Relative Importance of Bacterial and Mammalian Nitroreductases … · aerobic mutagenicity testing was performed by placing the inoculated agar plates into Gas Pak jars (BBL Microbiology

[CANCER RESEARCH 40, 4599-4605, December 1980]0008-5472/80/0040-OOOOS02.00

Relative Importance of Bacterial and Mammalian Nitroreductases for

Niridazole Mutagenesis

Jeffrey L. Blumer, Allen Friedman,1 LeRoy W. Meyer, Edward Fairchild, Leslie T. Webster, Jr.,2 and William T.Speck3

Divisions of Pediatrie Pharmacology and Infectious Disease. Department of Pediatrics, Rainbow Babies and Children s Hospital IJ. L. B.. A. F., L. W M., W. T. S.],and the Genetics Center Â¡J.L B.I and Department of Pharmacology Â¡J.L. B.. E. F., L. T. W.I, Case Western Reserve University School of Medicine, Cleveland,Ohio 44106

ABSTRACT

Niridazole is a nitrothiazole anthelmintic agent used to treatschistosomiasis. Its antibacterial activity was found to requirethe presence of the nitro group; a synthetic desnitro analogwas completely inactive. Niridazole was mutagenic for Salmonella tester strains TA1538, TA98, and TA100, suggesting thatit was both a frame-shift- and a base substitution-type mutagen.

It was effective under both aerobic and anaerobic conditions,while similar testing of the desnitro niridazole produced consistently negative results. Addition of rat liver S-9 fraction undereither aerobic or anaerobic conditions did not enhance muta-genicity. However, since bacterial killing limited the dose ofniridazole to 0.33 fig/plate in standard tester strains (1 /20 Kmfor the mammalian liver enzymes), further studies were performed using niridazole-resistant, histidine-dependent mutants

derived from strains TA98 and TA100. These mutants werefound to be nitroreductase deficient and to resist the mutageniceffects of niridazole, in the presence or absence of S-9, up to

concentrations of 10 jug/plate. In addition, even at niridazoleconcentrations of up to 100 /ig/plate, rat liver S-9 was ineffec

tive in enhancing the mutagenicity of niridazole. These resultssuggest that the mutagenicity of niridazole is dependent on itsaromatic nitro group and a specific bacterial nitroreductase.

INTRODUCTION

During the past 50 years, a variety of structurally diversenitroheterocycles have been synthesized and used as foodadditives (26), antibacterial agents (17), and antiparasiticagents (17, 18). A majority of these compounds are nitrofuranderivatives, among which nitrofurazone has been most extensively studied. The antimicrobial activity of these nitrofuranderivatives has been demonstrated for anaerobic and facultative aerobic bacteria. Studies on the mechanism of such antibacterial activity have shown that reduction of the nitro groupby bacterial pyridine nucleotide-dependent nitroreductases isessential (3, 13, 20). This metabolic pathway involves a 6-

electron reduction of the nitroaromatic parent compound to theamine with the formation of reactive nitroso and hydroxylamineintermediates (16). In most cases, the amine product has beenisolated.

In addition to their antibacterial activity, these nitroaromaticcompounds have also been identified as prokaryotic mutagens

1Present address: Department of Pediatrics, Children's Hospital of Philadel

phia, Philadelphia. Pa. 19104.1 Recipient of grants from WHO and the Rockefeller Foundation.3 Recipient of Grant 5R01-CA23692 and Research Career Development

Award 1KO-4-CA-00443 from the National Cancer Institute.

Received March 2, 1980; accepted August 22. 1980.

(21, 22) and mammalian carcinogens (9). Such extreme formsof toxicity require metabolic activation of the parent nitroaromatic compounds, and this activation is presumably catalyzedby the same enzyme which is responsible for their antibacterialeffects (3, 7, 13, 20). Consistent with this hypothesis is thedetection of covalently bound drug, presumably from nitrosoor hydroxylamine precursors, associated with macromoleculesof bacterial origin (20).

