g s y s t e m r e s u l t e d in a s u b s t a n t i a l decrease in m u t a g e n i c i t y . T h e r e f o r e t h e role o f s u l f o t r a n s f e r a s e is n o t resolved. H o w e v e r , 4 - a m i n o a z o b e n z e n e a n d p - a m i n o p h e n o l , c o m p o u n d s t h a t are k n o w n to inhibit t h e N , O - a c y l t r a n s f e r a s e c a t a l y z e d a d d u c t f o r m a t i o n b e t w e e n Nhydroxy-2-acetylaminofluorene and tRNA [ 9], markedly reduced mutagenic i t y , whereas also p a r a o x o n , an i n h i b i t o r of the d e - a c e t y l a t i o n r e a c t i o n , r e d u c e d m u t a g e n i c i t y considerably. I t s h o u l d be n o t e d t h a t these c o m p o u n d s s h o w e d no d e l e t e r i o u s e f f e c t on bacterial viability at the c o n c e n t r a t i o n s e m p l o y e d , w h e r e a s an i n h i b i t o r y e f f e c t on m i c r o s o m a l oxidizing c a p a c i t y a t t h e c o n c e n t r a t i o n s e m p l o y e d was virtually absent. These d a t a are in f a v o u r o f t h e c o n c e p t t h a t during i n c u b a t i o n of b e n z i d i n e w i t h r a t liver cells, a c e t y l a t e d p r o d u c t s are f o r m e d . This is also s u p p o r t e d b y t h e i n h i b i t o r y e f f e c t of h a r m i n e , p r e s e n t d u r i n g p r e - i n c u b a t i o n , o n the u l t i m a t e m u t a g e n i c a c t i v i t y (Table III). F u r t h e r m o r e , it was o b s e r v e d t h a t in a d i r e c t p l a t e assay r a t liver c y t o s o l
240
rever
rant
1200
-
1000
-
800
-
600
-
400
-
200
-
s
TA
1538/prate
--
-
e--
' 4'0 ' 6'0 incubation time (rain) Fig. 4. Mutagenic activation of benzidine by hepatic 9000-g supernatant after various pre-incubation times with, o---~--~, or without, ~--e---~, rat liver cells. Benzidine (500 uM final conc.) was pre-incubated either with or without rat liver cells for various times. Samples were immediately cooled on ice, quickly centrifuged in the cold, after which 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar. The remaining procedure was as described for Fig. 3. Values (revertants/plate), representing one typical experiment out of at least four, are means of determinations in triplicate. '
2()
TABLE I E F F E C T OF D I F F E R E N T POSTMITOCHONDRIAL LIVER FRA CTI O N S ON THE MUTAGENICITY OF BENZIDINE A F T E R PRE-INCUBATION WITH OR WITHOUT INTACT RAT LIVER CELLS Benzidine (500 uM) was pre-incubated with or without rat liver cells for 60 min as described in "Materials and Methods". After centrifugation the cell-free supernatant was tested for mutagenicity as follows: to 3 ml of molten topagar were added 0.5 ml of this supernatant, 0.5 ml of different sub-cellular liver fractions from phenobarbital pretreated rats, corresponding to 12.5 mg of liver tissue, in 0.15 M KC1 (including a NADPH regenerating system when indicated, consisting of 8 mM MgCl:, 4 mM NADP, 5 mM glucose 6-phosphate and 2.8 U/ml glucose-6-phosphate dehydrogenase), and 0.1 ml of bacteria (TA 1538), suspended in saline. Values, representing one typical experiment out of at least four, are means of 3 different determinations. Revertants TA 1538/plate Subcellular liver fraction
Pre-incubation with hepatocytes
Pre-incubation without hepatocytes
None 9000-g supernatant + NADPH 9000-g supernatant -- NADPH Microsomal fraction + NADPH (= A) 105 000-g supernatant (= B) A + B
23 1510 12 160 60 1390
21 161 15 37 35 182
241
TABLE II
EFFECT OF 4-AMINOAZOBENZENE, p-AMINOPHENOL, PARAOXON, S A L I C Y L A M I D E , A N D PAP + pNPS O N T H E M U T A G E N I C A C T I V A T I O N O F B E N Z I D I N E B Y H E P A T I C 9000-g S U P E R N A T A N T AFTER PRE-INCUBATION WITH INTACT RAT LIVER CELLS Benzidine (500 ~M) was pre-incubated with rat liver cells for 60 min as described in "Materials and Methods". After centrifugation, 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar, together with 0.5 ml 9000-g supernatant from liver of phenobarbital pretreated rats, corresponding to 12.5 mg of liver tissue, fortified with a NADPH regenerating system (= S-9 mix), 0.1 ml of bacteria (TA 1538) suspended in saline and the compounds under test, added as concentrated solutions in ethanol (salicylamide), DMSO (4-aminoazobenzene, paraoxon) or saline (p-aminophenol, PAP + pNPS) to reach the final concentrations in the topagar as indicated. Values are means of three different determinations. Addition
Relative mutagenicity (% of control)
None (= control) 4-Aminoazobenzene (50 ~M) 4-Aminoazobenzene (250 uM) p-Aminophenol (100 uM) p-Aminophenol (1 mM) Paraoxon (100 uM) Salicylamide (1 mM) PAP (20 uM) + pNPS (10 raM)
Exp. 1
Exp. 2
100 37 16 69 16 47 121 29
100 44 9
45 107 21
TABLE III E F F E C T OF HARMINE ON THE MUTAGENICITY OF BENZIDINE WHEN PRE-INCUBATED WITH R A T LIVER CELLS Benzidine (500 uM) was pre-incubated with rat liver cells for 60 rain in the presence of harmine. After centrifugation; 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar together with 0.5 ml S-9 mix and 0.1 ml of bacteria (TA 1538) suspended in saline. Values are means of 3 different determinations.
Conc. harmine (uM)
0 (= control) 100 500
242
Relative mutagenicity (% of control) Exp. 1
Exp. 2
100 69 47
100 84 53
alone markedly enhanced the liver cell mediated mutagenicity of benzidine (Fig. 5). Presumably, liver cells can excrete N-hydroxylated metabolite(s) that can be activated into an ultimate mutagenic form through non-oxidative pathways. The absence of an activation by cytosol alone in the case of liquid pre-incubation (Table I) may be a reflection of the hepatocellular formation of rather unstable products that are not detected under pre-incubation conditions.
