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Oxidation and protein binding of aromatic amines by rat liver microsomes
Chem-Blol Interactions
321
Elsevier Pubhshlng Company, Amsterdam Printed m The Netherlands
O X I D A T I O N A N D P R O T E I N B I N D I N G OF A R O M A T I C A M I N E S BY R A T LIVER MICROSOMES
BRITA BEIJE AND TORE HULTIN Department of Cell Physiology, The Wenner-Gren Institute, Stockholm (Sweden)
(Received November 23rd, 1970) (Revision accepted January 25th, 1971)
SUMMARY The binding of D M A and AF to protein by rat hver rmcrosomes was studied under various conditions and compared with the utilization of D M A for oxidative demethylatlon and N-oxide formation. Accordlng to current evidence only the former reaction involves cytochrome P-450 as oxygen activator. ( I ) When the binding was tested with detoxication lnhlbltors (CO, S K F 525-A, mercurials) known to differentiate between demethylation and N-oxidation, it gave an intermediate or blphaslc response. A similar result was obtained after graded, structural disorganization of the membranes by sonicatlon or cholate treatment While a major part of the binding approximated the demethylatlon in its susceptibility to inhibition, a minor, less well-defined part was more resistant. On the basis of the difference in CO susceptibility, the former type of binding was considered cytochromedependent, whale the latter may include cytochrome-Independent components. (2) The binding of D M A was not in all respects a reflection of the demethylation or N-oxide formation. For instance, the binding of D M A was not inhibited (but in fact stimulated) at high substrate concentrations or after pretreatment of the microsomes with moderate concentrations of trypsin, while the demethylatlon was markedly reduced under these conditions Similarly, the binding was not inhibited in microsomes after preincubation in plain medium, although the N-oxide formation greatly diminished after this treatment. These and other data suggest that the binding was related to intermediates In the oxygenation reactions, rather than to end products The posslblhty is discussed that cytochrome-dependent and cytochrome-independent binding are related to the enzyme-substrate complexes involved in C- and N-oxygenation, respectively Anomalous decomposition of these complexes may lead to a disconnection of bound reaction intermediates in the form of free radicals. (3) Under the conditions studied, the binding of AF was closely correlated with that of DMA. A.bbrevlatlons AF, 2-amlnofluorene; DMA, N-dlmethylandlne Chem-Btol Interactlons, 3 (1971) 321-336
322
BRITA BEIJE, TORE HULTIN
The covalent binding of aromatic amines to liver cell constatuents tn vtvo is generally correlated with the carclnogemc potency of the compounds and the susceptlblhty of the treated ammal to liver tumor Induction 1'2 ( c f refs 3 and 4). The binding is medaated through the detoxlcatlng enzyme system an the liver endoplasmac retlculum 5, but the mechanasm behind the formataon of reactave metabolltes as not clearly understood A slmalar banding can be demonstrated m i,ttro in macrosomal systems in the presence of N A D P H and oxygen 6,~ However, the in v i t r o reaction has a lower degree of chemical and baologacal specificity than the accumulataon of bound amane an long-term feedang experiments 6,7 During extended feedang of carcinogenic aromatic amines to rats there as a progressive ancrease an the amount of bound metabohtes in the liver, culminating after a few weeks I'2. At the same t~me the pattern of oxadatlve metabolism of the compounds shifts towards a higher proportion of N-oxygenation 8 It is generally held that thas metabolic path is specifically assocaated with the accumulative banding and, ultamately, with the carclnogenac process 9. This assumption is corroborated by experiments on animals wath inherent or experimentally induced differences in thear susceptaballty to liver carcanogenesls by aromatac amines In a number of Instances a close correlation has been observed between tumor ancldence, carcinogen banding and the tendency of the anamal to excrete N-oxygenated metabolac products a°-12 A s~milar correlation has been demonstrated by studying the amine oxidation pattern of preconditaoned animals m i'ttro in macrosomal systems, using the noncarclnogenic amine, D M A , as a model substrate j3-~5 In the interpretation of these experiments formaldehyde and N-oxide productaon were used as measures of C- and N-oxygenation, respectively. In vaew of these data ~t seemed desirable to reanvestigate the mechanasm and specaficity of banding of aromatic amanes to protein by asolated hver mlcrosomes The aim of the present experaments was to search for possible correlations between the banding actlvaty of the partacles under various experamental conditions and their activity of Co or N-oxygenation It wall be shown that the binding of D M A to protean was not in all respects correlated wath either oxadative demethylatlon or N-oxidation of this substrate. The experiments suggest that the banding of D M A m vztro is a composite reaction Whale a limited part may be related to N-oxygenataon, as has been postulated for the bandang of carcinogemc amines tn i'tvo, the greater part devmtes from the known forms of N-oxygenation 16,1v an beang dependent on cytochrome P-450 Under the condmons studied, the binding of AF largely paralleled that of D M A MATERIALS AND METHODS
Preparation of mtcrosomes
Male Sprague-Dawley rats (150-200 g) were starved overnaght and sacrificed by decapitation The livers were minced and homogenized in 2 5 vol. of 0 2 M potassaum phosphate buffer (pH 7 5) as recommended by ARRHENIUS14'18 For the preparatlon of mlcrosomes 6-ml samples of the 1 4 0 0 0 × g supernatant fraction were Chem -Bzol Interactions, 3 (1971) 321-336
OXIDATION AND BINDING OF AROMATIC AMINES
323
layered onto 5 ml of 0 3 M sucrose in buffer, and were centrifuged for 55 mm m a Splnco model L-2 centrifuge, rotor No 50, at 165000>'g The mlcrosome pellets were rinsed and suspended m buffer at a protein concentraUon of 10-15 mg/ml. Rough and smooth mlcrosomal subfract~ons were prepared by two different methods, described by FOUTS 19 and by BERGSTRANDAND DALLNER 2°.
Pretreatment of mlcrosomes Pretreatment with sodium cholate ~8,21 was for 1 h (0 °) at the concentraUons indicated m the figure legends For somcatlon successive 2-ml batches of-mlcrosomal suspensions were placed m ice-chilled test tubes and treated for 30 sec m a MSE ultrasomc disintegrator (20 kcycles/sec, 60 W) Samples of the recombined batches were withdrawn at intervals, and the remaining suspensmn was again divided in 2-ml batches before continuing sonlcatlon TM Pretreatment with trypsin was for 10 mm at 35 ° (refs 22 and 23) The reaction was stopped by the addition of a 3-fold excess of soybean trypsin Inhibitor Standard assay system The standard incubation system contained 0 05 m M NADP, 30 or 60 m M nlcotlnamlde, 5 m M glucose-6-phosphate, 0 125 Kornberg umts/ml glucose-6-phosphate dehydrogenase and 0 2 M potassium phosphate buffer (pH 7 5) Unless otherw~se stated, the D M A concentration was 5 m M These conditions were compatible with optimal demethylatlon at the mlcrosome concentration used (1-1 5 mg protein
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Fag 1 T i m e dependence o f protein binding ( L ) , N-oxxdatlon ( O ) a n d oxidative d e m e t h y l a t l o n (11) o f D M A by rat hver mlcrosomes T h e m l c r o s o m e s were prepared m 0 2 M p o t a s s m m p h o s p h a t e buffer (pH 7 5) a n d incubated aerobically at 35 ° In the s a m e buffer containing 0 05 m M N A D P , 30 m M m c o t m a m i d e , 5 m M glucose-6-phosphate, 0 125 K o r n b e r g u m t s glucose-6-phosphate deh y d r o g e n a s e per ml, a n d 5 m ~ / D M A T h e concentration o f m l c r o s o m a l protein was 1 45 m g / m l T h e N - o x l d e / H C H O ratio (O) is indicated on the inner right ordinate Detached, small m a n g l e s indicate binding in the absence o f a d d e d N A D P H - g e n e r a t l n g system (subtracted f r o m curve)
Chem-Btol Interactions, 3 (1971) 321-336
324
BRITA BEIJE~ TORE HULTIN
