Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Chem.-Biol. Interactions, 41 (1982) 297--312

297

Elsevier Scientific Publishers Ireland Ltd

D R U G RESIDUE F O R M A T I O N F R O M R O N I D A Z O L E , A 5-NITROIMIDAZOLE. III. STUDIES ON THE MECHANISM OF P R O T E I N ALKYLATION IN VITRO

G.T. MIWA*, S.B. WEST, J.S. WALSH, F.J. WOLF and A.Y.H. LU Department of Animal Drug Metabolism, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065 (U.S.A.)

(Received December 27th, 1981) (Revision received March 22nd, 1982) (Accepted March 22nd, 1982)

SUMMARY Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation are cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbony194Clabeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this c o m p o u n d is activated by microsomal enzymes. However, only a b o u t 5% of this reactive metabolite can be accounted for as proteinbound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.

INTRODUCTION Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is a 5nitroimidazole that was developed for the food producing animal industry Abbreviations: BSA,bovineserum albumin; TCA,trichloroaceticacid.

298 as an antiparasitic agent. Studies with radioactive drug dosed to food producing animals have shown the presence of persistent radioactive residues in animal tissues. These residues have not been fully characterized but are not due to the parent drug. We have previously shown that hepatic microsomes catalyze the formation of protein-bound residues in vitro [1,2]. Most of the enzyme activity is localized in the microsomal fraction of liver homogenates and additional studies have shown that the flavoprotein, NADPH-cytochrome P-450 reductase can carry out this activation reaction [2]. Goldman et al. [3] have, however, shown that gut microflora are capable of reductively metabolizing nitroimidazoles. Thus, gut bacteria and mammalian enzymes can potentially contribute to the metabolic activation of ronidazole required for protein alkylation. However, we have found (unpublished observations) that germfree rats do not form significantly higher levels of protein-bound residues for several tissues, except blood, when compared to normal rats, suggesting that the protein-bound residues can be produced by mammalian enzymes. Consequently, we have focused our attention on the mammalian hepatic enzymes. In this paper, the mechanism of ronidazole activation and protein alkylation has been investigated in vitro with both microsomal and purified NADPH-cytochrome P-450 reductase enzyme systems. The results of these studies demonstrate that protein cysteine thiols are the principal site of alkylation and that alkylation occurs with retention of the imidazole nucleus but with loss of the carbamoyl side chain. A mechanism is proposed to explain these results. MATERIALS AND METHODS Substrates The radiolabelled ronidazole substrates (Table I) 1-[l*C]methyl-5-nitroimidazole-2-methanol carbamate (I), 1-methyl-5-nitroimidazole-2-[l*C]methanol carbamate (II), 1-methyl-5-nitro-[4,5-14C]imidazole-2-methanol carbamate (III) and 1-methyl-5-nitroimidazole-2-methanol[14C]caxbamate (IV) along with the descarbamoyl ronidazole, 1-methyl-5-nitro-imidazole-2[l*C]methanol (V), were synthesized by Dr. Robert Ellsworth and Mr. Gregory Gatto at the Merck, Sharp and Dohme Research Laboratories (Rahway, NJ). All substrates were >97% pure both radiochemically and chemically and were originally prepared with a specific activity of 5.6-28 pCi/mg. High purity amino acids, bovine serum albumin (BSA) and dithiothreitol were obtained from Calbiochem (LaJolla, CA). Agarose-immobilized cysteine and methionine were obtained from the Sigma Chemical Co. (St. Louis, MO) and contained approx. 2--10 pmol of amino acid equivalent/ml of packed gel. Methyl methanethiolsulfonate (Sigma) was distilled under reduced pressure immediately before use. Agarose-ethane thiol was obtained from PL Biochemicals Inc. (Milwaukee, WI) and contained 2.3 pmol thiol equi-