DNA-excision repair Besides the application of bacterial mutagenicity as a parameter for the extra-hepatocellular appearance of proximate and ultimate mutagenic metabolites of benzidine, DNA-repair was used as an endpoint for genotoxic effects of benzidine within the liver cell. Exposure of the cells to benzidine caused a concentration dependent increase in [ 3 H ] T d R incorporation up to approximately 10 -s M benzidine (Fig. 6). Beyond this concentration an abrupt decline in incorporation was observed, possibly due to an injurious
revertants TA 1538/prate 2802~0 -
/ ~ o
200
/
~o
160 120 80 z~O
'
260
'
,-,oo'
'
6bo
benzidine conc ( ) J M )
Fig. 5. E f f e c t o f r a t liver c y t o s o l ( 1 0 5 0 0 0 g s u p e r n a t a n t ) a d d i t i o n o n t h e m u t a g e n i e a c t i v a t i o n o f b e n z i d i n e b y rat liver cells. T o 3 ml m o l t e n t o p a g a r were a d d e d : 0.5 ml cell s u s p e n s i o n (2.5 X 10 ~ viable cells/ml) or m e r e l y 0.5 ml s u s p e n s i o n b u f f e r , 0.5 ml h e p a t i c c y t o s o l f r o m p h e n o b a r b i t a l p r e t r e a t e d r a t s or 0.5 m l 0 . 1 5 M KCI, 0.1 ml b a c t e r i a (TA 1 5 3 8 ) s u s p e n d e d in saline, a n d 41 ul o f d i f f e r e n t freshly p r e p a r e d s o l u t i o n s of benzidine in DMSO. o---o---~ : liver cells. ~--a--n : h e p a t i c c y t o s o l , t---~--. : liver cells + h e p a t i c c y t o s o l . V a l u e s ( r e v e r t a n t s / p l a t e ) , r e p r e s e n t i n g o n e typical e x p e r i m e n t o u t of at least f o u r , are m e a n s o f d e t e r m i n a t i o n s in triplicate.
243
3 H-TdF~ i n c o r p o r a t i o n (% above control ) 50-
a
b
"/
40-
30
/
i
20
10 i
/
-10 -
-20
-30
i
10 -8
10 -7
I
10-6
i
10-5
i
i0-~
I
I
10-3
10 -7
I
i
10 .6 10 -5 benzidine
~
,
10 -z" 10 -3 conc(M)
Fig. 6. The effect of harmine and salicylamide on the enhanced incorporation of [3H]TdR into hepatocellular DNA, due to benzidine exposure. Liver cells (1 X 106 viable cells/ml) were incubated with different concentrations of benzidine, with or without addition of harmine (100 ~M) or salicylamide (1 raM). (a) incubation with e--e--e, or without, o---~---~,harmine; (b) incubation with e--e--e, or without, o---~--~, salicylamide. e f f e c t on t h e D N A - r e p a i r a p p a r a t u s as such, since cellular viability was u n a f f e c t e d u p t o at least 5 × 10 -4 M b e n z i d i n e . When t h e N - a c e t y l a t i o n and s u l f a t i o n c a p a c i t y of the liver cell were i n h i b i t e d b y h a r m i n e a n d s a l i c y l a m i d e respectively, the e n h a n c e d i n c o r p o r a t i o n o f [3H] T d R was r e d u c e d t o the level o b s e r v e d in the a b s e n c e of benzidine, suggesting t h a t b o t h N - a c e t y l a t i o n a n d sulfate c o n j u g a t i o n are involved in t h e g e n e r a t i o n o f r e a c t i v e m e t a b o l i t e s of b e n z i d i n e , leading to D N A - r e p a i r e f f e c t s w i t h i n t h e liver cell. I t s h o u l d be e m p h a s i z e d t h a t b o t h inhibitors h a d n o e f f e c t on t h e e n h a n c e d i n c o r p o r a t i o n o f [ 3 H ] T d R e v o k e d b y d i m e t h y l n i t r o s a m i n e a n d 4 - n i t r o q u i n o l i n e - l - o x i d e ( d a t a n o t s h o w n ) , thus e x c l u d i n g a deleterious action of harmine and salicylamide on DNA-repair mechanisms o f the liver cell. U n f o r t u n a t e l y we w e r e u n a b l e t o d e t e r m i n e t h e e f f e c t of 4 - a m i n o a z o b e n z e n e , an i n h i b i t o r of t h e N , O - a c y l a t i o n r e a c t i o n in vitro, on t h e b e n z i d i n e e v o k e d h e p a t o c e l l u l a r D N A - r e p a i r , since this c o m p o u n d itself is a c t i v a t e d b y t h e liver cell, t h e r e b y causing a s u b s t a n t i a l increase in [3H]TdR incorporation [ 17].
244
DISCUSSION It is k n o w n from in vitro studies t hat metabolic activation of benzidine can proceed via acetylation and h y d r o x y l a t i o n of the amino function, after which a subsequent N,O-acyltransfer or sulfate conjugation yields the ultimate reactive form [ 1 1 , 1 2 ] . In the present study isolated rat liver cells were f o u n d to be capable in activating benzidine to metabolites causing genotoxic effects both inside and outside the liver cell. Besides the f o r m a t i o n of metabolites that are directly genotoxic, data are presented regarding the extracellular appearance of metabolic products t h a t are n o t genotoxic as such, but can be m ore effectively activated by hepatic 9000-g supernatant than benzidine itself. F r o m our data, together with those obtained from in vitro studies by others, we c o m p o s e d a tentative scheme a b o u t the role of N-acetylation, N - h y d r o x y l a t i o n , N,O-sulfation and N,O-acylation in the metabolic activation of benzidine by the intact rat liver cell (Fig. 7). Being aware of the c o m p l e x i t y of the overall metabolism of benzidine, a t t e n t i o n was focussed
S-9 MIX
IN TA C T H EPATOCY TE
_1
Ar--NH2
1
O H II Ar--N--C--CH 3
0
Ar--NH2
1
0 H II ~_ A t - N - - C--CH a
1
0 II
Ar--~--~--CH O ,
O=S=O I
OH
I
3
Ar--y--C--CH3
/
/
OH
I
\
N,O-ocyltrons-
Ar--~H
ferose - C - - C H 3
o\ / II
OH
r- A r - - N H
I
OH
DNA-adducts ~----- Ar--NH
OH
=-
3
\ N,O-ocyltronsferase - C - C H 3
/
oII
Ar--NH
I
O--C--CH a II 0
O_C_CH 3 II 0 DNA- repair
0 II
Ar-N--C--CH
S.typhTA1538 mutation
Fig. 7. Tentative scheme for the metabolic activation of benzidine by isolated rat hepatocytes.