Niridazole (Chart 1) is a synthetic nitrothiazole antibioticwhich has been used extensively to treat schistosomiasis (18).Recent studies from several laboratories (11, 21, 22) havedemonstrated that niridazole is mutagenic for prokaryotic cells.In addition, Urman ef al. (27) have shown that the compoundis carcinogenic for mice. The metabolism of niridazole bybacteria has not been studied. However, in mammalian micro-somal systems, nitroreduction has not been demonstrable under aerobic conditions (5). Under anaerobic conditions, ratliver microsomes can reduce niridazole to a hydroxylaminemetabolite, but complete reduction to the amine has not beendemonstrated (15). More recently, the aerobic formation of atleast 4 niridazole metabolites has been found with rodent livermicrosomal systems (5, 6). One of these metabolites wasshown to be a glycol, which suggests the formation of a reactiveepoxide intermediate during aerobic metabolism (23). It ispossible that the formation of this alkene oxide is responsiblefor the mutagenic and/or carcinogenic effects of niridazoleobserved under more physiological conditions. Alternatively,the aerobic activation of niridazole to a mutagen and/or acarcinogen may proceed via nitroxide free radical formationwith the subsequent generation of Superoxide aniÃ³n as described for nitrofurazone (19).

The present study was designed to evaluate the role ofaromatic nitroreductase activity in the antibacterial and mutagenic effects of niridazole. It was found that nitroreduction wasan essential prerequisite for biological activity under both aerobic and anaerobic conditions. Addition of rat liver S-9 had noeffect on the dose-dependent mutagenicity of niridazole in

sensitive Salmonella typhimurium tester strains. Moreover,tester strains deficient in nitroreductase activity could not bemutagenized by niridazole in the presence of rat liver S-9 andoxygen. The significance of these results for mammalian mu-

tagenesis is discussed.

MATERIALS AND METHODS

Chemicals. Niridazole (Ciba-Giegy, Summit, N. J.), 2-nitro-fluorene (Eastman Kodak Co., Rochester, N. Y.), sodium azide(Fisher Scientific Co., Pittsburgh, Pa.), and 2-aminoanthracene(ICN, Irvine, Calif.) were dissolved in sterile dimethyl sulfoxide

DECEMBER 1980 4599

on March 6, 2020. © 1980 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

J. L. Blumer et al.

H2Câ€”CH24\

CIIo

Chart 1. Niridazole [1-(5'-nitro-2'-thiazolyl)-2-imidazolidinone].

(Fisher). 2-Chloroethyl isocyanate was obtained from Fluka(Hauppauge, N. Y.), and 2-aminothiazole was obtained from

Eastman.Bacterial Strains. S. typhimurium strains TA1535, TA1537,

TA1538, TA98, and TA100 was utilized for mutagenesis testing. Clinical isolates obtained from the diagnostic microbiologylaboratory of University Hospitals, Cleveland, Ohio, were utilized for determining the antibacterial activities of the testcompounds.

Mutagenesis Testing. The pour-plate incorporation methodfor quantitative determination of mutagenic activity was performed as initially described by Ames ef al (2). Compoundsinducing more than twice the number of spontaneous revert-

ants found on control plates were considered mutagenic. Anaerobic mutagenicity testing was performed by placing theinoculated agar plates into Gas Pak jars (BBL MicrobiologySystems, Baltimore, Md.) which were then incubated at 37Â°in

the dark for 14 hr. The plates were then removed from the jarsand incubated aerobically at 37Â°for an additional 34 hr (24).

Metabolic Activation. A liver enzyme metabolic activationmixture, S-9, derived from Aroclor-treated Sprague Dawleyrats, was utilized in mutagenesis testing as described previously by Ames ef a/. (1). The mammalian enzyme was considered to contribute significantly when the number of his* re-

vertants at any dose of niridazole was at least twice thatobserved in the absence of S-9 (12). Statistical significancewas determined by the standard Student's f test (25).