299 TABLE I S U B S T R A T E S T R U C T U R E S A N D 14C-LABEL P O S I T I O N S C,H3

C)2N~'./.~5 I~1 2 CH2-O--R

Compound

R

14C.labe 1 position O II

I

--CNH2

1--N---CH3

O IJ

II

--CNH 2

2--CH 2 -

O rl

III

--CNH 2

o

4,5-Ring

o

IV

--CNH=

II 2--C--

V

--H

2--CH~--

II

valents/ml of packed gel. Agarose gel (Sepharose 4B) was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). All other biochemicals were purchased from Sigma. [14C]Methyl methanethiosulfonate was prepared from sodium methanethiolsulfonate and [14C]methyliodide [4,5] and diluted with non-radioactive methyl methanethiolsulfonate to a specific activity of 1 mCi/mmol. Sodium methanethiolsulfonate was synthesized as described by Kenyon and Bruice [4]. E n z y m e sources Microsomes from untreated, adult (200--250 g), male Sprague--Dawley rats were prepared as described elsewhere [6]. NADPH-cytochrome P-450 reductase was purified from phenobarbital induced rat livers to a specific activity of 30 000--35 000 U/mg protein by published procedures [7,8]. One unit of reductase activity is defined as the amount of enzyme catalyzing the reduction of cytochrome c at an initial rate of 1 nmol/min at 23°C assayed under the conditions of Phillips and Langdon [9]. Protein binding assays The alkylation of microsomal proteins was measured with radiolabelled ronidazole as previously described [1]. All radioactive ronidazole samples

300 were diluted with non-radioactive ronidazole to a specific activity of 0.20-0.8 mCi/mmol and incubated either aerobically or anaerobically at 37°C for 30 min unless otherwise indicated. The incubation system contained 6 mg of microsomal protein, 3 ~mol of ronidazole, 0.3 mmol of potassium phosphate buffer (pH 7.4), 18 pmol of MgC12, 3 u m o l of NADP, 30 ~mol of glucose 6-phosphate and 2.1 units of glucose-6-phosphate dehydrogenase in a total volume of 3.0 ml. In some experiments, the effect of amino acids, immobilized mercaptans or BSA on the binding of ronidazole to microsomal proteins was also measured. The experimental details can be found in the appropriate Figure and Table legends. Following the incubations, the reactions were stopped by transferring the incubation mixture to test tubes containing 0.8 ml of 3.0 M trichloroacetic acid (TCA). The precipitated proteins were extensively washed with TCA until the radioactivity in 0.5 ml of the TCA supernatant was no greater than twice the background level. The pellets were then solubilized with 1.0 ml of 1 N NaOH and analyzed for protein concentrations and radioactive protein-bound product [ 1].

BSA cysteine modification The cysteine thiol of BSA was selectively modified with methyl methanethiolsulfonate according to Smith et al. [10]. BSA (1.56 mM) in 0.1 M phosphate buffer (pH 7.4), was mixed with a 30-fold mole excess of freshly distilled methyl methanethiolsulfonate and allowed to react for 30 min at room temperature. The modified BSA was passed through a Sephadex G-25 column, equilibrated with 0.1 M phosphate buffer, (pH 7.4), to separate the modified BSA from the other reaction products. Half of this modified BSA was then reduced with a 5-fold mole excess of dithiothreitol for 30 min at 4°C to regenerate the unmodified BSA. This sample was again passed through a Sephadex G-25 column to isolate the BSA free of other reaction products. These BSA samples were used to determine the effect of blocking BSA cysteine thiols on the alkylation of this protein by ronidazole (Table III). Aliquots (36 mg) of the treated BSA protein were each incubated with 6 mg of liver microsomes as described above. Following the incubations, the microsomes were sedimented by centrifugation at 100 000 X g for 30 min. The supernatant, containing the BSA, was transferred to test tubes containing 2.4 ml of 3M TCA and the BSA-bound residues assayed as described previously [ 1 ]. Determination o f reactive cysteine thiol residues in microsomes Microsomal cysteine thiols were quantitated with [14C] methyl methanethiolsulfonate. To a microsomal suspension (1.5 mg protein at 30 mg/ml) was added 2.8 ul of a methanol solution o f [14C]methyl methanethiolsulfonate (0.21 mM, 1 mCi/mmol). The mixture was allowed to react for 30 min at room temperature and then the microsomes were passed through a Sephadex G-100 column (1 X 16 cm) to separate the microsomes from the unreacted thiol reagent.

301 An aliquot of the microsomal suspension was then assayed for modified cysteine thiols by liquid scintillation counting while another aliquot was assayed for protein.