245
on the involvement of only 1 amino function. Our data are in favour of the liver cell mediated formation of metabolites that are directly mutagenic to S. typhimurium and are presumably formed through N-acetylation, N-Hydroxylation, N,O-acyltransfer and/or deacetylation. It has been reported [22] t h a t in the case of N-hydroxy-2-acetylaminofluorene the N,O-acyltransferase catalyzed mutagenic activation is unlikely to proceed via the formation of a N-acetoxy derivative, in contrast to the formation of reactive metabolites leading to tRNA adducts. In the case of benzidine, however, the inhibitory effect of 4-aminoazobenzene and paminophenol (Table II), compounds that can serve as acceptors for the acyl moiety, suggests that an intramolecular transfer of the acyl group to yield the N-acetoxy derivative may be involved in its mutagenic activation. Whether this type of metabolite can actually be regarded as a mutagen, either directly or after additional transformation, including bacterial activation pathways as suggested by Weeks et al. [22], is still an open question. Furthermore, the inhibitory effect of paraoxon on the observed mutagenicity is in favour of the involvement of a deacetylation reaction in the mutagenic activation of benzidine. With respect to the release of benzidine metabolites from rat liver cells that require additional metabolic factors to become mutagenic, indications are presented t h a t at least 2 types of metabolic products are involved. One is likely to be a N-acetylated product, requiring both a microsomal and a cytosolic factor to become mutagenic, the other is activated by cytosol alone and is presumably a N-acetylated N-hydroxy derivative. Since it was found that the proposed N-acetylated metabolite was considerably more mutagenic than benzidine itself after metabolic activation by rat liver 9000-g supernatant, one may speculate about the impact of this p h e n o m e n o n on the estimation of the genotoxic potential of benzidine. In this connection, the observed increase in mutagenic activity of benzidine after metabolic activation by intact liver cells, compared to the 9000-g supernatant from these cells (Fig. 1), might be an indication of a better Nacetylation capacity of the intact cell. It can be concluded from Fig. 1 that in the case of benzidine, destruction of the biochemical organization of the intact liver cell results in a decreased activating capacity. This observation is not a general p h e n o m e n o n since no mutAgenicity was found in the hepatocyte mediated assay with benzo[a] pyrene [19]. These examples may illustrate the important value of isolated hepatocytes as metabolizing system in the evaluation of the genotoxic potential of premutagenic and precarcinogenic compounds. In terms of the extrahepatic genotoxicity of benzidine, the extrahepatocellular appearance of a Nacetylated N-hydroxy metabolite may also be important, since the enzyme that is proposed to be involved in its mutagenic activation is localized not only in liver but also in various other tissues such as kidney, stomach, small intestine and colon [9]. Our data on hepatocellular DNA-repair are in favour of the role of both N-acetylation and sulfation in the formation of DNA adducts that give rise
246
t o D N A - r e p a i r (Figs. 6a,b). Since i n h i b i t i o n o f the sulfation r e a c t i o n had no e f f e c t o n bacterial m u t a g e n i c i t y o f benzidine, the role of sulfate esters in this r e s p e c t is q u e s t i o n a b l e , p r e s u m a b l y because of their c h e m i c a l reactivity a n d / o r h y d r o p h i l i c properties. F r o m these d i s c r e p a n t findings it m a y be c o n c l u d e d t h a t the same m e t a b o l i c p r o d u c t s o f b e n z i d i n e are n o t necessarily involved in the g e n e r a t i o n o f m u t a g e n i c effects within Salmonella a n d the f o r m a t i o n o f D N A a d d u c t s w i t h i n the liver cell. It remains an intriguing q u e s t i o n w h i c h o f these g e n o t o x i c p a r a m e t e r s is a c t u a l l y related to the f o r m a t i o n o f c a r c i n o g e n i c m e t a b o l i t e s of benzidine by the liver cell. ACKNOWLEDGEMENTS T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e t h e excellent technical assistance o f Mrs. E.W.M. Yih-van de H u r k , Mrs Th. van H o e k - K o n a n d Mr P.J.L. van G e m e r t . Financial s u p p o r t was given b y t h e D u t c h F o u n d a t i o n f o r Medical R e s e a r c h ( F U N G O ) a n d b y t h e L a b o u r I n s p e c t o r a t e , D u t c h Ministry of Social Affairs. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
T.J. Haley, Clin. Toxicol., 8 (1975) 13. J.W. Cramer, J.A. Miller and E.C. Miller, J. Biol. Chem., 235 (1960) 885. E.C. Miller, J.A. Miller and H.A. Hartmann, Cancer Res., 21 (1961) 815. J.A. Miller and E.C. Miller, Prog. Exp. Tumor Res., 11 (1969) 273. J.R. DeBaun, E.C. Miller and J.A. Miller, Cancer Res., 30 {1970} 577. J.R. DeBaun, J.Y.R. Smith, E.C. Miller and J.A. Miller, Science, 167 (1970) 184. H. Bartsch, M. Dworkin, J.A. Miller and E.C. Miller, Biochim. Biophys. Acta, 286 (1972) 272. H. Bartsch, C. Dworkin, E.C. Miller and J.A. Miller, Biochim. Biophys. Acta, 304 (1973) 42. C.M. King, Cancer Res., 34 (1974) 1503. C.C. Irving and R. Wiseman Jr., Cancer Res., 31 (1971) 1645. K.C. Morton, C.M. King and K.P. Baetcke, Cancer Res., 39 (1979) 3107. K.C. Morton, F.A. Beland, F.E. Evans, N.F. Futlerton and F.F. Kadlubar, Cancer Res., 40 (1980) 751. R.M.E. Brouns, R.P. Bos, R. van Doorn and P.Th. Henderson, Arch. Toxicol., 45 (1980) 53. R.M.E. Brouns, R. van Doorn, R.P. Bos, L.J.S. Muileners and P.Th. Henderson, Toxicology, 19 (1981) 67. M.N. Berry and D.S. Friend, J. Cell Biol., 43 (1969) 506. P.O. Seglen, Exp. Cell Res., 82 (1973) 391. R.M.E. Brouns, M. Poot, R. de Vrind, Th. van Hoek-Kon, P.Th. Henderson and Ch.M.A. Kuyper, Mutat. Res., 64 (1979) 425. B.N. Ames, J. McCann and E. Yamasaki, Mutat. Res., 31 (1975) 347. R.M.E. Brouns, R.P. Bos, P.J.L. van Gemert, E.W.M. Yih-van de Hurk and P.Th. Henderson, Mutat. Res., 62 (1979) 19. E.E. Wright, J.L. Bird and J.M. Feldman, Res. Commun. Chem. Pathol. Pharmacol., 24 (1979) 259. B. Andersson, M. Berggren and P. Moldeus, Drug Metab. Dispos., 6 (1978) 611. C.E. Weeks, W.T. Allaben, S.C. Louie, E.J. Lazear and C.M. King, Cancer Res., 38 (1978) 613.