per ml) ~4. The tubes were incubated aerobically for 40 mln at 35 ° All reactions were essentially linear over this period (Fig. 1). For the determination of oxidative demethylatlon and N-oxide formation the final volumes were 2.0 ml, and the reaction was terminated by the addluon of 1 0 ml 0.9 M perchloric acid. In the binding assay ~4C-labelled DMA (or AF) was used as substrate Final volumes were usually 0 2 ml After incubation, the tubes were returned to the lcebath, and 50-/zl samples were placed on filter paper disks, which were plunged into icecold 1 0 ~ (w/v) tnchloroacetlc acid 24 The binding and oxidation reactions were run in parallel on the same materml The presented data refer to at least 3 independent experiments. Anal)'ttcal methods Formaldehyde was determined by the method of NASH25, as modified by ARRHENIUS 1'* DMA N-oxide was deterrmned as described by ZIEGLER AND PETTIT 21 Protein was measured by the method of LOWRY et al. z6, using bovine serum albumin as a standard. Determmatton of bound ra&oacttve amines The filter paper disks with the radioactive samples were treated for 30 nun with ice-cold 1 0 ~ trlchloroacetlc acid, and then for 15 mln with hot 9 6 ~ ethanol The disks were rinsed in methanol, transferred to a Sohxlet continual extractor, and extracted for 18-20 h with methanol, and for 2 h wlth ether The filter disks were dried and counted m a Packard Tricarb hquld scintillation counter with a counting efficiency for ~4C of 45 ~ . The sclntdlation hquid contained 0 5 ~ (w/v) PPO and 0.015 ~ (w/v) dlmethylPOPOP in toluene Chemtcals Glucose-6-phosphate, NADP, nlcotmamlde, p-hydroxymercuribenzoate, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and trypsin (EC 3 4.4 4, 2 times crystallized) were from the Sigma Chemical C o , St Louis, Mo ; MeHgOH from the Casco AB, Stockholm, Sweden; SKF 525-A from the Smith, Kline & French Labs, Philadelphia, P a , soybean trypsin inhibitor from The Serva Entwicklungslabor, Heidelberg, Germany, PPO and dlmethylPOPOP from the Packard Instrument C o , La Grange, Ill. Uniformly labeled [14C]anxhne and [9-~4C]acetylamlnofluorene were supplied by the Radlochemlcal Centre, Amersham, England, and the NEN Chemicals GmbH, Frankfurt am Main, Germany, respectively From [14C]anlline, ring-labeled [14C]DMA was synthesized by heating with methanol and methyl iodide z7, and diluted w~th carrier to a specific radioactivity of 80-100 mCl/mole From ~4C-labeled acetylamlnofluorene, [~4C]AF was prepared by hydrolysis in 5 M HCI for 2 5 h at 100°, and adjusted to a specific radioactivity of 80 mCl/mole. Chem -Btol Interactions, 3 (1971) 321-336
325
OXIDATION AND BINDING OF AROMATIC AMINES RESULTS
Dependence o f oxidation and bmdmg on enzyme and substrate concentration
In confirmatmn of earher data 18, the rate of D M A demethylatlon by rat hver mlcrosomes culmmated at the fairly low D M A concentration of 5 mM, and then gradually decreased (Fig 2a) As a contrast, the formation of N-oxide increased to a concentration of at least 50 m M 18 The binding of labeled D M A to protein followed the latter kmd of pattern, mcreasmg m a nearly hnear fashion over the range of 1-50 rnM (Fig. 2a). 15C
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Fig 2a Effect o f substrate concentration on protein binding ( A ) , N-oxidation (@) a n d oxidative d e m e t h y l a t l o n (11) o f D M A by rat liver m i c r o s o m e s I n c u b a t i o n was as in Fig 1, b u t wzth 1 14 m g m i c r o s o m a l protein per ml T h e i n c u b a t i o n time was 40 m l n Fig 2b E n z y m e dependence o f protein binding ( A ) , N-oxidation (@) a n d oxidative demethylaUon (11) o f D M A by rat liver m l c r o s o m e s I n c u b a t m n was as in Fig 1, b u t with an i n c u b a t i o n time o f 40 m l n a n d with m l c r o s o m e s a d d e d as Indicated
Fig 2b shows the enzyme dependence of the various reactions The D M A concentration was 5 raM, t e close to the demethylatlon optimum. To facilitate comparison with the experiments just described the final data have been calculated per mg mlcrosomal protein. With increasing concentration of mlcrosomes, both the demethylatlon and binding per mg protein gradually decreased. However, the binding decreased at a considerably more rapid rate. The binding product had not the character of a mlcrosomal enzyme-substrate complex 28'29 This is already indicated by the method ofprocesslng the protein samplefor counting It is further supported by the t~me dependence of the reaction 6,7 and by the appearance of a high proportion of bound amine m the soluble fraction of the incubation system 6 (Table I) Chem-Btol Interactions, 3 (1971) 321-336
326
BRITA BEIJE, TORE HULTIN
TABLE I D I S T R I B U T I O N OF P R O T E I N - B O U N D
DMA
BETWEEN SEDIMENTABLE AND SOLUBLE FRACTIONS
The incubation system was as described In Fig 1, with 5 m M D M A and 1 14 mg mlcrosomal protern per ml. Soluble protein, when added, was taken from the post-microsomal supernatant F r o m the suspensions incubated with [14C]DMA, samples were immediately withdrawn for fixation, while the rest was centrifuged for 60 mln at 150 0 0 0 × g in 0 5-ml tubes The a m o u n t of proteinb o u n d [~4C]DMA was determined separately in the mlcrosomal and soluble fractions The data indicate nmoles of D M A b o u n d or oxidized per mg mlcrosomal protein in the incubation system
Soluble protein (mg/ml)
Binding of [x'*C]DMA Total Mtcrosomal suspension sediment
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HCHO formed
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96 9 88 8
0 62 0 71
The amount of bound amine in the soluble fraction was only moderately increased by the presence of soluble protein as acceptor of reactive material (Table I) This would suggest that a great part of the radioactive protein had been emitted as such from the microsomes Similar binding experiments were carried out with the liver carcinogen, AF. For solubility reasons, lower substrate concentrations had to be used in these experiments. However, the appearance of the substrate and enzyme dependence curves was basically similar to that In the D M A experiments (Figs. 2c and 2d). The above experiments indicate a clear difference between binding and demethylation In enzyme and substrate dependence, and emphasize the importance of a high substrate/enzyme ratio for the binding reaction. 25
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Fig 2c The effect of substrate concentration on protein binding of A F by rat liver mlcrosomes. Experimental conditions were as in Fig 2a, except that [x4C]AF, at the concentrations indicated, was used as substrate The protein concentration was 1 1 mg/ml Fig 2d Enzyme dependence of the protein binding o f A F by rat liver mlcrosomes Experimental conditions were as in Fig 2b, except that 0 05 m M [14C]AF was used as substrate
Chem-Btol Interactions, 3 (1971) 321-336
OXIDATIONAND BINDINGOF AROMATICAMINES
327
Distribution of oxtdlzmg and bmdmg acttwtws between rough and smooth mtcrosomal membranes Two basically different subfractlonatlon methods were used in these experiments 19,2°. They gave essentially the same picture of the distribution of the oxidizing and banding actxvmes between the rough and smooth membranes (Fig. 3). In both cases, but particularly with the preparations according to FOUTS~9, the specific activity of the oxidative demethylatlon of D M A was markedly higher m the smooth than in the rough membrane fraction a9, while the formation of N-oxide was more uniformly distributed. As in the unfractmnated mlcrosomes, the activity of demethylatlon already culminated at a substrate concentration of 5 mM, while the formation of N-oxide showed a continual increase. With both kinds of preparations the activity of N-oxide formation was appreciably higher than in fresh whole mlcrosomes Like the N-oxide formation, the bmdmg of labeled D M A to protein was a b o u t eqmvalent in the two types of submlcrosomal membranes, after due correction for the hlgher proportion of ribosomal protein in the rough fraction ~9,2° (Fig 3) In this case also the results were independent of the method of fractlonatlon Thus, the subfractlonatton data add to the evidence obtained in the previous section of a clear distraction between the demethylatmg and binding activities. Oxldatton and binding by mlcrosomes modified m l'ltro Treatment of rat liver microsomes with ultrasonic vibrations or with cholate progressively reduces their activity of oxidative demethylatton, while the formation of N-oxide first markedly increases, then gradually decreases 18.z~. In the experiments shown m Figs 4 and 5a these characteristic effects were compared with the activity of the same m~crosomes of D M A binding It appears that a great part of the binding was as sensitive to these kinds of structural damage as the demethylatlon, while a quantitatively poorly defined rest fraction seemed to be more resistant. Similar results were obtained on the binding of AF (Fig 5b) Treatment of liver mmcrosomes with trypsin strongly inhibits oxidative demethylatlon under condmons when neither N A D P H - c y t o c h r o m e e reductase nor cytochrome P-450 are noticeably impaired, although the former enzyme is progressively detached from the particles 23. In conformity with the previous experiments, the N-oxide formation was initially stimulated under these conditions (Fig 6) In trypsin experiments, the D M A binding also clearly deviated from the demethylatlon pattern and was nearly unaffected by the treatment Premcubatlon of the microsomes at 35 ° in the absence of N A D P H led to a marked reduction of subsequent N-oxide formation iv, while oxidative demethylatlon was slightly enhanced (Fig 7) This effect was also observed with somcated mlcrosomes (not illustrated), as ff mechanically disorganized membrane sites were pamally restored by the premcubatlon in plato medium Under these conditions the binding activity of the particles was almost unaffected or showed a slight initial stimulation. The presence of N A D P H prevented the seemingly membrane-consohdatlng effects of prelncubatlon 17 (Fig 7) It is concluded that the oxidative demethylatlon of D M A is more dependent on Chem-Btol Interactions, 3 (1971) 321-336
328
BRITA BEIJE, TORE HULTIN