Binding to immobilized mercaptans Sepharose 4B and the agarose-immobilized mercaptans (Table IV) were each (5 ml of packed gel) treated for 10 min with 50 ml of 20 mM dithiothreitol in 0.1 M Tris buffer (pH 8.0), containing 0.3 M NaC1 and 1 mM EDTA to completely reduce any disulfides to thiols on the gels. The gels were then filtered dry and the treatment with dithiothreitol repeated. The gels were washed five times with 50 ml of distilled water, filtered dry and resuspended in 10 ml of 10 mM potassium phosphate buffer (pH 7.4). One milliliter of a slurry of these gels was added to reaction mixtures containing II and incubated with rat liver microso:nes or purified reductase as described above for the protein binding assay. In some experiments with the purified reductase, BSA was also added as a protein source for trapping the reactive metabolite(s) of ronidazole. The reactions were stopped by passing the incubation mixture through a Pasteur pipette containing a glass wool plug as a filter. The filtrate, containing the proteins, was collected in a test tube containing 0.8 ml of 3 M TCA while the agarose gels were collected on the glass wool. The TCA precipitated proteins were washed and analyzed for bound residue [1] while the agarose gels were serially rinsed with 50 ml of water, 25 ml 0.5 M NaC1, 25 ml water, 25 ml 0.5 NaC1, 25 ml water. The last wash contained no detectable radioactivity. The gels were completely dried in an oven (100°C), transferred to scintillation vials and counted in 5.5 ml Dimulene-30 (Packard).

RESULTS

Effect o f amino acids on ronidazole protein binding To examine which of the amino acid residues on proteins is the probable target for alkylation, eighteen amino acids were tested for their effect on the covalent binding of ronidazole metabolite(s) to microsomal proteins (Table II). Of the amino acids tested only cysteine produced a reduction in the amount of ronidazole residue b o u n d to protein demonstrating a highly specific interaction between the reactive metabolite(s) and cysteine. Anaerobic incubation resulted in approximately two-fold greater binding of ronidazole metabolites relative to aerobic incubation. However, the degree o f inhibition by cysteine was comparable under these two conditions. Methionine does not inhibit the binding of ronidazole to microsomal proteins. These data indicate that the sulfhydryl moiety is essential for the inhibition of protein binding. This conclusion is also supported by the fact that other sulfhydryl-containing c o m p o u n d s such as glutathione and Nacetyl cysteine are also effective in inhibiting binding of ronidazole metabolite(s) to microsomal proteins [ 1].

302 TABLE II E F F E C T OF AMINO ACIDS ON PROTEIN-BOUND PRODUCT FORMATION CATALYZED BY LIVER MICROSOMES Conditions a

Control + cysteine + methionine + phenylalanine + tyrosine + tryptophan + lysine + histidine + arginine + aspartic acid + glutamic acid + serine + threonine + proline + glycine + alanine + valine + leucine + isoleucine

Relative activity (% control)b

Anaerobic

Aerobic

100 24 100 105 101 118 102 93 93 101 109 97 102 113 94 101 103 111 108

100 29 112 94 88 97 89 99 97 89 105 94 99 85 85 99 94 89 90

a[14C] Ronidazole (II) was incubated with rat liver microsomes and the protein-bound radioactivity assayed as described in Materials and Methods. All amino acids were tested at 1 mM concentration except for tyrosine which was tested at 0.5 raM. bAnaerobic and aerobic incubations were carried out as previously described [1]. Control values (100%) correspond to 1.72 and 0.98 nmol/mg/30 rain of bound product equivalent to ronidazole for the anaerobic and aerobic incubations, respectively.

Kinetics of cysteine inhibition of ronidazole protein binding The inhibition of protein binding by cysteine does not result from the inhibition of ronidazole metabolism [1]. However, a competition between protein cysteine residues and the added cysteine for a common reactive i n t e r m e d i a t e ( S c h e m e I) m a y e x p l a i n t h e i n h i b i t i o n b y c y s t e i n e . T h i s c a n b e s h o w n f r o m t h e a n a l y s i s o f S c h e m e I: ks [protein]

,p,

k7 [ c y s ] Scheme I w h e r e P is t h e r e a c t i v e m e t a b o l i t e o f S; P ' is t h e p r o t e i n - b o u n d m e t a b o l i t e ; P " is t h e c y s t e i n e - b o u n d m e t a b o l i t e . T h e r a t e o f p r o t e i n b i n d i n g i n t h e