247
STUDIES ON T H E METABOLIC ACT IV A T IO N OF BENZIDINE BY I S OLATED R A T H E P A T O C Y T E S
R.M.E. BROUNS, R.P. BOS, R. VAN DOORN and P.TH. HENDERSON Institute of Pharmacology and Toxicology, University of Nijmegen, Nijmegen (The Ne the rlands) (Received October 26th, 1981) (Accepted February 7th, 1982)
SUMMARY Isolated rat liver cells were shown t o metabolize the aromatic amine benzidine to reactive products which are mutagenic to S a l m o n e l l a t y p h i m u r i u m TA 1538 and which give rise t o DNA excision repair within the liver cells. Intact rat liver cells are shown to be m ore active in the form at i on of mutagenic metabolites than the 9000-g supernatant from these cells. Data are presented which are in favour of the role of N-acetylation in this respect. F u r t h e r m o r e , indications are presented t hat a sulfation reaction is involved in the generation of DNA m odi f yi ng metabolites, whereas f o r m a t i o n of mutagenic products is likely to proceed via deacetylation and/ or N,Oacyltransfer. Finally, data are given a b o u t the extrahepatocellular appearance of premutagenic metabolites which are more prone to metabolic activation by additional metabolic factors in the Salmonella assay than benzidine itself. The impact of these observations on the estimation of the genotoxic potential o f benzidine will be discussed.
INTRODUCTION The aromatic amine benzidine was epidemiologically shown to be a h u man carcinogen, producing urinary bladder tumors. In experimental animals it also produces tumors, f or example, in the liver of mice and hamsters, and in the liver, intestine and m a m m a r y gland of rats [1]. During the last 2 decades much information has become available a b o u t the mechanisms t h a t can be held responsible for the carcinogenic effects of arylamines and arylamides. An essential initial step in the bioactivation of Abbreviations: DMSO, dimethylsulphoxide, MEM, minimal essential medium; TdR, thymidine.
0300-483Xl8210000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.
23 5
aromatic amines and amides is claimed to be N-hydroxylation, mediated by monooxygenases, mainly localized in the endoplasmic reticulum of the liver [2--4]. For the generation of the ultimate reactive intermediates that bind covalently to cellular constituents and that are believed to represent the actual carcinogens, transferase catalyzed reactions appear to be implicated. In this respect sulfotransferase [ 4 - 6 ] , N,O-acyltransferase [ 7 - 9 ] , and glucuronyltransferase [10] catalyzed conjugation of the N-hydroxy moiety have been postulated. Only recently, in vitro studies showed that metabolic activation of benzidine can proceed via N-acetylation and subsequent N-hydroxylation leading to the formation of N-hydroxy-N,N'-diacetylbenzidine [11]. Furthermore, the role of both sulfotransferase and N,O-acyltransferase in the formation of metabolites leading to adduct formation and mutations have been indicated [11,12]. Recently we showed that the isolated rat hepatocyte, representing a morphological and biochemical entity, is able to metabolize the aromatic amines 2-aminofluorene and 2-acetylaminofluorene to genotoxic products, causing DNA-repair effects within the liver cell and mutations in S. typhimurium [ 13,14]. In the present study we investigated the role of hepatocellular metabolism of benzidine in the formation of reactive metabolites, using DNA-repair effects within the liver cell and bacterial mutagenicity as parameters for genotoxicity. MATERIALS AND METHODS
Chemicals [3H]Thymidine ([3H] TdR), specific activity 23 Ci/mmol was purchased from the Radiochemical Centre (Amersham, England). Collagenase type I (EC 3.4.24.3) was bought from Sigma Chemical Co. (St. Louis, USA). Eagle's minimal essential medium (MEM), including Hank's salts, and foetal calf serum were obtained from Biomed (Gibco). Benzidine, harmine-HC1, salicylamide and 4-aminoazobenzene (= p-phenylazoaniline) were from Aldrich Europe (Beerse, Belgium). All other reagents, obtained from different sources, were of the highest purity obtainable. Isolation of rat liver cells Parenchymal liver cells were isolated from adult male Wistar rats under sterile conditions by means of a collagenase perfusion method [15,16]. Twenty-four hours before the isolation procedure was started the animals were deprived of food. In the final cell preparation more than 95% of the cells were in a viable state as judged by the Trypan Blue exclusion method. Measurement o f hepatocellular DNA-repair Incubation of the hepatocytes, suspended in Eagle's MEM, supplemented with Hank's salts, 10% foetal calf serum, 0.01% streptomycin and 100 U/ml
236
penicillin G with the compounds to be investigated and subsequent measurem e n t of [3H] TdR incorporation into DNA via liquid scintillation counting was essentially as described previously [ 17].