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Fig 3 Distribution of D M A binding and oxadlzmg actwltles between rough (A, C) and smooth (B, D) mlcrosomal subfractlons, prepared by the methods of FOUTS19 (A, B) and BERGSTRANDAND DALLNER2° (C, D) The incubation system was as described in Fig 2a The concentrations ofmlcrosomal protein (mg/ml) were 1 25 (A), 1 06 (B) and 1 44 (C, D) The symbols indicate A , proteinbound [14C]DMA, @, II, D M A N-oxide and HCHO formed, O, N-oxlde/HCHO ratio (right ordinate) Detached, small triangles indicate binding m the absence of added NADPH-generatlng system (subtracted from curves)
Chem-B:ol Interactions, 3 (1971) 321-336
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OXIDATION AND BINDING OF AROMATIC AMINES
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Fig 4 Effect of ultrasomc vibrations on the D M A binding and oxtdlzlng actlvxtles of rat hver m~crosomes The m~crosomes were somcated at 20 kcycles/sec before being added to the incubation system (Fig 2b) at a concentratmn of 1 6 mg protein per ml. The symbols mdacate A, protein-bound [14C]DMA (left ordinate), O, II, DMA N-oxide and HCHO formed (right o r d i n a t e ) O , , N-oxide/ HCHO ratio (tuner side of right ordinate). Detached, small triangles indicate bmdmg m the absence of added NADPH-generatmg system (subtracted from curve)
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Fig. 5. Effect of cholate treatment on the activity of rat liver m~crosomes of binding and oxidizing D M A (A) and of binding A F (B) The mlcrosomes were treated for 1 h (0°) with sodmm cholate at the concentrations indicated, before being added to the mcubaUon system (Fig 2) at a concentraUon of 1.25 (A) and 1 95 (B) mg protein per ml The concentration of D M A (A) was 5 mM, and that of A F (B) 0 05 m M Symbols indicate A, protein-bound [14C]DMA (A, left ordinate) and [t4C]AF (B), 0 , II, DMA N-oxide and HCHO formed (A, right outer ordinate), O, N-oxide/ HCHO ratio (A, right tuner ordinate) Detached, small triangles indicate binding m the absence of added NADPH-generatmg system (subtracted from curves)
Chem-Btol Interactions, 3 (1971) 321-336
330
BRITA BEIJE, TORE HULTIN
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Fig 6 Effect of trypsin on the D M A b i n d i n g a n d oxidizing activities of rat hver m i c r o s o m e s The m i c r o s o m e s were treated for 10 m i n (35 °) with trypsin at the c o n c e n t r a t i o n s indicated, after which trypsin i n h i b i t o r was a d d e d The i n c u b a t i o n system was as described in Fig 2b The m l e r o s o m e c o n c e n t r a t i o n was 1 22 mg p r o t e i n per ml The sym bol s are the s a me as In Fig 4 Fig 7 Effect o f p r e i n c u b a t l o n on D M A b i n d i n g and o x i d a t i o n by rat liver m i c r o s o m e s The microsomes were p r e i n c u b a t e d at 35 °, for the periods indicated, in 0 2 M p o t a s s i u m p h o s p h a t e buffer (pH 7 5) a n d were tested in the s t a n d a r d assay system (Fig 2b) at a c o n c e n t r a t i o n of 1 6 mg p r o t e i n per ml The sy mbols are the same as in Fig 3 The smgle values m a r k e d out with an a r r o w refer to m i c r o s o m e s p r e l n c u b a t e d for 30 mln in a N A D P H - s u p p h e d system
the structural Integrtty of the mlcrosomes than the N-oxide formation 2~. In this respect the binding reactton occupies an intermediate position Depending on the nature and extent of the mlcrosomal dIsorgantzatlon, the binding may be unaffected, more or less inhibited, or even slightly stimulated
Effects of detoxlcatlon mhtbttors on oxidation and bmdmg The demethylanon of aromatic amines by rat liver mlcrosomes ts strongly Inhibited by sulfhydryl reagents 6'Is Under these conditions, the formation of D M A N-oxide passes through an initial phase of stimulation, and ts only inhibited at relatively high concentrations 18 When the binding of D M A to protein was measured m the same system, it followed an intermediate course (Fig 8a) While the sensitivity of one part of the blndmg reaction to p-hydroxymercumbenzoate approached that of the demethylatlon, another part was considerably more resistant, and m this respect more resembled the N-oxide formation. Simdar results were obtamed with M e H g O H , and also when A F (Fig 8b) was used as a substrate Carbon monoxide mterferes with mlcrosomal detoxicatton at the level of cytochrome P-450 It decreases the demethylanon of D M A but not the formation of N-oxide 17. As IS shown by Table II, the binding of D M A to protem was markedly mhlbited by 40% CO, although slgmficantly less than the demethylation. In an analogous experiment with 80% CO, the blndmg was inhibited by 50% and the demethylatlon by 69 % Chem-Blol Interactions, 3 (1971) 321-336
331
OXIDATION AND BINDING OF AROMATIC AMINES
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Fig 8a Effects o f p - h y d r o x y m e r c u r i b e n z o a t e on D M A bi ndi ng a nd oxldaUon I n c u b a t i o n was as in Fig 2b, with a m l c r o s o m e c o n c e n t r a t i o n of 1 22 mg prot e i n per ml, a nd p - h y d r o x y m e r c u r i b e n zoate a d d e d as indicated The same symbols are used as m Fig 4 Fig 8b Effects of M e H g O H on the b i n d i n g of A F I n c u b a t i o n was as in F i g 2d, with a m l c r o s o m e c o n c e n t r a t i o n o f 1 4 m g protein per ml and M e H g O H a dde d as i n d i c a t e d
Like CO, SKF 525-A can be used for dlscrtmmatmg between the demethylation of D M A and the formation of N-oxide 17 In this case also the binding was inhibited, but less than the demethylatlon (Fig. 9) 2 0 100
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Fig 9 Effects of S K F 525-A on D M A binding an d o x i d a t i o n 1ncubatmn was as in Fig 2b, w i t h a m l c r o s o m e c o n c e n t r a t i o n of 1 1 m g p r o t e i n per m l a n d S K F 525-A a d d e d as i ndi c a t e d Symbols, e x p l a i n e d in Fig 3, represent the m e a n values of 3 e x p e r i m e n t s Vertical bars i ndi c a t e 1 S D from the m e a n
Chem-Btol Interacttons, 3 (1971) 321-336
332
BRITA BEIJE, TORE HULTIN
T A B L E II EFFECT OF C O
ON THE BINDING AND OXIDATION OF
DMA
Incubation was as in Fig. 1, with 5 mM DMA and 1 38 mg mlcrogomal protein per ml DMA was plpetted as a separate droplet in the flasks, and was mixed with the rest of the system after a few minutes of gassing The gas p h a s e contained 96 ~ N2 and 4 ~o 02 (controls), or 40 ~o CO, 56 ~o N2 and 4 ~ O2 (CO-treated) Figures indicate nmoles of DMA bound or oxidized per mg mlcrosomal protein, and represent mean values of 4 experiments ± 1 S.D
Control CO-treated Percent inhibition
[I'*C]DMA bound
N-oxtde formed
HCHO formed
N-oxtde/ H CH O
274 ± 1 7 209 ±24 24 ± 3 5
4 7 3 -- 5 8 658 ± 87 None
962-t- 37 484 ±72 49 ± 5
048 ± 005 142 ± 0 4
These experiments indicate t h a t the b i n d i n g o f D M A to p r o t e i n is heterogeneous. W h i l e a m a j o r p a r t o f the b i n d i n g a p p r o x i m a t e l y parallels the oxidative dem e t h y l a t l o n in its susceptibility to d e t o x l c a t m n mhlbltors, the r e m a i n i n g p a r t is m o r e resistant A l t h o u g h thts p a r t is clearly indicated m the present experiments, it remains quantitatively a n d qualitatively less clearly defined DISCUSSION
W h e n a r o m a t i c amines are m e t a b o l i z e d by liver e n d o p l a s m l c m e m b r a n e s in vn,o or m I,ttro, there is a tendency for reactive derivatives to a p p e a r , charactertzed by thelr ability to f o r m covalent b o n d s with protein, R N A a n d other cell constituents l-v. Little is k n o w n at present a b o u t the chemical nature o f the reactive c o m p o u n d s a n d the enzymattc m e c h a m s m s by which they are p r o d u c e d 9.18,30 The zn vttro b i n d i n g has a strict r e q u i r e m e n t for N A D P H a n d oxygen, suggesting that it has basic functions in c o m m o n with the m i x e d oxldase system responsible for the N- a n d C-oxygenation o f a r o m a t i c amines 6,7,21 N - O x y g e n a t m n o f D M A to N - o x i d e m vttro constitutes a f r a g m e n t a r y micros o m a l r e a c t m n m the sense that it takes place without d e m o n s t r a b l e need o f cytoc h r o m e P-450 for oxygen activation 16,17. Some a r g u m e n t s have previously been raised for a preferential involvement o f N - o x y g e n a t i o n in the f o r m a t i o n o f reactive m e t a b ohtes m vitro in the f o r m o f free c a r b o n radicals ls,3° This would be c o m p a t i b l e with current m vtvo d a t a indicating a correlation between the protein binding o f carcmogenlc amines a n d the f o r m a t m n o f N - h y d r o x y l a t e d excretion p r o d u c t s z,s-xz. Alternatively, the a p p e a r a n c e o f reactive metabolltes tn vttro m a y take place at some, s u p p o s e d l y later~S,.stage in the o x i d a t i o n sequence, characterized by a direct involvem e n t o f c y t o c h r o m e P-450. Binding resulting from this type o f r e a c t m n w o u l d be in essential respects c o m p a r a b l e to reactions revolving C-oxygenation, e g . o x i d a t i v e d e m e t h y l a t l o n . The present experiments m a y help to distinguish between these two alternatives. Relationship o f D M A
bindmg to demethylatton and N - o x t d e f o r m a t t o n
The b i n d i n g o f D M A to p r o t e i n b y the m l c r o s o m a l system used in the p r e s e n t Chem-Btol lnteracttons, 3 (1971) 321-336