303 presence of cysteine is then: dP'__ dt = v

-

{

Vm[S ] + [S]

Km

k~ } k~ + k~ [cys]

wherek'. = ks[protein]

(1)

rearrangement of Eqn. (1) yields: v =

Ym is] Km (1 + [cys]/Ki) + [S](1 + [cys]/Ki)

where Ki -

k; k7

(2)

Equation (2) is in the form of an equation for noncompetitive inhibition [11]. Thus, non-competitive inhibition kinetics would be expected for a mechanism in which the added cysteine competes with proteins for a c o m m o n reactive metabolite (P in Scheme I). The reciprocal transformation of Eqn. (2) is: + [cys]/Ki)1/[S]

1/v = Km/Vm(1

+, 1/Vm(1 + [cys]/Ki)

which demonstrates that double reciprocal plots of the protein binding rate and ronidazole concentration should yield a family of straight lines with both slopes and intercepts on the ordinate sensitive to the cysteine concentration. Figure 1 is a double reciprocal plot of the inhibition of ronidazole protein

I

I

I

I

I

T.. 2o~

I

/v

~5

0

I i

I 2

I

I

I

3

4

5

t/[Ronidazole-I (mM) -1 Fig. 1. Double reciprocal plot of the inhibition of microsomal protein alkylation by cysteine. Liver microsornes (6 mg) were anaerobically incubated with variable concentrations of ronidazole, III, (0.2--3.0 m M ) and the indicated concentrations of cysteine for 30 rain at 37°C. The protein-bound ronidazole metabolites were assayed as previously described [1 ].

304 binding by cysteine. Cysteine increases both the slope and intercept as expected from Eqn (3). Furthermore, only the Vm parameter is sensitive to the cysteine concentration while the K m remains constant. The Ki determined by nonlinear regression analysis of the data is 0.18 mM. Further evidence supporting microsomal cysteine alkylation by ronidazole can be obtained by analysis of the Ki term. The Ki term is composed of the rate constants k7 and ks (Eqns. (1) and (2)) for the reactions between the reactive metabolite and exogenous cysteine or microsomal protein, respectively. Assuming that the microsomal cysteine and exogenous cysteine thiols are equally reactive (e.g. ks = kT), the Ki term (0.18 mM) should be equal to the microsomal cysteine concentration in the incubation. The microsomal cysteine thiol concentration was determined by cysteine modification with [14C] methyl methanethiolsulfonate. The cysteine content determined in this way was 0.10 mM. Doubling the methyl methanethiolsulfonate concentration gave the same value. The close agreement between the Ki and protein cysteine concentrations is remarkable considering that they were obtained by completely independent methods. These data argue favorably that protein cysteine thiols are the principal site of ronidazole alkylation. More direct evidence for the specificity of the reactive metabolite of ronidazole for protein cysteine thiols was obtained from studies in which these groups were specifically blocked with methyl methanethiolsulfonate (Table III). Blocking the cysteine thiol of BSA caused a 76% reduction in protein-bound residues relative to the untreated BSA while unblocking the BSA cysteine thiol with dithiothreitol resulted in complete restoration of protein alkylation. The small (24%) residual binding to the blocked BSA samples was not due to contamination of the BSA with microsomal proteins. Samples in which microsomes were incubated without BSA present resulted in only 0.2% of the microsomal protein-bound residue being carried over into the BSA

T A B L E III EFFECT OF BSA CYSTEINE MODIFICATION ON PROTEIN ALKYLATION

Treatment a

Product bound to BSA b (nmol/mg/30 rain)

% Untreated BSA

Microsomes alone

0.001 0.502 0.12 0.542

0.2 100 24 108

+ untreated BSA + blocked BSA + unblocked BSA

aMicrosomes (6 rag) were anaerobically incubated, either alone or w i t h u n t r e a t e d , b l o c k e d or u n b l o c k e d BSA ( 3 6 mg). T h e u n b l o c k e d B S A was p r e p a r e d b y d i t h i o t h r e i t o l r e d u c t i o n of the b l o c k e d B S A as d e s c r i b e d in Materials a n d Methods. bThe BSA was separated from the microsomal proteins by centrifugation. T h e ronidazole residue bound to B S A was assayed as described in Materials a n d Methods.