Measurement o f mutagenicity Experiments on mutagenicity were basically performed according to the m e t h o d described by Ames et al. [18]. Salmonella typhimurium TA 1538 was used as bacterial tester strain. Thereto, an overnight nutrient broth culture was centrifuged and the bacterial pellet was resuspended in saline at a concentration of approximately 5 X l 0 s bacteria/ml. The experimental procedure was as follows: a suspension of liver cells in HEPES buffer (Seglen), 2.5 X 106 viable cells/ml, was added to 3 ml of molten topagar, together with the compounds under test and the bacteria, and, when indicated, supplemented with a postmitochondrial fraction from rat liver. In the case of pre-incubation (see below), cell free supernatant was added to the topagar together with the test compounds, bacteria and different subcellular liver fractions as an additional activating system. Detailed experimental conditions are given in the legends. The topagars supplemented with the different components were poured onto minimal agar petri dishes. After 48 h at 37°C the number of histidine prototrophic colonies was counted on each dish.
Pre-incubation o f liver cells Suspensions of freshly isolated hepatocytes, 2.5 X 106 viable cells/ml, were incubated in 50-ml erlenmeyer flasks in a shaking water bath (Infors WTR1) at 37°C together with the c o m p o u n d under test, added as a concentrated solution in dimethylsulphoxide (DMSO). The final a m o u n t of DMSO did not exceed 10 t~l/ml incubation volume. After various time intervals 2-ml aliquots were taken and centrifuged immediately. Next 0.5-ml aliquots of the supernatant were added to the molten topagar together with the other components. Detailed descriptions are given in the legends. RESULTS
Mutagenic effects after activation o f benzidine by hepatocytes and hepatocellular 9000-g supernatant When intact rat liver cells were used as metabolizing system, it was found that benzidine could be activated to metabolites exerting mutagenic effects in S. typhimurium TA 1538. The mutagenicity, as a function of the benzidine concentration, is presented in Fig. 1. When, instead of intact cells, the 9000-g supernatant of the same cells, prepared by ultrasonic treatment as described previously [ 19], was given as a metabolizing component, fortified with an external NADPH regenerating system, mutagenicity was considerably lower.
237
revertants TA1538/prate 200 160
120
/o/e/e
80
/"
40
'
2 0' 0
'
4 60
benzidine
'
cone (,uM)
Fig. 1. Mutagenic activation of benzidine by intact rat liver cells, o - ~ - - o , or 900O-g supernatant from these cells after ultrasonic treatment, ----.---. To 3 ml of molten topagar were added: 0.5 ml cell suspension (2.5 x 104 viable cells/ml) or 0.5 ml 9000-g supernatant, equivalent to 2.5 x 10' cells, fortified with a NADPH regenerating system, consisting of 8 mM MgCl2, 4 mM NADP, 5 mM glucose 6-phosphate and 2.8 U/ml glucose-6-phosphate dehydrogenase, 0.1 ml of bacteria (TA 1538) suspended in saline and 41 ul of different freshly prepared solutions of benzidine in DMSO. Values (revertants/ plate), representing one typical experiment out of at least four, are means of determinations in triplicate.
Effect of modified hepatocellular N-acetylation and sulfation The involvement of N-acetyltransferase and sulfotransferase catalyzed reactions in the metabolic activation of benzidine was demonstrated by Morton et al. [11,12]. It was now studied whether liver cell mediated mutagenicity of benzidine could be affected by modifying these transferase catalyzed reactions. For that purpose harmine was used to inhibit N-acetylation [20], whereas the sulfation reaction was antagonized by salicylamide [21]. The influence of these modifying agents on the activation of benzidine by isolated rat liver cells to mutagenic species is presented in Fig. 2. Mutagenicity was considerably reduced by harmine (500 t~M), whereas inhibition of the sulfation reaction by salicylamide (1 mM) had no influence. Stimulation of the sulfate conjugation even resulted in a marked decrease of mutagenicity. It should be remarked that none of these treatments had any effect on bacterial survival, nor did they reduce hepatocellular viability. It cannot fully be excluded by these experiments that the strong reduction in mutagenicity by harmine is partly due to an interference by this c o m p o u n d with the N-oxidation of benzidine. However, we did not find any inhibitory effect of 0.5 mM harmine on microsomal monooxygenase activity, as measured in vitro with aniline or aminopyrine. On the contrary, it was reported [20] that the N-acetylating capacity in vitro is inhibited by about 90% in the presence of 0.5 mM harmine.
238
rever tants TA153B/prate 160 O
O
120
8O
z~o
r '
:25o
4~o benzidine
'
660
conc (/uM)
Fig. 2. Mutagenic activation of benzidine by intact rat liver cells in the presence of harmine (500 uM), e---o-.; salieylamide (1 mM), o---o---o ; Control, o--o--o. To 3 ml of molten topagar were added: 0.5 ml cell suspension (2.5 x 106 viable cells/ml), 0.1 ml bacteria (TA 1538) suspended in saline and the compounds under test, added as concentrated solutions in ethanol (salicylamide), DMSO (4-aminoazobenzene) or saline (harmineHC1) to reach the final concentrations in the topagar as indicated. Values (revertants/ plate), representing one typical experiment out of at least four, are means of determinations in triplicate.