OXIDATIONAND BINDING OF AROMATICAMINES
333
experiments differed m several respects from the concomitant N-oxide formatmn. (1) It was partially inhibited by CO and S K F 525-A (2) It was more susceptible to graded mlcrosomal damage by ultrasomc wbratlons or detergent (3) It was more readily inhibited by mercurials. (4) It was not repressed when the mlcrosomes were preincubated In plain medmm. In these respects the D M A binding partially resembled the oxidative demethylatlon of the same substrate. However, unlike demethylatlon, the binding was not inhibited (but to the contrary progressively stimulated) by mcreased substrate concentrations Furthermore, it was not markedly reduced by trypsm under conditions when the demethylation was strongly inhibited. It also showed a slight but clear difference from the demethylatlon in its distribution between the two mlcrosomal subunits It should be noted that under the various conditions tested the binding of D M A and A F were closely correlated, although, for solubdlty reasons, the A F binding had to be measured at considerably lower concentrations
Bmdmg mechamsm In the interpretation of these data it may be useful to refer to a tentative scheme for the N- and C-oxygenation of DMA, recently proposed by ARRHENIUS3°. In the formation of N-oxide this scheme postulates an intermediate complex between D M A and a flavoprotem enzyme. Within this complex the D M A nitrogen is adduced to flavoprotein-actlvated oxygen in the form of an ammonium cation radical. In oxidative demethylation a ternary enzymatic complex is postulated involving D M A , flavoprotein and cytochrome P-450. Within this complex the corresponding a m m o n i u m radical is adduced to a cytochrome-actlvated oxygen molecule. This scheme is partially based on HORNER'S31'3z observation that complexes involving a m m o n i u m radical cations are formed between D M A and organic peroxides Such adducts assume the character of carbon radicals by the release of a labillzed hydrogen atom The position of the carbon radical will depend on the constitution and environment of the molecules involved According to ARRHENIUS' concept 18, N-oxygenation is an indication of a disturbed flavoproteln-cytochrome P-450 interaction, and is expected to be the main source of carbon radical emission The present experiments indicate that the binding of D M A to protein m vitro IS a composite reaction: (1) When measured under comparable condmons, the binding was less susceptible to Inhibition by CO or S K F 525-A than oxidative demethylatlon (Table II, Fig. 9) (2) The mercurial inhibition curves were biphaslc, with about the same proportionality between the components as indicated in the experiments just mentioned (Fig 8) (3) Evidence of heterogeneity was also prowded by the experiments on structurally disorganized mlcrosomes (Fig 4) The binding data are consistent with ARRHENIUS' scheme 3°, if it is assumed that free radicals are released tn vitro not only from the binary complex supposed to be mvolved in N-oxygenation 18, but also (and mainly) from the ternary, cytochrome P-450-dependent complex postulated as intermediate in C-oxygenation
Specificity of bmdmg The difference in chemical and biological specificity between the binding of
Chem-Btol Interactions, 3 (1971) 321-336
334
BRITA BEIJE, TORE HULTIN
a r o m a t i c amines m vtvo a n d tn vitro 6"7 IS o f interest m this context A m o n g the m a n i f o l d integrating factors, which m a y c o n t r i b u t e to the greater selectivity tn w v o , the p r o p o r t i o n o f c y t o c h r o m e - l n d e p e n d e n t binding m a y be one o f i m p o r t a n c e . Current evidence suggests t h a t the selective binding o f carcinogenic amines in longt e r m 3 feeding experiments is specifically related to N - h y d r o x y l a t l o n 8'9 This r e a c t i o n (as analyzed m vitro) has been f o u n d CO-resistant 33. It ~s therefore an Interesting possibility that, in c o n t r a s t to the in vitro binding, the selective b l n d l n g tn vtvo is p r e d o m i n a n t l y o f a c y t o c h r o m e - m d e p e n d e n t type Esters 9 (or even m o r e distant derivatives 34) o f N - h y d r o x y l a t e d c a r c m o g e m c amines have been held responsible for the b i n d i n g reaction m vtvo u n d e r c o n d i t i o n s o f e x t e n d e d feeding. By way o f a m i d o m u m ~ons, the esters are s u p p o s e d to f o r m electrophlhc c a r b o n l u m ions by a m e c h a n i s m 9 partmlly a n a l o g o u s to t h a t a l r e a d y discussed ~8,30 Since ester f o r m a t i o n is a p p a r e n t l y n o t involved in the m vitro binding, the present as well as earher 6'7 d a t a p o i n t to a m o r e direct t y p e o f reaction u n d e r these conditions By analogy, it seems an obvious p o s s l b d l t y that the in VlVO b i n d i n g m a y also involve a c a p t u r e o f reactive o x y g e n a t i o n l n t e r m e d m t e s p r i o r to the form a t i o n o f the h y d r o x y l a t e d end p r o d u c t Independence o f b m d m g o f the f o r m a t i o n o f end p r o d u c t s
The c y t o c h r o m e - d e p e n d e n t release o f reactive m e t a b o h t e s m w t r o seems to involve an a n o m a l o u s d e c o m p o s i t i o n o f the e n z y m e - s u b s t r a t e complex at the cytoc h r o m e P-450 level, thereby rendering the overall d e m e t h y l a t t o n system hable to persistent d a m a g e This is suggested by the fact that the increase in binding activity at high D M A c o n c e n t r a t i o n s is associated with a progressive inhibition o f the dem e t h y l a t l o n ~s ( F i g 1). Thus, the c y t o c h r o m e - d e p e n d e n t binding is i n d e p e n d e n t o f the terminal step in the demethylat~on reaction This is further illustrated by the trypsin experiments, which clearly indicate that the f o r m a t i o n o f reactive m e t a b o h t e s m a y continue with great efficiency, a l t h o u g h the d e m e t h y l a t m g function o f the system IS a b o h s h e d (Fig. 6). ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by the Swedish C a n c e r Society O u r t h a n k s are due to Dr. ERIK ARRHENIUS for n u m e r o u s respiring discussions, a n d to Dr. STEN ORRENIUS for help with the C O - i n h i b i t i o n experiments. REFERENCES 1 E C MILLERAND J A MILLER,The presence and significance of bound ammoazodyes in the livers of rats fed p-dlmethylamlnoazobenzene, Cancer Res, 7 (1947) 468-480 2 J M PRICE, E C MILLER,J A MILLER AND G M WEBER, Studies on the mtracellular composition of livers from rats fed various amlnoazodyes, Cancer Res, 9 (1949) 398--402 3 H TERAYAMAAND K KUSAMA,Study on the nature of binding of dlmethylamlnoazobenzene with hver protein of rats fed the carcinogen, Gann, 49 0958) 97-103 4 J J ROBERTSAND G P WARWICK,The covalent binding of metabohtes of dlmethylamlnoazobenzene, fl-naphthylamlne and aniline to nucleic acids in vlvo, Intern J Cancer, 1 (1966) 179-196. Chem-Btol Interactions, 3 (1971) 321-336
OXIDATION AND BINDING OF AROMATIC AMINES 5
335
q" HULTIN, T h e distribution o f p r o t e i n - b o u n d azo dye in subfractlons o f liver c y t o p l a s m fractions,
Exptl Cell R e s , 10 (1956) 697-703 6 7 8
9
10
11
12 13 14 15
16 17 18 19 20 21
22 23 24 25 26 27 28
29
T HULTIN, Reactions o f l'*C-labeled carcinogenic azo dyes with rat liver proteins, Exptl Cell Res, 13 (1957) 47-59 T HULTIN, Reactions o f 2-amlnofluorene a n d related aromatic a m i n e s with liver proteins in vitro, EAptl Cell Res, 18 (1959) 112-125 J W fRAMER,J A MILLER AND E C MILLER, N - h y d r o x y l a t l o n A new m e t a b o h c reaction observed in the rat with the carcinogen 2-acetylammofluorene, J Blol Chem, 235 (1960) 885-888 E C MILLER AND J A MILLER, Stu&es on the m e c h a m s m o f activation o f aromatic a m i n e a n d a m l d e carcinogens to ultimate carcinogenic electrophihc reactants, Ann N Y Acad Scl, 163 (1969) 731-750 A MARGRETH, P D LOTLIKAR, E C MILLER AND J A M~LLER, T h e effects o f hepatotoxac agents a n d o f liver g r o w t h on the urinary excretion o f the N - h y d r o x y m e t a b o h t e o f 2-acetyla m m o f l u o r e n e by rats, Cancer Res, 24 (1964) 920-925 P D LOTLIKAR,M ENEMOTO,E C MILLER AND J A MILLER,T h e effects o f adrenalectomy, h y p o p h y s e c t o m y , a n d castration on the urinary m e t a b o h t e s o f 2-acetylammofluorene in the rat, Cancer Res, 24 (1964) 1835-1844 J A MILLER, J W fRAMER AND E C MILLER,T h e N- a n d rlng-hydroxylatlon o f 2-acetyla m l n o f l u o r e n e during carclnogenesas in the rat, Cancer Res, 20 (1960) 950-962 T HULTIN AND E ARRHENIUS,A dual effect o f carcinogenic amanes on protein m e t a b o l i s m m hver, Advan Enzyme Regulation, 3 (1965) 389-404 E ARRHEN1US, Effects on hepatic m l c r o s o m a l N- a n d C-oxygenation o f aromatic a m i n e s by in vlvo cortlcosterold or a m l n o f l u o r e n e treatment, diet, or stress, Cancer Res, 28 (1968) 264-273 E ARRHENIUS, Correlation o f N- a n d C-oxygenation o f a r o m a t i c a m i n e s with conditions which increase the carclnogemclty o f these c o m p o u n d s , in D SHUCAR ( E d ) , Biochemical Aspects o f Antlmetabohtes and of Drug HydroAylatton, Syrup Fed Europ Btochem Soc , Vol 16, A c a d e m i c Press, L o n d o n , 1959, pp 209-225 H KAMPFFMEYERAND M KIES~, T h e effect