305 binding assay. The residual binding to the blocked BSA probably results from the release of the blocking agent that may occur when methyl methanethiolsulfonate treated BSA is exposed to microsomal thiols during the incubation. Smith et al. [10] have indicated that this m a y occur in coupled enzyme assays in which only one of the enzymes has been treated. Studies with immobilized mercaptan The specificity of the ronidazole for nucleophilic sulfhydryls could also be evaluated in studies employing immobilized mercaptans (Table IV}. Liver microsomes catalyze the formation of a reactive ronidazole metabolite that is capable of alkylating immobilized mercaptans such as agarose-cysteine and agarose-ethane thiol. Immobilized methionine, in contrast, does not bind more radioactivity than the agarose gel alone. The same results are also observed when the purified NADPH-cytochrome P-450 reductase is used as a source of activating enzyme. The agarose-methionine controls show very little binding of radioactivity relative to agarose~thane thiol, closely mirroring the results expected from the amino acid inhibition studies shown in Table II. The binding of ronidazole radioactivity to immobilized mercaptans TABLE IV B I N D I N G O F [2-14C] R O N I D A Z O L E MICROSOMAL PROTEINS Conditions a

TO IMMOBILIZED

MERCAPTANS

Binding (nmol/30 min) b

AND

% Inhibition of microsomal binding

Agarose gel

Microsomes

-0.51 0.64 3.64 10.8

9.4 11.2 8.5 8.0 8.5

0 0 10 15 10

10.7 -9.74

0 -9

-8.06

-25

A. Microsomal activation Microsomes alone + agarose + agarose-met + agarose-cys + agarose-ethanethiol

B. NADPH-cytochrome P-450 reductase activation Reductase + BSA c + agarose-met + BSA + agarose-met

+ Agarose-ethanethiol + B S A + agarose-ethanethiol

-0.19 0.15 1.59

1.44

a [i4C ]Ronidazole, II,was anaerobically incubated with livermicrosomes (6 mg) or N A D P H cytochrome P-450 reductase (4000 units, 0.16 rag) and the agarose gels indicated. The agarose gels were separated from the microsomes by filtration and the bound radioactivity in both the gels and microsomes assayed as described in Materials and Methods. bResults are expressed as nmol equivalent to II bound to the total amount of microsomal and B S A proteins or agarose gel. C B S A (6 m g ) was added to some incubations with the purified reductase as a protein trap for reactive metabolites of II.

306 provides, therefore, further evidence for the specificity of the ronidazole metabolite for the sulfhydryl function. The presence of these immobilized mercaptans did not, however, appreciably inhibit the binding of the ronidazole metabolite to either microsomal protein or BSA. This is in marked contrast to the results observed for free cysteine (Table II and Fig. 1) and clearly illustrates a major difference in reactivity between free and immobilized cysteine. These data demonstrate that immobilized cysteine is no longer capable of competing with the microsomal cysteine residues for the ronidazole metabolite. BSA was also used as a source of immobilized cysteine in order to rule out any artifactual results that might have been obtained when agarose gels were introduced into the incubation mixture. Table V demonstrates that the addition of BSA to the incubation mixture results in a BSA concentrationdependent increase in binding to this protein without affecting the a m o u n t b o u n d to the microsomal proteins. When these data are normalized to the amount of protein incubated from the two sources, it can be seen that the specific activity of the bound residues in BSA protein is inversely related to the BSA concentration while the specific activity of the microsomal b o u n d residues is independent of the a m o u n t of BSA added.

TABLE V E F F E C T OF BSA CONCENTRATION ON MICROSOMAL PROTEIN-BOUND PRODUCT FORMATION a BSA (rag/incubation)

0 3 9 18 36 72

Total protein-bound product (nmol/incubation)

Spec. act. (nmol/mg protein)

BSA b

Microsomes

BSA

Microsomes

0 1.97 9.90 10.3 12.9 18.6

2.80 3.00 2.77 2.77 2.77 2.50

-1.04 1.27 0.66 0.40 0.28

1.40 1.50 1.39 1.38 1.38 1.25

Mean (S.D.)