Influence o f pre-incubation and additional metabolic activation on mutagenic potential of benzidine Besides the observation t h a t upon incubation of benzidine with rat liver cells metabolic products are formed that exert direct mutagenic effects in S. typhimurium, evidence was obtained that liver cell mediated metabolism of benzidine gives rise to the formation of products that are not mutagenic per se but need an additional activation to become mutagenic. The experimental approach was based on a pre-incubation of benzidine with liver cells, whereafter the extracellular medium was subjected .to an additional metabolic activation according to the standard Ames-assay with rat liver 9000-g supernatant. In Fig. 3 the effect of pre-incubation with rat liver cells on the mutagenic activation by rat liver 9000-g supernatant is visualized. Due to pre-incubation with intact cells a 4-fold increase in the a m o u n t of revertant colonies was found. Furthermore, the observed difference in mutagenicity was found to be dependent upon the pre-incubation time, as illustrated in Fig. 4. In order to obtain information a b o u t the nature of the premutagenic products released by the hepatocytes, it was investigated which metabolic factors were required for the additional mutagenic activation. As can be seen from Table I, a combined action of both microsomes and cytosol was essential in this respect, whereas NADPH was also required. These findings suggest the involvement of a microsomal oxidative step, followed by either a sulfotransferase or N,O-acyltransferase catalyzed reaction [11,12] in the
239
revertants 1200
TA 1 5 3 8 / p l . a t e
lOOO
BOO
6OO 4OO
200
400 6~)0 benzidine conc. ()JM)
Fig. 3. Mutagenic activation of benzidine by hepatic 9000-g supernatant after preincubation with, o--c--~, or without, ,--a--o, intact rat liver cells. Benzidine (final conc. on the axis) was pre-incubated either with or without rat liver cells for 60 min as indicated in "Materials and Methods". After removal of the cells by centrifugation, 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar, together with 0.5 ml 9000-g supernatant from liver of phenobarbital pretreated rats (75 mg/kg, i.p. for 5 days), corresponding to 12.5 mg of liver tissue, fortified with a NADPH regenerating system (see legend to Fig. 1), and 0.1 ml of bacteria (TA 1538) suspended in saline. Values (revertants/plate), representing one typical experiment out of at least four, are means oi determinations in triplicate. g e n e r a t i o n o f t h e m u t a g e n i c species. F r o m T a b l e II it can be seen t h a t s a l i c y l a m i d e , a s u b s t r a t e t h a t is k n o w n t o a n t a g o n i z e t h e s u l f a t i o n r e a c t i o n [ 21] had n o i n h i b i t o r y e f f e c t on t h e m u t a g e n i c i t y data, whereas s u p p l e m e n t a t i o n w i t h a P A P S g e n e r a t i n g s y s t e m r e s u l t e d in a s u b s t a n t i a l decrease in m u t a g e n i c i t y . T h e r e f o r e t h e role o f s u l f o t r a n s f e r a s e is n o t resolved. H o w e v e r , 4 - a m i n o a z o b e n z e n e a n d p - a m i n o p h e n o l , c o m p o u n d s t h a t are k n o w n to inhibit t h e N , O - a c y l t r a n s f e r a s e c a t a l y z e d a d d u c t f o r m a t i o n b e t w e e n Nhydroxy-2-acetylaminofluorene and tRNA [ 9], markedly reduced mutagenic i t y , whereas also p a r a o x o n , an i n h i b i t o r of the d e - a c e t y l a t i o n r e a c t i o n , r e d u c e d m u t a g e n i c i t y considerably. I t s h o u l d be n o t e d t h a t these c o m p o u n d s s h o w e d no d e l e t e r i o u s e f f e c t on bacterial viability at the c o n c e n t r a t i o n s e m p l o y e d , w h e r e a s an i n h i b i t o r y e f f e c t on m i c r o s o m a l oxidizing c a p a c i t y a t t h e c o n c e n t r a t i o n s e m p l o y e d was virtually absent. These d a t a are in f a v o u r o f t h e c o n c e p t t h a t during i n c u b a t i o n of b e n z i d i n e w i t h r a t liver cells, a c e t y l a t e d p r o d u c t s are f o r m e d . This is also s u p p o r t e d b y t h e i n h i b i t o r y e f f e c t of h a r m i n e , p r e s e n t d u r i n g p r e - i n c u b a t i o n , o n the u l t i m a t e m u t a g e n i c a c t i v i t y (Table III). F u r t h e r m o r e , it was o b s e r v e d t h a t in a d i r e c t p l a t e assay r a t liver c y t o s o l
240
rever
rant
1200
-
1000
-
800
-
600
-
400
-
200
-
s
TA
1538/prate
--
-
e--
' 4'0 ' 6'0 incubation time (rain) Fig. 4. Mutagenic activation of benzidine by hepatic 9000-g supernatant after various pre-incubation times with, o---~--~, or without, ~--e---~, rat liver cells. Benzidine (500 uM final conc.) was pre-incubated either with or without rat liver cells for various times. Samples were immediately cooled on ice, quickly centrifuged in the cold, after which 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar. The remaining procedure was as described for Fig. 3. Values (revertants/plate), representing one typical experiment out of at least four, are means of determinations in triplicate. '
2()
TABLE I E F F E C T OF D I F F E R E N T POSTMITOCHONDRIAL LIVER FRA CTI O N S ON THE MUTAGENICITY OF BENZIDINE A F T E R PRE-INCUBATION WITH OR WITHOUT INTACT RAT LIVER CELLS Benzidine (500 uM) was pre-incubated with or without rat liver cells for 60 min as described in "Materials and Methods". After centrifugation the cell-free supernatant was tested for mutagenicity as follows: to 3 ml of molten topagar were added 0.5 ml of this supernatant, 0.5 ml of different sub-cellular liver fractions from phenobarbital pretreated rats, corresponding to 12.5 mg of liver tissue, in 0.15 M KC1 (including a NADPH regenerating system when indicated, consisting of 8 mM MgCl:, 4 mM NADP, 5 mM glucose 6-phosphate and 2.8 U/ml glucose-6-phosphate dehydrogenase), and 0.1 ml of bacteria (TA 1538), suspended in saline. Values, representing one typical experiment out of at least four, are means of 3 different determinations. Revertants TA 1538/plate Subcellular liver fraction
Pre-incubation with hepatocytes
Pre-incubation without hepatocytes
None 9000-g supernatant + NADPH 9000-g supernatant -- NADPH Microsomal fraction + NADPH (= A) 105 000-g supernatant (= B) A + B
23 1510 12 160 60 1390
21 161 15 37 35 182
241
TABLE II
EFFECT OF 4-AMINOAZOBENZENE, p-AMINOPHENOL, PARAOXON, S A L I C Y L A M I D E , A N D PAP + pNPS O N T H E M U T A G E N I C A C T I V A T I O N O F B E N Z I D I N E B Y H E P A T I C 9000-g S U P E R N A T A N T AFTER PRE-INCUBATION WITH INTACT RAT LIVER CELLS Benzidine (500 ~M) was pre-incubated with rat liver cells for 60 min as described in "Materials and Methods". After centrifugation, 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar, together with 0.5 ml 9000-g supernatant from liver of phenobarbital pretreated rats, corresponding to 12.5 mg of liver tissue, fortified with a NADPH regenerating system (= S-9 mix), 0.1 ml of bacteria (TA 1538) suspended in saline and the compounds under test, added as concentrated solutions in ethanol (salicylamide), DMSO (4-aminoazobenzene, paraoxon) or saline (p-aminophenol, PAP + pNPS) to reach the final concentrations in the topagar as indicated. Values are means of three different determinations. Addition
Relative mutagenicity (% of control)
None (= control) 4-Aminoazobenzene (50 ~M) 4-Aminoazobenzene (250 uM) p-Aminophenol (100 uM) p-Aminophenol (1 mM) Paraoxon (100 uM) Salicylamide (1 mM) PAP (20 uM) + pNPS (10 raM)
Exp. 1
Exp. 2
100 37 16 69 16 47 121 29
100 44 9
45 107 21
TABLE III E F F E C T OF HARMINE ON THE MUTAGENICITY OF BENZIDINE WHEN PRE-INCUBATED WITH R A T LIVER CELLS Benzidine (500 uM) was pre-incubated with rat liver cells for 60 rain in the presence of harmine. After centrifugation; 0.5 ml of the cell free supernatant was added to 3 ml of molten topagar together with 0.5 ml S-9 mix and 0.1 ml of bacteria (TA 1538) suspended in saline. Values are means of 3 different determinations.