o f carbon m o n o x i d e on the hydroxylatlon o f aniline a n d N - e t h y l a n l h n e by m l c r o s o m a l enzymes, Arch Exptl Pathol Pharmacol, 250 (1965) 1-8 D M ZIEOLER ANO F H PETTIT, Mlcrosomal oxadases, I T h e Isolation a n d d m l k y l a r y l a m l n e oxygenase actavaty o f pork liver m I c r o s o m e s , Biochemistry, 5 (1966) 2932-2938 E ARRHENIUS, Effects o f various tn vitro conditions on hepatic mlcrosomal N- a n d C-oxygenation o f aromatic amines, Chem-Btol lnteracnons, 1 (1969/70) 361-380 J R FOUTS, T h e m e t a b o l i s m o f drugs by subtractions o f hepatic macrosomes, Btochem Blophys Res Commun, 6 (1961) 373-378 A BERGSTRAND AND G DALLNER,Isolation o f r o u g h a n d s m o o t h m l c r o s o m e s f r o m rat liver by m e a n s o f a commercially available centrifuge, Anal Btochem, 29 (1969) 351-356 D M ZIEGLER AND F H PETTIT, F o r m a t i o n o f an intermediate N-oxide m the oxidative demethylat~on o f N , N - d l m e t h y l a m h n e catalyzed by hver m l c r o s o m e s , Blochem Blophys Res Commnn, 15 (1964) 188-193 U OSTNER ANn T HULTIN, T h e use o f proteolytlc e n z y m e s m the study o f ribosomal structure Btochtrn Btoph)'s Acta, 154 (1968) 376-387 S ORRENIUS, A BERG AND L ERNSTER, Effects o f trypsin on the electron t r a n s p o r t systems o f laver macrosomes, Fur J Bzochem, 11 (1969) 193-200 R J MANS AND G D NOVELLI, M e a s u r e m e n t o f the incorporation o f ra&oactave a m i n o acids into protein by a filter-paper disk m e t h o d , Arch Blochem Blophys, 94 (1961) 48-53 T NASH, T h e c o l o n m c t n c esttmataon o f formaldehyde by m e a n s o f the H a n t z s c h reaction, Btochem J , 55 (1953) 416-421 O H LOWRY, N J ROSEBROUGH,A L FARR AND R J RANDALL,Protein m e a s u r e m e n t s wath the F o h n phenol reagent, J Blol Chem, 193 (1951) 265-275 A E HOULEHAN,Verfahren zur Darstellung yon N-Alkylderwaten des A n l h n s , Chem Zentr, 93 IV (1922) 375 H REMMER, J SCHENKMAN, R W ESTABRDOK, H SASAME,J GILLETTE, S NARASIMHULU, D Y COOPER AND O ROSENTHAL,D r u g interaction wath hepatac macrosomal cytochrome, Mol Pharmacol, 2 (1966) 187-190 S ORRENIUS AND L ERNSTER,Interaction between hver m~crosomes a n d c o m p o n e n t s capable o f u n d e r g o i n g enzymic hydroxylatlon, Life Scl, 6 (1967) 1473-1482
Chem-Blol Interactions, 3 (1971) 321-336
336 30 31 32
33 34
BRITA BEIJE, TORE HULTIN E ARRHENIUS, Carclnogemc amines Some aspects of their metabolism and interaction with mlcrosomal functions of liver cells, Thesis, Stockholm, 1968 L HORNER, Autooxidation of various orgamc substances, m W O LUNDBERG(Ed), Autooxldatton and Anttoxtdants, Vol l, Intersclence, New York, 1961, pp 379-396 L HORNER AND H. JLrNKERMANN,StudieD zum Ablauf der Substitution, VII Beltrag zum Kmetlschen Nachwels yon Durchgangsradlkalen im System Tertlares Amln-Dlbenzoylperoxyd, Ann Chem, 591 (1955) 53-68 H UEHLEKE,General biological aspects of N-hydroxylatlon, FEBS Symposmm, 16 (1969) 97-107 E C MILLER, D McKECHNIE, M M POIRIER AND J A MILLER, Inhibition of amino acid incorporation tn vitro by metabohtes of 2-acetylammofluorene and by certain nltroso compounds, Proc Soc Exptl Btol Med, 120 (1965)538-541
Chem-Blol Interactions, 3 (1971) 321-336
321
Elsevier Pubhshlng Company, Amsterdam Printed m The Netherlands
O X I D A T I O N A N D P R O T E I N B I N D I N G OF A R O M A T I C A M I N E S BY R A T LIVER MICROSOMES
BRITA BEIJE AND TORE HULTIN Department of Cell Physiology, The Wenner-Gren Institute, Stockholm (Sweden)
(Received November 23rd, 1970) (Revision accepted January 25th, 1971)
SUMMARY The binding of D M A and AF to protein by rat hver rmcrosomes was studied under various conditions and compared with the utilization of D M A for oxidative demethylatlon and N-oxide formation. Accordlng to current evidence only the former reaction involves cytochrome P-450 as oxygen activator. ( I ) When the binding was tested with detoxication lnhlbltors (CO, S K F 525-A, mercurials) known to differentiate between demethylation and N-oxidation, it gave an intermediate or blphaslc response. A similar result was obtained after graded, structural disorganization of the membranes by sonicatlon or cholate treatment While a major part of the binding approximated the demethylatlon in its susceptibility to inhibition, a minor, less well-defined part was more resistant. On the basis of the difference in CO susceptibility, the former type of binding was considered cytochromedependent, whale the latter may include cytochrome-Independent components. (2) The binding of D M A was not in all respects a reflection of the demethylation or N-oxide formation. For instance, the binding of D M A was not inhibited (but in fact stimulated) at high substrate concentrations or after pretreatment of the microsomes with moderate concentrations of trypsin, while the demethylatlon was markedly reduced under these conditions Similarly, the binding was not inhibited in microsomes after preincubation in plain medium, although the N-oxide formation greatly diminished after this treatment. These and other data suggest that the binding was related to intermediates In the oxygenation reactions, rather than to end products The posslblhty is discussed that cytochrome-dependent and cytochrome-independent binding are related to the enzyme-substrate complexes involved in C- and N-oxygenation, respectively Anomalous decomposition of these complexes may lead to a disconnection of bound reaction intermediates in the form of free radicals. (3) Under the conditions studied, the binding of AF was closely correlated with that of DMA. A.bbrevlatlons AF, 2-amlnofluorene; DMA, N-dlmethylandlne Chem-Btol Interactlons, 3 (1971) 321-336
322
BRITA BEIJE, TORE HULTIN
The covalent binding of aromatic amines to liver cell constatuents tn vtvo is generally correlated with the carclnogemc potency of the compounds and the susceptlblhty of the treated ammal to liver tumor Induction 1'2 ( c f refs 3 and 4). The binding is medaated through the detoxlcatlng enzyme system an the liver endoplasmac retlculum 5, but the mechanasm behind the formataon of reactave metabolltes as not clearly understood A slmalar banding can be demonstrated m i,ttro in macrosomal systems in the presence of N A D P H and oxygen 6,~ However, the in v i t r o reaction has a lower degree of chemical and baologacal specificity than the accumulataon of bound amane an long-term feedang experiments 6,7 During extended feedang of carcinogenic aromatic amines to rats there as a progressive ancrease an the amount of bound metabohtes in the liver, culminating after a few weeks I'2. At the same t~me the pattern of oxadatlve metabolism of the compounds shifts towards a higher proportion of N-oxygenation 8 It is generally held that thas metabolic path is specifically assocaated with the accumulative banding and, ultamately, with the carclnogenac process 9. This assumption is corroborated by experiments on animals wath inherent or experimentally induced differences in thear susceptaballty to liver carcanogenesls by aromatac amines In a number of Instances a close correlation has been observed between tumor ancldence, carcinogen banding and the tendency of the anamal to excrete N-oxygenated metabolac products a°-12 A s~milar correlation has been demonstrated by studying the amine oxidation pattern of preconditaoned animals m i'ttro in macrosomal systems, using the noncarclnogenic amine, D M A , as a model substrate j3-~5 In the interpretation of these experiments formaldehyde and N-oxide productaon were used as measures of C- and N-oxygenation, respectively. In vaew of these data ~t seemed desirable to reanvestigate the mechanasm and specaficity of banding of aromatic amanes to protein by asolated hver mlcrosomes The aim of the present experaments was to search for possible correlations between the banding actlvaty of the partacles under various experamental conditions and their activity of Co or N-oxygenation It wall be shown that the binding of D M A to protean was not in all respects correlated wath either oxadative demethylatlon or N-oxidation of this substrate. The experiments suggest that the banding of D M A m vztro is a composite reaction Whale a limited part may be related to N-oxygenataon, as has been postulated for the bandang of carcinogemc amines tn i'tvo, the greater part devmtes from the known forms of N-oxygenation 16,1v an beang dependent on cytochrome P-450 Under the condmons studied, the binding of AF largely paralleled that of D M A MATERIALS AND METHODS
Preparation of mtcrosomes
Male Sprague-Dawley rats (150-200 g) were starved overnaght and sacrificed by decapitation The livers were minced and homogenized in 2 5 vol. of 0 2 M potassaum phosphate buffer (pH 7 5) as recommended by ARRHENIUS14'18 For the preparatlon of mlcrosomes 6-ml samples of the 1 4 0 0 0 × g supernatant fraction were Chem -Bzol Interactions, 3 (1971) 321-336
OXIDATION AND BINDING OF AROMATIC AMINES
323
layered onto 5 ml of 0 3 M sucrose in buffer, and were centrifuged for 55 mm m a Splnco model L-2 centrifuge, rotor No 50, at 165000>'g The mlcrosome pellets were rinsed and suspended m buffer at a protein concentraUon of 10-15 mg/ml. Rough and smooth mlcrosomal subfract~ons were prepared by two different methods, described by FOUTS 19 and by BERGSTRANDAND DALLNER 2°.