2.77 (0.16)

1.38 (0.08)

aLiver microsomes (6 mg protein) were incubated anaerobically with ['4C]ronidazole, II, in the presence of an NADPH-generating system and variable concentrations of BSA. Assays were carried out in triplicate. After incubation, triplicate samples were pooled, kept on ice and then centrifuged at 42 000 rev./min in the 75 Ti rotor for 1 h. Both supernatant and mierosomal suspension were treated with TCA and the precipitated proteins extensively washed with TCA followed by absolute ethanol. Covalent-bound metabolite was determined in both soluble (containing mostly BSA and a small amount of protein released from microsomes during centrifugation) and microsomal fractions as described previously [1 ]. bExpressed as total nmol equivalent to ronidazole bound per incubation. Values are corrected for microsomal protein contamination and BSA recoveries.

307 The fact that BSA does not inhibit microsomal protein alkylation provides a convenient m e t h o d for estimating the fraction of the reactive metabolites generated that alkylates microsomal proteins. The quantity o f reactive metabolite that does not alkylate microsomal proteins was assumed to be equal to the a m o u n t bound to BSA at infinite BSA concentration. This was determined from a double reciprocal plot of the data in Table V. Such a plot reveals that approx. 48 nmol of ronidazole metabolite can be bound at infinite BSA concentration. That is, about 48 molecules o f ronidazole metabolite(s) were capable of diffusing into the aqueous medium for every 2.8 molecules that alkylated microsomal proteins under these conditions. The partition ratio for covalent binding to microsomal proteins (pathway e relative to pathway c of Fig. 2) was estimated from the equation: partition ratio = m a x i m u m bound to [BSA] = + bound to microsomes

48 + 2.8 -

bound to microsomes

18

2.8

These calculations reveal that for every 18 molecules of reactive metabolite formed, only one molecule alkylates the microsomal protein. The remaining 17 molecules diffuse to the surrounding medium and are capable o f alkylating BSA. Activating Enzyme System (Microsomes or NADPH-Cytochrome P450 IReductase /

o

= Non Reactive Metabolites

Ronidazole b=

[Reactive Metabolite'l

/

Microsome or Reductose Alkylation

Cysteine Adduct (s)

e Diffusion

Aqueous

Medium

[Reactive Metobolite'l

BS Cy ine Hydration l Alkylation Adduct (s) Product (s}

Immobilized Mercopton Adduct (s)

Fig. 2. Disposition of ronidazole. Ronidazole is activated through reductive m e t a b o l i s m by microsomes or NADPH~cytochrome P-450 reductase. The pathways for the d i s p o s i t i o n of t h e reactive metabolite(s) are shown and discussed in t h e text.

308 TABLE VI PROTEIN-BINDING OF SPECIFICALLY RADIOLABELLED RONIDAZOLE SUBSTRATESa Substrate

Protein binding (nmol/mg/30 min) Anaerobic

Aerobic

Expt. 1

I II III

1.76 1.73 1.91

1.08 1.36 1.28

Expt. 2

III IV V

1.45 0.21 0.21

0.63 0.04 0.04

aRonidazole substrates, specifically radiolabelled in the positions shown in Table I, were incubated with rat liver microsomal preparations. The protein-bound drug residues were determined after exhaustively washing the microsomal proteins as described in Materials and Methods.

Studies with specifically radiolabeied ronidazoles Structural information about the protein bound ronidazole residues was obtained through the use of several, specifically radiolabelled ronidazole compounds. Table VI summarizes these studies in which both anaerobic and aerobic incubations of these compounds were conducted and the resulting protein-bound radioactivity quantitated and expressed as nmol equivalent to ronidazole bound/mg protein. In Expt. 1, the specific activity of the bound residues is found to be independent of the 14C-label position so that the N-methyl (I), the 2-methylene (II) and the 4,5-ring (III) labelled substrates produce the same quantity of bound residues. These data demonstrate that the bound residues retain these four carbon atoms and suggest that the imidazole nucleus is retained in the bound residue. When identical studies are done with the radiolabel in the carbamoyl group (IV), considerably less apparent binding is observed relative to the 4,5-ring labeled substrate (Expt. 2). Moreover, descarbamoyl ronidazole (V), which lacks the carbamoyl group, binds to microsomal proteins to a much smaller extent. DISCUSSION

Nitroimidazoles are effective antiprotozoal agents that are also relatively specific against anaerobic bacteria [12]. They are also mutagenic [13,14] and, in the case of ronidazole, form protein-bound products [ 1 ]. The reactive metabolite leading to protein-bound residues may, however, be distinct from that leading to mutagenesis since no significant binding occurs to RNA or DNA [1] under these conditions. Furthermore, nucleotides do not effectively compete for proteins as targets for ronidazole alkylation [1].