Antibacterial Activity. The minimum inhibitory concentrationof the test compound was determined by the agar dilutionmethod using Mueller-Hinton agar (BBL Microbiology Systems)

containing concentrations of the test compounds ranging from0 to 64 jug/ml (the limit of solubility for niridazole and desnitroniridazole) in 2-fold dilutions. A control plate without addedchemicals was included in each series. Approximately 104

organisms from an actively growing culture were added to theplates by a replicating device, allowing 25 strains to be testedsimultaneously. Plates were then incubated in the dark at 37Â°

for 24 hr. The median inhibitory concentration value was defined as the lowest concentrations of the chemical that permitted growth following incubation.

Synthesis of 1-(2-Thiazolyl)-2-imidazolidinone (Desnitro

Niridazole). The procedure used was that of Wilhelm et al.(30). Dropwise addition of 6.9 g (0.066 mol) of 2-chloroethyl

isocyanate to a refluxing suspension of 6.5 g (0.065 mol)(recrystallized once from benzene/petroleum ether) in anhydrous diethyl ether gave a white precipitate which was filteredoff after 2 hr. Recrystallization from methanol gave 8.0 g (60%)of crystalline /V-(2-thiazolyl)-A/'-(2-chloroethyl)urea, m.p. 141to 143Â°.Nuclear magnetic resonance spectra were consistent

with the structure of the desired compound, and mass spectralanalysis gave M+ 205.0054 (calculated for CeHeNsOSCI20.00777). Five g (0.048 mol) of A/-(2-thiazoly)-/V'-(2-chloro-

ethyOurea were refluxed for 4 hr in 100 ml of water. Filtration

of the cooled reaction mixture gave a precipitate that wasrecrystallized from dimethylformamide to give 1.5 g (19%) ofwhite crystals, m.p. 209 to 212Â°. Nuclear magnetic resonance

spectra were consistent with the desired compound 1-(2-thia-zolyl)-2-imidazolidinone M +.

C6H7N3OS

Calculated: 169.0310Found: 169.0295

Determination of Bacterial and Liver Nitroreductase Activities. Bacterial nitroreductase activities were determined bythe method of McCalla ef al. (20) with nitrofurazone used asthe substrate. Anaerobic reactions were performed in Thun-

berg tubes purged with N2 which had been scrubbed by passage through Feiser's solution. For determinations of bacterial

niridazole nitroreductase, anaerobic cuvets were used, andniridazole was used in place of nitrofurazone. Niridazole nitroreductase activity was determined by the procedure of Felleref al. (15) as modified in this laboratory (6).

RESULTS

Antibacterial Activity of Niridazole. Niridazole demonstrated antibacterial activity against a variety of clinical isolates.Table 1 lists the niridazole median inhibitory concentrations fora variety of gram-negative organisms. Organisms were considered sensitive if the required median inhibitory concentrationwas < 64 /Â¿g/ml.In agreement with previous studies (10, 20),Salmonella species were found to be sensitive to the antibacterial effects of niridazole.

The importance of the aromatic nitro group to the antibacterial activity of niridazole is strongly suggested by the resultsobtained with desnitro niridazole (Table 1). In each case whereniridazole effectively inhibited bacterial growth, the desnitroderivative was ineffective.

Mutagenic Activity of Niridazole in Salmonella TesterStrains. Chart 2 shows the dose-dependent mutagenicity of

niridazole for 3 bacterial tester strains. Strain TA1538 appeared to be the most sensitive of the 3 strains. Qualitativelysimilar results have been reported by McCann ef al. (22),suggesting that niridazole acts predominantly as a frame-shift

mutagen.

Table 1

Antibacterial activity of niridazole and desnitro niridazoleGram-negative bacteria were grown overnight in Mueller-Hinton broth and

then planted on Mueller-Hinton agar plates containing either niridazole or desnitro

niridazole in concentrations from 0.5 to 64 fig/plate. Plates were incubatedovernight and read the next day.