Conc. harmine (uM)
0 (= control) 100 500
242
Relative mutagenicity (% of control) Exp. 1
Exp. 2
100 69 47
100 84 53
alone markedly enhanced the liver cell mediated mutagenicity of benzidine (Fig. 5). Presumably, liver cells can excrete N-hydroxylated metabolite(s) that can be activated into an ultimate mutagenic form through non-oxidative pathways. The absence of an activation by cytosol alone in the case of liquid pre-incubation (Table I) may be a reflection of the hepatocellular formation of rather unstable products that are not detected under pre-incubation conditions.
DNA-excision repair Besides the application of bacterial mutagenicity as a parameter for the extra-hepatocellular appearance of proximate and ultimate mutagenic metabolites of benzidine, DNA-repair was used as an endpoint for genotoxic effects of benzidine within the liver cell. Exposure of the cells to benzidine caused a concentration dependent increase in [ 3 H ] T d R incorporation up to approximately 10 -s M benzidine (Fig. 6). Beyond this concentration an abrupt decline in incorporation was observed, possibly due to an injurious
revertants TA 1538/prate 2802~0 -
/ ~ o
200
/
~o
160 120 80 z~O
'
260
'
,-,oo'
'
6bo
benzidine conc ( ) J M )
Fig. 5. E f f e c t o f r a t liver c y t o s o l ( 1 0 5 0 0 0 g s u p e r n a t a n t ) a d d i t i o n o n t h e m u t a g e n i e a c t i v a t i o n o f b e n z i d i n e b y rat liver cells. T o 3 ml m o l t e n t o p a g a r were a d d e d : 0.5 ml cell s u s p e n s i o n (2.5 X 10 ~ viable cells/ml) or m e r e l y 0.5 ml s u s p e n s i o n b u f f e r , 0.5 ml h e p a t i c c y t o s o l f r o m p h e n o b a r b i t a l p r e t r e a t e d r a t s or 0.5 m l 0 . 1 5 M KCI, 0.1 ml b a c t e r i a (TA 1 5 3 8 ) s u s p e n d e d in saline, a n d 41 ul o f d i f f e r e n t freshly p r e p a r e d s o l u t i o n s of benzidine in DMSO. o---o---~ : liver cells. ~--a--n : h e p a t i c c y t o s o l , t---~--. : liver cells + h e p a t i c c y t o s o l . V a l u e s ( r e v e r t a n t s / p l a t e ) , r e p r e s e n t i n g o n e typical e x p e r i m e n t o u t of at least f o u r , are m e a n s o f d e t e r m i n a t i o n s in triplicate.
243
3 H-TdF~ i n c o r p o r a t i o n (% above control ) 50-
a
b
"/
40-
30
/
i
20
10 i
/
-10 -
-20
-30
i
10 -8
10 -7
I
10-6
i
10-5
i
i0-~
I
I
10-3
10 -7
I
i
10 .6 10 -5 benzidine
~
,
10 -z" 10 -3 conc(M)
Fig. 6. The effect of harmine and salicylamide on the enhanced incorporation of [3H]TdR into hepatocellular DNA, due to benzidine exposure. Liver cells (1 X 106 viable cells/ml) were incubated with different concentrations of benzidine, with or without addition of harmine (100 ~M) or salicylamide (1 raM). (a) incubation with e--e--e, or without, o---~---~,harmine; (b) incubation with e--e--e, or without, o---~--~, salicylamide. e f f e c t on t h e D N A - r e p a i r a p p a r a t u s as such, since cellular viability was u n a f f e c t e d u p t o at least 5 × 10 -4 M b e n z i d i n e . When t h e N - a c e t y l a t i o n and s u l f a t i o n c a p a c i t y of the liver cell were i n h i b i t e d b y h a r m i n e a n d s a l i c y l a m i d e respectively, the e n h a n c e d i n c o r p o r a t i o n o f [3H] T d R was r e d u c e d t o the level o b s e r v e d in the a b s e n c e of benzidine, suggesting t h a t b o t h N - a c e t y l a t i o n a n d sulfate c o n j u g a t i o n are involved in t h e g e n e r a t i o n o f r e a c t i v e m e t a b o l i t e s of b e n z i d i n e , leading to D N A - r e p a i r e f f e c t s w i t h i n t h e liver cell. I t s h o u l d be e m p h a s i z e d t h a t b o t h inhibitors h a d n o e f f e c t on t h e e n h a n c e d i n c o r p o r a t i o n o f [ 3 H ] T d R e v o k e d b y d i m e t h y l n i t r o s a m i n e a n d 4 - n i t r o q u i n o l i n e - l - o x i d e ( d a t a n o t s h o w n ) , thus e x c l u d i n g a deleterious action of harmine and salicylamide on DNA-repair mechanisms o f the liver cell. U n f o r t u n a t e l y we w e r e u n a b l e t o d e t e r m i n e t h e e f f e c t of 4 - a m i n o a z o b e n z e n e , an i n h i b i t o r of t h e N , O - a c y l a t i o n r e a c t i o n in vitro, on t h e b e n z i d i n e e v o k e d h e p a t o c e l l u l a r D N A - r e p a i r , since this c o m p o u n d itself is a c t i v a t e d b y t h e liver cell, t h e r e b y causing a s u b s t a n t i a l increase in [3H]TdR incorporation [ 17].