Pretreatment of mlcrosomes Pretreatment with sodium cholate ~8,21 was for 1 h (0 °) at the concentraUons indicated m the figure legends For somcatlon successive 2-ml batches of-mlcrosomal suspensions were placed m ice-chilled test tubes and treated for 30 sec m a MSE ultrasomc disintegrator (20 kcycles/sec, 60 W) Samples of the recombined batches were withdrawn at intervals, and the remaining suspensmn was again divided in 2-ml batches before continuing sonlcatlon TM Pretreatment with trypsin was for 10 mm at 35 ° (refs 22 and 23) The reaction was stopped by the addition of a 3-fold excess of soybean trypsin Inhibitor Standard assay system The standard incubation system contained 0 05 m M NADP, 30 or 60 m M nlcotlnamlde, 5 m M glucose-6-phosphate, 0 125 Kornberg umts/ml glucose-6-phosphate dehydrogenase and 0 2 M potassium phosphate buffer (pH 7 5) Unless otherw~se stated, the D M A concentration was 5 m M These conditions were compatible with optimal demethylatlon at the mlcrosome concentration used (1-1 5 mg protein
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Fag 1 T i m e dependence o f protein binding ( L ) , N-oxxdatlon ( O ) a n d oxidative d e m e t h y l a t l o n (11) o f D M A by rat hver mlcrosomes T h e m l c r o s o m e s were prepared m 0 2 M p o t a s s m m p h o s p h a t e buffer (pH 7 5) a n d incubated aerobically at 35 ° In the s a m e buffer containing 0 05 m M N A D P , 30 m M m c o t m a m i d e , 5 m M glucose-6-phosphate, 0 125 K o r n b e r g u m t s glucose-6-phosphate deh y d r o g e n a s e per ml, a n d 5 m ~ / D M A T h e concentration o f m l c r o s o m a l protein was 1 45 m g / m l T h e N - o x l d e / H C H O ratio (O) is indicated on the inner right ordinate Detached, small m a n g l e s indicate binding in the absence o f a d d e d N A D P H - g e n e r a t l n g system (subtracted f r o m curve)
Chem-Btol Interactions, 3 (1971) 321-336
324
BRITA BEIJE~ TORE HULTIN
per ml) ~4. The tubes were incubated aerobically for 40 mln at 35 ° All reactions were essentially linear over this period (Fig. 1). For the determination of oxidative demethylatlon and N-oxide formation the final volumes were 2.0 ml, and the reaction was terminated by the addluon of 1 0 ml 0.9 M perchloric acid. In the binding assay ~4C-labelled DMA (or AF) was used as substrate Final volumes were usually 0 2 ml After incubation, the tubes were returned to the lcebath, and 50-/zl samples were placed on filter paper disks, which were plunged into icecold 1 0 ~ (w/v) tnchloroacetlc acid 24 The binding and oxidation reactions were run in parallel on the same materml The presented data refer to at least 3 independent experiments. Anal)'ttcal methods Formaldehyde was determined by the method of NASH25, as modified by ARRHENIUS 1'* DMA N-oxide was deterrmned as described by ZIEGLER AND PETTIT 21 Protein was measured by the method of LOWRY et al. z6, using bovine serum albumin as a standard. Determmatton of bound ra&oacttve amines The filter paper disks with the radioactive samples were treated for 30 nun with ice-cold 1 0 ~ trlchloroacetlc acid, and then for 15 mln with hot 9 6 ~ ethanol The disks were rinsed in methanol, transferred to a Sohxlet continual extractor, and extracted for 18-20 h with methanol, and for 2 h wlth ether The filter disks were dried and counted m a Packard Tricarb hquld scintillation counter with a counting efficiency for ~4C of 45 ~ . The sclntdlation hquid contained 0 5 ~ (w/v) PPO and 0.015 ~ (w/v) dlmethylPOPOP in toluene Chemtcals Glucose-6-phosphate, NADP, nlcotmamlde, p-hydroxymercuribenzoate, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and trypsin (EC 3 4.4 4, 2 times crystallized) were from the Sigma Chemical C o , St Louis, Mo ; MeHgOH from the Casco AB, Stockholm, Sweden; SKF 525-A from the Smith, Kline & French Labs, Philadelphia, P a , soybean trypsin inhibitor from The Serva Entwicklungslabor, Heidelberg, Germany, PPO and dlmethylPOPOP from the Packard Instrument C o , La Grange, Ill. Uniformly labeled [14C]anxhne and [9-~4C]acetylamlnofluorene were supplied by the Radlochemlcal Centre, Amersham, England, and the NEN Chemicals GmbH, Frankfurt am Main, Germany, respectively From [14C]anlline, ring-labeled [14C]DMA was synthesized by heating with methanol and methyl iodide z7, and diluted w~th carrier to a specific radioactivity of 80-100 mCl/mole From ~4C-labeled acetylamlnofluorene, [~4C]AF was prepared by hydrolysis in 5 M HCI for 2 5 h at 100°, and adjusted to a specific radioactivity of 80 mCl/mole. Chem -Btol Interactions, 3 (1971) 321-336
325
OXIDATION AND BINDING OF AROMATIC AMINES RESULTS
Dependence o f oxidation and bmdmg on enzyme and substrate concentration
In confirmatmn of earher data 18, the rate of D M A demethylatlon by rat hver mlcrosomes culmmated at the fairly low D M A concentration of 5 mM, and then gradually decreased (Fig 2a) As a contrast, the formation of N-oxide increased to a concentration of at least 50 m M 18 The binding of labeled D M A to protein followed the latter kmd of pattern, mcreasmg m a nearly hnear fashion over the range of 1-50 rnM (Fig. 2a). 15C
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Fig 2b shows the enzyme dependence of the various reactions The D M A concentration was 5 raM, t e close to the demethylatlon optimum. To facilitate comparison with the experiments just described the final data have been calculated per mg mlcrosomal protein. With increasing concentration of mlcrosomes, both the demethylatlon and binding per mg protein gradually decreased. However, the binding decreased at a considerably more rapid rate. The binding product had not the character of a mlcrosomal enzyme-substrate complex 28'29 This is already indicated by the method ofprocesslng the protein samplefor counting It is further supported by the t~me dependence of the reaction 6,7 and by the appearance of a high proportion of bound amine m the soluble fraction of the incubation system 6 (Table I) Chem-Btol Interactions, 3 (1971) 321-336
326
BRITA BEIJE, TORE HULTIN
TABLE I D I S T R I B U T I O N OF P R O T E I N - B O U N D
DMA
BETWEEN SEDIMENTABLE AND SOLUBLE FRACTIONS
The incubation system was as described In Fig 1, with 5 m M D M A and 1 14 mg mlcrosomal protern per ml. Soluble protein, when added, was taken from the post-microsomal supernatant F r o m the suspensions incubated with [14C]DMA, samples were immediately withdrawn for fixation, while the rest was centrifuged for 60 mln at 150 0 0 0 × g in 0 5-ml tubes The a m o u n t of proteinb o u n d [~4C]DMA was determined separately in the mlcrosomal and soluble fractions The data indicate nmoles of D M A b o u n d or oxidized per mg mlcrosomal protein in the incubation system
Soluble protein (mg/ml)
Binding of [x'*C]DMA Total Mtcrosomal suspension sediment
Soluble fraction
None 1 31
28 6 35 1
12 0 17.5
17 7 18 3
N-oxide formed
HCHO formed
N-oxide~ HCHO
60 1 63 4
96 9 88 8
0 62 0 71
The amount of bound amine in the soluble fraction was only moderately increased by the presence of soluble protein as acceptor of reactive material (Table I) This would suggest that a great part of the radioactive protein had been emitted as such from the microsomes Similar binding experiments were carried out with the liver carcinogen, AF. For solubility reasons, lower substrate concentrations had to be used in these experiments. However, the appearance of the substrate and enzyme dependence curves was basically similar to that In the D M A experiments (Figs. 2c and 2d). The above experiments indicate a clear difference between binding and demethylation In enzyme and substrate dependence, and emphasize the importance of a high substrate/enzyme ratio for the binding reaction. 25
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Fig 2c The effect of substrate concentration on protein binding of A F by rat liver mlcrosomes. Experimental conditions were as in Fig 2a, except that [x4C]AF, at the concentrations indicated, was used as substrate The protein concentration was 1 1 mg/ml Fig 2d Enzyme dependence of the protein binding o f A F by rat liver mlcrosomes Experimental conditions were as in Fig 2b, except that 0 05 m M [14C]AF was used as substrate
Chem-Btol Interactions, 3 (1971) 321-336
OXIDATIONAND BINDINGOF AROMATICAMINES
327