Median inhibitoryconcentrationOrganismEscherichia

coliSalmonellaAerobacterKlebsiellaEnterobacterSerratiaProteusProvidenciaPseudomonas

aeruginosaNeisseriameningitidisHaemophilus

influenzae bTotal

isolatestested193222232111(fig/ml)Desnitroniri-Niridazoledazole321632643232>64>64>64>64>64>64>64>64>64>64>64>64>64>64>64>64

4600 CANCER RESEARCH VOL. 40




D 0.1 Q2 Q3

[NIRIDAZOLE]jug/piateChart 2. Dose-dependent effect of niridazole on reversion of standard Sal

monella tester strains TA1535 (â€¢),TA1537 (A), and TA1538 (â€¢)determined ina plate incorporation assay as described under "Materials and Methods." No

bacterial toxicity was observed over the dose ranges tested.

Two additional tester strains (TA98 and TA100) were studiedbecause of their increased sensitivity and spectrum of response(22). Niridazole was more effective in causing reversion tohistidine independence in strain TA98 than it was in the parental strain TA1538 (Table 2; Chart 2). Likewise, TA100 displayedgreater niridazole sensitivity than did its parent strain TA1535(Table 2; Chart 2). This mutagenic activity in strain TA100suggests that niridazole may also act to cause base substitutions. At any given dose, the number of revertants per platewas substantially greater with TA100 than it was with TA98(Table 2); however, the percentage of increase above background was similar in the 2 strains. In all subsequent work,TA98 and TA100 were used because of their greater sensitivityto niridazole than were the TA1535 and TA1538 Salmonellatester strains.

Bacterial killing was apparent in both strains at doses ofniridazole above 0.33 /xg. The killing was first assessed byvisual inspection of the agar plates. Bacterial colonies appearing abnormally small or plates containing regions of opacitywere considered suggestive of killing. Colonies were thenchosen from these regions and streaked upon fresh minimalmedium. Killing was confirmed by the failure of these selectedcolonies to grow.

Effect of O? and Rat Liver S-9 on the Mutagenic Activity ofNiridazole. Chart 3 depicts the effects of O2 and the additionof an S-9 fraction derived from Aroclor-treated rats on the

mutagenicity of 0.33 Â¡ugof niridazole. Similar effects wereobserved at all doses except when bacterial killing was apparent. Anaerobiosis increased the number of revertants by 37and 93% in strains TA98 and TA100, respectively.

Under aerobic conditions, the addition of rat liver S-9 resulted in a 43% increase in the number of TA98 revertants anda 13% increase in TA100 revertants. Anaerobic incubation inthe presence of S-9 increased the number of revertants by

63% in TA98, but similar treatment of TA100 resulted in aslight (15%) decrease.

None of the observed changes was statistically significantexcept for the effect of anaerobiosis on the number of TA100revertants in the absence of S-9 (p < 0.05). These results

suggest that rat liver enzymes can contribute very little to the

observed mutagenicity of niridazole when drug concentrationsare maintained below that which causes bacterial killing.

Roles of Aromatic Nitro Group in Niridazole Mutagenesis.The desnitro derivative of niridazole was synthesized in orderto evaluate the role of the aromatic nitro group in the antibacterial and mutagenic effects of niridazole. As shown in Table 1,the nitro group is required for antibacterial activity. Chart 4depicts the mutagenic effects of desnitro niridazole in Salmonella tester strains TA98 and TA100. Over a 4-log dose range,

no bacterial killing and/or mutagenic activity could be demonstrated.

Role of Aromatic Nitroreductase Activity in the Antibacterial and Mutagenic Activity of Niridazole. To assess the roleof bacterial nitroreductase in the antibacterial and mutageniceffects of niridazole, several niridazole-resistant but histidine-

dependent strains were developed from both TA98 and TA100.One mutant line from each strain was then selected for furtherstudy. These were designated TA98NR101 and TA100NR3,respectively.

Bacterial nitroreductase activity was assessed under bothaerobic and anaerobic conditions using nitrofurazone as thesubstrate. Anaerobiosis resulted in an approximate 2-fold stim-

Table 2

Effect of niridazole on reversions in TA98 and TA100 tester strains

Salmonella strains TA98 and TA100 were plated in the presence of variousconcentrations of niridazole as described in the legend to Chart 2. Three standardmutagens were used as controls and gave expected results: 2-nitrofluorene. adirect frame-shift mutagen; sodium azide, a direct base substitution mutagen;and 2-aminoanthracene, an agent requiring metabolism by mammalian enzymesin order to cause revertants.