244
DISCUSSION It is k n o w n from in vitro studies t hat metabolic activation of benzidine can proceed via acetylation and h y d r o x y l a t i o n of the amino function, after which a subsequent N,O-acyltransfer or sulfate conjugation yields the ultimate reactive form [ 1 1 , 1 2 ] . In the present study isolated rat liver cells were f o u n d to be capable in activating benzidine to metabolites causing genotoxic effects both inside and outside the liver cell. Besides the f o r m a t i o n of metabolites that are directly genotoxic, data are presented regarding the extracellular appearance of metabolic products t h a t are n o t genotoxic as such, but can be m ore effectively activated by hepatic 9000-g supernatant than benzidine itself. F r o m our data, together with those obtained from in vitro studies by others, we c o m p o s e d a tentative scheme a b o u t the role of N-acetylation, N - h y d r o x y l a t i o n , N,O-sulfation and N,O-acylation in the metabolic activation of benzidine by the intact rat liver cell (Fig. 7). Being aware of the c o m p l e x i t y of the overall metabolism of benzidine, a t t e n t i o n was focussed
S-9 MIX
IN TA C T H EPATOCY TE
_1
Ar--NH2
1
O H II Ar--N--C--CH 3
0
Ar--NH2
1
0 H II ~_ A t - N - - C--CH a
1
0 II
Ar--~--~--CH O ,
O=S=O I
OH
I
3
Ar--y--C--CH3
/
/
OH
I
\
N,O-ocyltrons-
Ar--~H
ferose - C - - C H 3
o\ / II
OH
r- A r - - N H
I
OH
DNA-adducts ~----- Ar--NH
OH
=-
3
\ N,O-ocyltronsferase - C - C H 3
/
oII
Ar--NH
I
O--C--CH a II 0
O_C_CH 3 II 0 DNA- repair
0 II
Ar-N--C--CH
S.typhTA1538 mutation
Fig. 7. Tentative scheme for the metabolic activation of benzidine by isolated rat hepatocytes.
245
on the involvement of only 1 amino function. Our data are in favour of the liver cell mediated formation of metabolites that are directly mutagenic to S. typhimurium and are presumably formed through N-acetylation, N-Hydroxylation, N,O-acyltransfer and/or deacetylation. It has been reported [22] t h a t in the case of N-hydroxy-2-acetylaminofluorene the N,O-acyltransferase catalyzed mutagenic activation is unlikely to proceed via the formation of a N-acetoxy derivative, in contrast to the formation of reactive metabolites leading to tRNA adducts. In the case of benzidine, however, the inhibitory effect of 4-aminoazobenzene and paminophenol (Table II), compounds that can serve as acceptors for the acyl moiety, suggests that an intramolecular transfer of the acyl group to yield the N-acetoxy derivative may be involved in its mutagenic activation. Whether this type of metabolite can actually be regarded as a mutagen, either directly or after additional transformation, including bacterial activation pathways as suggested by Weeks et al. [22], is still an open question. Furthermore, the inhibitory effect of paraoxon on the observed mutagenicity is in favour of the involvement of a deacetylation reaction in the mutagenic activation of benzidine. With respect to the release of benzidine metabolites from rat liver cells that require additional metabolic factors to become mutagenic, indications are presented t h a t at least 2 types of metabolic products are involved. One is likely to be a N-acetylated product, requiring both a microsomal and a cytosolic factor to become mutagenic, the other is activated by cytosol alone and is presumably a N-acetylated N-hydroxy derivative. Since it was found that the proposed N-acetylated metabolite was considerably more mutagenic than benzidine itself after metabolic activation by rat liver 9000-g supernatant, one may speculate about the impact of this p h e n o m e n o n on the estimation of the genotoxic potential of benzidine. In this connection, the observed increase in mutagenic activity of benzidine after metabolic activation by intact liver cells, compared to the 9000-g supernatant from these cells (Fig. 1), might be an indication of a better Nacetylation capacity of the intact cell. It can be concluded from Fig. 1 that in the case of benzidine, destruction of the biochemical organization of the intact liver cell results in a decreased activating capacity. This observation is not a general p h e n o m e n o n since no mutAgenicity was found in the hepatocyte mediated assay with benzo[a] pyrene [19]. These examples may illustrate the important value of isolated hepatocytes as metabolizing system in the evaluation of the genotoxic potential of premutagenic and precarcinogenic compounds. In terms of the extrahepatic genotoxicity of benzidine, the extrahepatocellular appearance of a Nacetylated N-hydroxy metabolite may also be important, since the enzyme that is proposed to be involved in its mutagenic activation is localized not only in liver but also in various other tissues such as kidney, stomach, small intestine and colon [9]. Our data on hepatocellular DNA-repair are in favour of the role of both N-acetylation and sulfation in the formation of DNA adducts that give rise
246
t o D N A - r e p a i r (Figs. 6a,b). Since i n h i b i t i o n o f the sulfation r e a c t i o n had no e f f e c t o n bacterial m u t a g e n i c i t y o f benzidine, the role of sulfate esters in this r e s p e c t is q u e s t i o n a b l e , p r e s u m a b l y because of their c h e m i c a l reactivity a n d / o r h y d r o p h i l i c properties. F r o m these d i s c r e p a n t findings it m a y be c o n c l u d e d t h a t the same m e t a b o l i c p r o d u c t s o f b e n z i d i n e are n o t necessarily involved in the g e n e r a t i o n o f m u t a g e n i c effects within Salmonella a n d the f o r m a t i o n o f D N A a d d u c t s w i t h i n the liver cell. It remains an intriguing q u e s t i o n w h i c h o f these g e n o t o x i c p a r a m e t e r s is a c t u a l l y related to the f o r m a t i o n o f c a r c i n o g e n i c m e t a b o l i t e s of benzidine by the liver cell. ACKNOWLEDGEMENTS T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e t h e excellent technical assistance o f Mrs. E.W.M. Yih-van de H u r k , Mrs Th. van H o e k - K o n a n d Mr P.J.L. van G e m e r t . Financial s u p p o r t was given b y t h e D u t c h F o u n d a t i o n f o r Medical R e s e a r c h ( F U N G O ) a n d b y t h e L a b o u r I n s p e c t o r a t e , D u t c h Ministry of Social Affairs. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
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