Distribution of oxtdlzmg and bmdmg acttwtws between rough and smooth mtcrosomal membranes Two basically different subfractlonatlon methods were used in these experiments 19,2°. They gave essentially the same picture of the distribution of the oxidizing and banding actxvmes between the rough and smooth membranes (Fig. 3). In both cases, but particularly with the preparations according to FOUTS~9, the specific activity of the oxidative demethylatlon of D M A was markedly higher m the smooth than in the rough membrane fraction a9, while the formation of N-oxide was more uniformly distributed. As in the unfractmnated mlcrosomes, the activity of demethylatlon already culminated at a substrate concentration of 5 mM, while the formation of N-oxide showed a continual increase. With both kinds of preparations the activity of N-oxide formation was appreciably higher than in fresh whole mlcrosomes Like the N-oxide formation, the bmdmg of labeled D M A to protein was a b o u t eqmvalent in the two types of submlcrosomal membranes, after due correction for the hlgher proportion of ribosomal protein in the rough fraction ~9,2° (Fig 3) In this case also the results were independent of the method of fractlonatlon Thus, the subfractlonatton data add to the evidence obtained in the previous section of a clear distraction between the demethylatmg and binding activities. Oxldatton and binding by mlcrosomes modified m l'ltro Treatment of rat liver microsomes with ultrasonic vibrations or with cholate progressively reduces their activity of oxidative demethylatton, while the formation of N-oxide first markedly increases, then gradually decreases 18.z~. In the experiments shown m Figs 4 and 5a these characteristic effects were compared with the activity of the same m~crosomes of D M A binding It appears that a great part of the binding was as sensitive to these kinds of structural damage as the demethylatlon, while a quantitatively poorly defined rest fraction seemed to be more resistant. Similar results were obtained on the binding of AF (Fig 5b) Treatment of liver mmcrosomes with trypsin strongly inhibits oxidative demethylatlon under condmons when neither N A D P H - c y t o c h r o m e e reductase nor cytochrome P-450 are noticeably impaired, although the former enzyme is progressively detached from the particles 23. In conformity with the previous experiments, the N-oxide formation was initially stimulated under these conditions (Fig 6) In trypsin experiments, the D M A binding also clearly deviated from the demethylatlon pattern and was nearly unaffected by the treatment Premcubatlon of the microsomes at 35 ° in the absence of N A D P H led to a marked reduction of subsequent N-oxide formation iv, while oxidative demethylatlon was slightly enhanced (Fig 7) This effect was also observed with somcated mlcrosomes (not illustrated), as ff mechanically disorganized membrane sites were pamally restored by the premcubatlon in plato medium Under these conditions the binding activity of the particles was almost unaffected or showed a slight initial stimulation. The presence of N A D P H prevented the seemingly membrane-consohdatlng effects of prelncubatlon 17 (Fig 7) It is concluded that the oxidative demethylatlon of D M A is more dependent on Chem-Btol Interactions, 3 (1971) 321-336
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Chem-B:ol Interactions, 3 (1971) 321-336
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OXIDATION AND BINDING OF AROMATIC AMINES
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Fig. 5. Effect of cholate treatment on the activity of rat liver m~crosomes of binding and oxidizing D M A (A) and of binding A F (B) The mlcrosomes were treated for 1 h (0°) with sodmm cholate at the concentrations indicated, before being added to the mcubaUon system (Fig 2) at a concentraUon of 1.25 (A) and 1 95 (B) mg protein per ml The concentration of D M A (A) was 5 mM, and that of A F (B) 0 05 m M Symbols indicate A, protein-bound [14C]DMA (A, left ordinate) and [t4C]AF (B), 0 , II, DMA N-oxide and HCHO formed (A, right outer ordinate), O, N-oxide/ HCHO ratio (A, right tuner ordinate) Detached, small triangles indicate binding m the absence of added NADPH-generatmg system (subtracted from curves)
Chem-Btol Interactions, 3 (1971) 321-336
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BRITA BEIJE, TORE HULTIN
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Fig 6 Effect of trypsin on the D M A b i n d i n g a n d oxidizing activities of rat hver m i c r o s o m e s The m i c r o s o m e s were treated for 10 m i n (35 °) with trypsin at the c o n c e n t r a t i o n s indicated, after which trypsin i n h i b i t o r was a d d e d The i n c u b a t i o n system was as described in Fig 2b The m l e r o s o m e c o n c e n t r a t i o n was 1 22 mg p r o t e i n per ml The sym bol s are the s a me as In Fig 4 Fig 7 Effect o f p r e i n c u b a t l o n on D M A b i n d i n g and o x i d a t i o n by rat liver m i c r o s o m e s The microsomes were p r e i n c u b a t e d at 35 °, for the periods indicated, in 0 2 M p o t a s s i u m p h o s p h a t e buffer (pH 7 5) a n d were tested in the s t a n d a r d assay system (Fig 2b) at a c o n c e n t r a t i o n of 1 6 mg p r o t e i n per ml The sy mbols are the same as in Fig 3 The smgle values m a r k e d out with an a r r o w refer to m i c r o s o m e s p r e l n c u b a t e d for 30 mln in a N A D P H - s u p p h e d system
the structural Integrtty of the mlcrosomes than the N-oxide formation 2~. In this respect the binding reactton occupies an intermediate position Depending on the nature and extent of the mlcrosomal dIsorgantzatlon, the binding may be unaffected, more or less inhibited, or even slightly stimulated
Effects of detoxlcatlon mhtbttors on oxidation and bmdmg The demethylanon of aromatic amines by rat liver mlcrosomes ts strongly Inhibited by sulfhydryl reagents 6'Is Under these conditions, the formation of D M A N-oxide passes through an initial phase of stimulation, and ts only inhibited at relatively high concentrations 18 When the binding of D M A to protein was measured m the same system, it followed an intermediate course (Fig 8a) While the sensitivity of one part of the blndmg reaction to p-hydroxymercumbenzoate approached that of the demethylatlon, another part was considerably more resistant, and m this respect more resembled the N-oxide formation. Simdar results were obtamed with M e H g O H , and also when A F (Fig 8b) was used as a substrate Carbon monoxide mterferes with mlcrosomal detoxicatton at the level of cytochrome P-450 It decreases the demethylanon of D M A but not the formation of N-oxide 17. As IS shown by Table II, the binding of D M A to protem was markedly mhlbited by 40% CO, although slgmficantly less than the demethylation. In an analogous experiment with 80% CO, the blndmg was inhibited by 50% and the demethylatlon by 69 % Chem-Blol Interactions, 3 (1971) 321-336
331
OXIDATION AND BINDING OF AROMATIC AMINES
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Fig 8a Effects o f p - h y d r o x y m e r c u r i b e n z o a t e on D M A bi ndi ng a nd oxldaUon I n c u b a t i o n was as in Fig 2b, with a m l c r o s o m e c o n c e n t r a t i o n of 1 22 mg prot e i n per ml, a nd p - h y d r o x y m e r c u r i b e n zoate a d d e d as indicated The same symbols are used as m Fig 4 Fig 8b Effects of M e H g O H on the b i n d i n g of A F I n c u b a t i o n was as in F i g 2d, with a m l c r o s o m e c o n c e n t r a t i o n o f 1 4 m g protein per ml and M e H g O H a dde d as i n d i c a t e d
Like CO, SKF 525-A can be used for dlscrtmmatmg between the demethylation of D M A and the formation of N-oxide 17 In this case also the binding was inhibited, but less than the demethylatlon (Fig. 9) 2 0 100
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Fig 9 Effects of S K F 525-A on D M A binding an d o x i d a t i o n 1ncubatmn was as in Fig 2b, w i t h a m l c r o s o m e c o n c e n t r a t i o n of 1 1 m g p r o t e i n per m l a n d S K F 525-A a d d e d as i ndi c a t e d Symbols, e x p l a i n e d in Fig 3, represent the m e a n values of 3 e x p e r i m e n t s Vertical bars i ndi c a t e 1 S D from the m e a n
Chem-Btol Interacttons, 3 (1971) 321-336
332
BRITA BEIJE, TORE HULTIN
T A B L E II EFFECT OF C O
ON THE BINDING AND OXIDATION OF
DMA
Incubation was as in Fig. 1, with 5 mM DMA and 1 38 mg mlcrogomal protein per ml DMA was plpetted as a separate droplet in the flasks, and was mixed with the rest of the system after a few minutes of gassing The gas p h a s e contained 96 ~ N2 and 4 ~o 02 (controls), or 40 ~o CO, 56 ~o N2 and 4 ~ O2 (CO-treated) Figures indicate nmoles of DMA bound or oxidized per mg mlcrosomal protein, and represent mean values of 4 experiments ± 1 S.D
Control CO-treated Percent inhibition
[I'*C]DMA bound
N-oxtde formed
HCHO formed
N-oxtde/ H CH O
274 ± 1 7 209 ±24 24 ± 3 5
4 7 3 -- 5 8 658 ± 87 None
962-t- 37 484 ±72 49 ± 5
048 ± 005 142 ± 0 4
These experiments indicate t h a t the b i n d i n g o f D M A to p r o t e i n is heterogeneous. W h i l e a m a j o r p a r t o f the b i n d i n g a p p r o x i m a t e l y parallels the oxidative dem e t h y l a t l o n in its susceptibility to d e t o x l c a t m n mhlbltors, the r e m a i n i n g p a r t is m o r e resistant A l t h o u g h thts p a r t is clearly indicated m the present experiments, it remains quantitatively a n d qualitatively less clearly defined DISCUSSION