309 0

CH 3

O2N. I N . A / U , , , , ~ _ _ N / ~ "0 NH2

[

2eo

O=N ~ _ _ / N~ 0

NHz

2e-

y

o

HO--U.

N-

A

" ~

~. ~

'r NH 2

CH~ .N. ~

,

CIH3 N. A

.

"

~-~---N T3t-a

~-'-N ilx

y HON.

S-.~<

"

Protein

Protein

1Y b

2e-

"

CH 3 ' ~-N 3t"

0

CH3 L----N 3t'O

CH3 - - Protein

I b

_~

Protein

Fig. 3. Proposed mechanism for ronidazole activation and alkylation of protein cysteine

thiols.

Thus it appears that mutagenesis may occur by a mechanism not involving the formation of stable alkylation products of ronidazole. In contrast, protein residue formation results in stable adducts that are readily quantitated [1,2]. Moreover, these protein-bound adducts appear to retain the intact imidazole nucleus but form with the loss of the carbamoyl side chain. A proposed mechanism consistent with these observations is illustrated in Fig. 3. The initial step is the NADPH-dependent enzymatic reduction of the parent nitroimidazole, VII, to the aminoimidazole, X. Formation of the putative Michael acceptor intermediate Xa would be facilitated by loss of the carbamoyl group thus explaining the almost complete absence of protein binding when this group is not present as in substrate V (Table VI). Furthermore, the apparent loss in protein binding when the radiolabel is in the carbamoyl carbon (substrate IV) argues strongly that protein binding must occur with loss of the carbamoyl group. Thus, alternative mechanisms involving the nucleophilic attack at the 4-position of the nitrosoimidazole or the hydroxylamine to give the 4-substituted hydroxylamine or amine products, would require subsequent loss of the carbamoyl group

310 and are, therefore, less attractive alternatives to the proposed mechanism. The proposed mechanism shows the six electron reduction to the aminoimidazole, X, although the nitrosoimidazole, VIII, or hydroxylamine, IX, cannot be excluded as potential precursors to alternate Michael acceptor intermediates such as IXa, Thus, reduction to either the nitrosoimidazole or hydroxylamine could ameliorate the strong electron withdrawing properties of the nitro group and permit the delocalization of the imidazole nitrogen electron pair to form the cation IXa in a fashion analogous to the aminoimidazole. This alternative, however, seems untenable in view of the stability of the N-acetyl derivative of the aminoimidazole, X, in aqueous solutions (Walsh and Miwa, unpublished data). The available evidence, therefore, is most consistent with the mechanism proposed in Fig. 3 although several other alternative mechanisms including nucleophilic attack at the 4-position, cannot be unequivocally ruled out at this time. The site of alkylation on tissue macromolecules appears to be the thiol of cysteine since this is the only amino acid (Table II) that causes the inhibition of ronidazole protein binding. The inhibition is kinetically consistent with that expected for competition between the added cysteine and the alkylation site on the protein (Fig. 1). Furthermore, immobilized thiols, such as cysteine, trap the intermediate during metabolism while immobilized methionine does not (Table IV). Finally, the blocking of the cysteine thiols on BSA with methyl methanethiolsulfonate causes inhibition of BSA alkylation which is restored when the blocking agent is removed with dithiothreitol (Table III). The scheme represented in Fig. 2 illustrates the possible disposition of the reactive intermediate. The lifetime of the intermediate is sufficiently long to permit diffusion (pathway e) from the site of enzymic activation to immobilized cysteine on agarose (pathway i) or BSA (pathway f). The immobilization of the nucleophilic cysteine thiol prevents direct competition between the immobilized cysteine and those cysteine residues that are alkylated in the microsomal proteins explaining the absence of inhibition of microsomal alkylation when a source of immobilized cysteine is added to the system (Table V). In contrast, free cysteine is sufficiently small to permit the direct competition (pathway d) with microsomal cysteine (pathway c) for the intermediate and results in the observed inhibition of microsomal alkylation (Fig. 1 and Table II). In the absence of thiol containing nucleophiles, the reactive intermediate appears to decompose through reaction with water (pathway h) yielding a stable metabolite (Walsh and Miwa, unpublished data). Indeed, the formation of this metabolite is markedly increased when thiols are omitted from the incubation system. The partition ratio calculated from the data in Table V reveals that only one molecule out of approximately eighteen molecules of the reactive metabolite actually alkylates the microsomal protein by pathway c. The remaining seventeen molecules are capable of diffusing away from the activating enzyme into the media (pathway e) where they can be trapped and quantitated as BSA-adducts (.pathway f). Alternatively, the reactive