W h e n a r o m a t i c amines are m e t a b o l i z e d by liver e n d o p l a s m l c m e m b r a n e s in vn,o or m I,ttro, there is a tendency for reactive derivatives to a p p e a r , charactertzed by thelr ability to f o r m covalent b o n d s with protein, R N A a n d other cell constituents l-v. Little is k n o w n at present a b o u t the chemical nature o f the reactive c o m p o u n d s a n d the enzymattc m e c h a m s m s by which they are p r o d u c e d 9.18,30 The zn vttro b i n d i n g has a strict r e q u i r e m e n t for N A D P H a n d oxygen, suggesting that it has basic functions in c o m m o n with the m i x e d oxldase system responsible for the N- a n d C-oxygenation o f a r o m a t i c amines 6,7,21 N - O x y g e n a t m n o f D M A to N - o x i d e m vttro constitutes a f r a g m e n t a r y micros o m a l r e a c t m n m the sense that it takes place without d e m o n s t r a b l e need o f cytoc h r o m e P-450 for oxygen activation 16,17. Some a r g u m e n t s have previously been raised for a preferential involvement o f N - o x y g e n a t i o n in the f o r m a t i o n o f reactive m e t a b ohtes m vitro in the f o r m o f free c a r b o n radicals ls,3° This would be c o m p a t i b l e with current m vtvo d a t a indicating a correlation between the protein binding o f carcmogenlc amines a n d the f o r m a t m n o f N - h y d r o x y l a t e d excretion p r o d u c t s z,s-xz. Alternatively, the a p p e a r a n c e o f reactive metabolltes tn vttro m a y take place at some, s u p p o s e d l y later~S,.stage in the o x i d a t i o n sequence, characterized by a direct involvem e n t o f c y t o c h r o m e P-450. Binding resulting from this type o f r e a c t m n w o u l d be in essential respects c o m p a r a b l e to reactions revolving C-oxygenation, e g . o x i d a t i v e d e m e t h y l a t l o n . The present experiments m a y help to distinguish between these two alternatives. Relationship o f D M A
bindmg to demethylatton and N - o x t d e f o r m a t t o n
The b i n d i n g o f D M A to p r o t e i n b y the m l c r o s o m a l system used in the p r e s e n t Chem-Btol lnteracttons, 3 (1971) 321-336
OXIDATIONAND BINDING OF AROMATICAMINES
333
experiments differed m several respects from the concomitant N-oxide formatmn. (1) It was partially inhibited by CO and S K F 525-A (2) It was more susceptible to graded mlcrosomal damage by ultrasomc wbratlons or detergent (3) It was more readily inhibited by mercurials. (4) It was not repressed when the mlcrosomes were preincubated In plain medmm. In these respects the D M A binding partially resembled the oxidative demethylatlon of the same substrate. However, unlike demethylatlon, the binding was not inhibited (but to the contrary progressively stimulated) by mcreased substrate concentrations Furthermore, it was not markedly reduced by trypsm under conditions when the demethylation was strongly inhibited. It also showed a slight but clear difference from the demethylatlon in its distribution between the two mlcrosomal subunits It should be noted that under the various conditions tested the binding of D M A and A F were closely correlated, although, for solubdlty reasons, the A F binding had to be measured at considerably lower concentrations
Bmdmg mechamsm In the interpretation of these data it may be useful to refer to a tentative scheme for the N- and C-oxygenation of DMA, recently proposed by ARRHENIUS3°. In the formation of N-oxide this scheme postulates an intermediate complex between D M A and a flavoprotem enzyme. Within this complex the D M A nitrogen is adduced to flavoprotein-actlvated oxygen in the form of an ammonium cation radical. In oxidative demethylation a ternary enzymatic complex is postulated involving D M A , flavoprotein and cytochrome P-450. Within this complex the corresponding a m m o n i u m radical is adduced to a cytochrome-actlvated oxygen molecule. This scheme is partially based on HORNER'S31'3z observation that complexes involving a m m o n i u m radical cations are formed between D M A and organic peroxides Such adducts assume the character of carbon radicals by the release of a labillzed hydrogen atom The position of the carbon radical will depend on the constitution and environment of the molecules involved According to ARRHENIUS' concept 18, N-oxygenation is an indication of a disturbed flavoproteln-cytochrome P-450 interaction, and is expected to be the main source of carbon radical emission The present experiments indicate that the binding of D M A to protein m vitro IS a composite reaction: (1) When measured under comparable condmons, the binding was less susceptible to Inhibition by CO or S K F 525-A than oxidative demethylatlon (Table II, Fig. 9) (2) The mercurial inhibition curves were biphaslc, with about the same proportionality between the components as indicated in the experiments just mentioned (Fig 8) (3) Evidence of heterogeneity was also prowded by the experiments on structurally disorganized mlcrosomes (Fig 4) The binding data are consistent with ARRHENIUS' scheme 3°, if it is assumed that free radicals are released tn vitro not only from the binary complex supposed to be mvolved in N-oxygenation 18, but also (and mainly) from the ternary, cytochrome P-450-dependent complex postulated as intermediate in C-oxygenation
Specificity of bmdmg The difference in chemical and biological specificity between the binding of
Chem-Btol Interactions, 3 (1971) 321-336
334
BRITA BEIJE, TORE HULTIN
a r o m a t i c amines m vtvo a n d tn vitro 6"7 IS o f interest m this context A m o n g the m a n i f o l d integrating factors, which m a y c o n t r i b u t e to the greater selectivity tn w v o , the p r o p o r t i o n o f c y t o c h r o m e - l n d e p e n d e n t binding m a y be one o f i m p o r t a n c e . Current evidence suggests t h a t the selective binding o f carcinogenic amines in longt e r m 3 feeding experiments is specifically related to N - h y d r o x y l a t l o n 8'9 This r e a c t i o n (as analyzed m vitro) has been f o u n d CO-resistant 33. It ~s therefore an Interesting possibility that, in c o n t r a s t to the in vitro binding, the selective b l n d l n g tn vtvo is p r e d o m i n a n t l y o f a c y t o c h r o m e - m d e p e n d e n t type Esters 9 (or even m o r e distant derivatives 34) o f N - h y d r o x y l a t e d c a r c m o g e m c amines have been held responsible for the b i n d i n g reaction m vtvo u n d e r c o n d i t i o n s o f e x t e n d e d feeding. By way o f a m i d o m u m ~ons, the esters are s u p p o s e d to f o r m electrophlhc c a r b o n l u m ions by a m e c h a n i s m 9 partmlly a n a l o g o u s to t h a t a l r e a d y discussed ~8,30 Since ester f o r m a t i o n is a p p a r e n t l y n o t involved in the m vitro binding, the present as well as earher 6'7 d a t a p o i n t to a m o r e direct t y p e o f reaction u n d e r these conditions By analogy, it seems an obvious p o s s l b d l t y that the in VlVO b i n d i n g m a y also involve a c a p t u r e o f reactive o x y g e n a t i o n l n t e r m e d m t e s p r i o r to the form a t i o n o f the h y d r o x y l a t e d end p r o d u c t Independence o f b m d m g o f the f o r m a t i o n o f end p r o d u c t s
The c y t o c h r o m e - d e p e n d e n t release o f reactive m e t a b o h t e s m w t r o seems to involve an a n o m a l o u s d e c o m p o s i t i o n o f the e n z y m e - s u b s t r a t e complex at the cytoc h r o m e P-450 level, thereby rendering the overall d e m e t h y l a t t o n system hable to persistent d a m a g e This is suggested by the fact that the increase in binding activity at high D M A c o n c e n t r a t i o n s is associated with a progressive inhibition o f the dem e t h y l a t l o n ~s ( F i g 1). Thus, the c y t o c h r o m e - d e p e n d e n t binding is i n d e p e n d e n t o f the terminal step in the demethylat~on reaction This is further illustrated by the trypsin experiments, which clearly indicate that the f o r m a t i o n o f reactive m e t a b o h t e s m a y continue with great efficiency, a l t h o u g h the d e m e t h y l a t m g function o f the system IS a b o h s h e d (Fig. 6). ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by the Swedish C a n c e r Society O u r t h a n k s are due to Dr. ERIK ARRHENIUS for n u m e r o u s respiring discussions, a n d to Dr. STEN ORRENIUS for help with the C O - i n h i b i t i o n experiments. REFERENCES 1 E C MILLERAND J A MILLER,The presence and significance of bound ammoazodyes in the livers of rats fed p-dlmethylamlnoazobenzene, Cancer Res, 7 (1947) 468-480 2 J M PRICE, E C MILLER,J A MILLER AND G M WEBER, Studies on the mtracellular composition of livers from rats fed various amlnoazodyes, Cancer Res, 9 (1949) 398--402 3 H TERAYAMAAND K KUSAMA,Study on the nature of binding of dlmethylamlnoazobenzene with hver protein of rats fed the carcinogen, Gann, 49 0958) 97-103 4 J J ROBERTSAND G P WARWICK,The covalent binding of metabohtes of dlmethylamlnoazobenzene, fl-naphthylamlne and aniline to nucleic acids in vlvo, Intern J Cancer, 1 (1966) 179-196. Chem-Btol Interactions, 3 (1971) 321-336
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BRITA BEIJE, TORE HULTIN E ARRHENIUS, Carclnogemc amines Some aspects of their metabolism and interaction with mlcrosomal functions of liver cells, Thesis, Stockholm, 1968 L HORNER, Autooxidation of various orgamc substances, m W O LUNDBERG(Ed), Autooxldatton and Anttoxtdants, Vol l, Intersclence, New York, 1961, pp 379-396 L HORNER AND H. JLrNKERMANN,StudieD zum Ablauf der Substitution, VII Beltrag zum Kmetlschen Nachwels yon Durchgangsradlkalen im System Tertlares Amln-Dlbenzoylperoxyd, Ann Chem, 591 (1955) 53-68 H UEHLEKE,General biological aspects of N-hydroxylatlon, FEBS Symposmm, 16 (1969) 97-107 E C MILLER, D McKECHNIE, M M POIRIER AND J A MILLER, Inhibition of amino acid incorporation tn vitro by metabohtes of 2-acetylammofluorene and by certain nltroso compounds, Proc Soc Exptl Btol Med, 120 (1965)538-541
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