311 metabolite could also react with ot her thiol sources in the incubation media such as free cysteine, glutathione, or agarose immobilized thiols. Th e s to ic hi om et r y between the quantity o f microsomal protein alkylated and the a m o u n t of ronidazole metabolized indicates t hat only one molecule, for every t w e n t y molecules of ronidazole metabolized, alkylates microsomal proteins [1]. The similarity between the partition ratios calculated from the a m o u n t o f ronidazole metabolized and from the a m o u n t o f ronidazole t hat has alkylated BSA, at infinite BSA concentration, indicates, therefore, that essentially all of the ronidazole metabolized goes to the f o r m a t i o n of reactive metabolites that are capable of alkylating proteins. Thus, little or n o n e of the ronidazole is metabolized through a nonreactive p a t h w a y (pathway a). Based on these data we conclude t ha t the reductive metabolism of ronidazole by microsomal enzymes results p r e d o m i n a n t l y in the f o r m a t i o n o f a reactive intermediate that specifically alkylates the thiol of cysteine amino acid residues on protein macromolecules. ACKNOWLEDGEMENTS We are grateful to Dr. R o b e r t Ellsworth and Mr. Gregory G at t o for t he synthesis o f t he radiolabelled ronidazole substrates and to Dr. Holly Mertel for assistance in the preparation of radioactive m e t h y l methanethiolsulfonate. We would also like to acknowledge Dr. Leonard Oppenheimer and Thomas Capizzi for assistance on the design and analysis of experi m ent shown in Fig. 1 and Mrs. Donna Gibson for her assistance in the preparation of this manuscript. REFERENCES 1 S.B. West, P.G. Wislocki, K.M. Fiorentini, R. Alvaro, F.J. Wolf and A.Y.H. Lu, Drugresidue formation from ronidazole, a 5-nitroimidazole. I. Characterization o f in vitro protein alkylation, Chem.-Biol. Interact., 41 (1982) 265. 2 S.B. West, P.G. Wislocki, F.J. Wolf and A.Y.H. Lu, Drug-residue formation from ronidazole, a 5°nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro, Chem.-Biol. Interact., 41 (1982) 281. 3 R.L. Koch and P. Goldman, The anaerobic metabolism of metronidazole forms N(2-hydroxyethyl)-oxamicacid, J. Pharmacol. Exp. Therap., 208 (1979) 406. 4 G.L. Kenyon and T.W. Bruice, Novel sulfhydryl reagents, in: C.H.W. Hirs (Ed.), Methods in Enzymology, Vol. 47, Academic Press, 1977, pp. 407--430. 5 S.F. Currier and H.G. Mautner, Interaction o f analogues o f Coenzyme A with choline acetyltransferase, Biochemistry, 16 (1977) 1944. 6 A.Y.H. Lu and W. Levin, Partial purification of cytochromes P450 and P448 from rat liver mierosomes, Biochem. Biophys. Res. Commun., 46 (1972) 1334. 7 J.P. Dignam and H.W. Strobel, NADPH-cytochrome P450 reductase from rat liver. Purification by affinity chromatography and characterization, Biochemistry, 16 (1977) 1116. 8 Y. Yasukochi and B.S.S. Masters, Some properties o f a detergent-solubilized NADPHcytochrome c (cytochrome P450)reductase purified by biospeeifie affinity chromatography, J. Biol. Chem., 251 (1976) 5337. 9 A.H. Phillips and R.E. Langdon, Hepatic triphosphopyridine nucleotide-cytochrome c

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