CompoundNone2-nitrofluoreneSodium

azide2-AminoanthraceneNiridazoleDose

(/ig/plate)10.001.001.000.030.100.331.003.3010.00Revertants/plateTA98218702654129447153a144Â°101aTA1001394521341786702130905a158a

Killing observed; see text for details.

TA98 TAIOO

Ã•Ãœ

Chart 3. Effect of O2 and rat liver S-9 on the mutagenic activity of niridazole.Salmonella tester strains TA98 and TA100 were added to plates of minimalnutrient agar containing 0.33 fig of niridazole in the presence or absence of S-9fraction fom Aroclor-induced rats and then incubated for 24 hr at 37Â° under

aerobic or anaerobic conditions. Bars, mean Â± S.E. of at least 4 separateexperiments.

DECEMBER 1980 4601



J. L. Blumer et al.

TA 98

LU

40

30

20

IO

A

A

TA 100

U 160

UJ

120

80

40

AO

A

8

o

t

0.1 1.0 10.0 100.0[DESNITRO NIRIDAZOLEjjug/plote

Chart 4. Dose-dependent effect of desnitro niridazole on reversion in TA98and TA100 tester strains. Salmonella tester strains TA98 (top) and TA100(ooffom) were plated on minimal nutrient agar containing 1 to 100 fig of desnitroniridazole. Plates were incubated for 24 hr at 37Â°either aerobically (A, â€¢)or

anaerobically (A. O). Studies were performed both in the presence (A, A) andabsence (â€¢.O) of S-9 fraction from Aroclor-treated rats. In the absence of drug,there were 21 and 30 aerobic and 23 and 27 anaerobic TA98 revertants in theabsence and presence of S-9. Likewise, for TA100, there were 120 and 106aerobic and 139 and 188 anaerobic revertants.

ulation of the initial rate of reducÃase activity in TA98 andTA100NR3 but had no effect in the other 2 strains (Chart 5).The initial rate of nitroreduction in mutant strains was one-thirdto one-seventh of that in the parental strains.

Niridazole nitroreduction was assessed under anaerobicconditions (Table 3). ReducÃase activily was delecled in bolhTA98 and TA100 bul was noi demonslrable in eilher of Iheresislant mutants. This suggested lhal an inabilily lo reduclivelyactivate niridazole was, al least in part, responsible for Iheresislance of slrains TA98NR101 and TA1OONR3. Table 3 alsoshows lhal ral liver S-9 conlains a subslanlial amounl ofanaerobic niridazole nitroreduclase aclivily. Nevertheless, S-9did noi significanlly enhance Ihe number of niridazole-induced

reversions in TA98 and TA100 (Chart 3).Table 4 shows the mulagenic response of the nilroreduclase-

deficienl slrains to slandard chemical mulagens in Ihe presence and absence of ral liver S-9. In Ihe absence of S-9, bolh

TA98NR101 and TA100NR3 gave Ihe expected response to2-aminoanlhracene (Table 2), and Ihe addilion of S-9 fraction

increased Ihe number of revertanls in both slrains. With sodiumazide, strain TA100NR3 gave resulls indislinguishable fromIhose oblained wilh slrain TA100 (Table 2). The response ofTA98NR101 lo 2-nilrofluorene was considerably blunted whencompared lo lhal of the parental strain TA98 (Table 2), con-sislenl wilh Ihe observed deficiency in nilroreduclase. Never-

Iheless, Ihe number of revertants was substantially increased(â€”5-fold) by the addilion of ral liver S-9.

Under aerobic condilions, Ihe nilroreduclase-deficient mu

tants were resistanl lo bolh the anlibaclerial effecls of niridazole over a 4-log dose range (dala noi shown) and the muta-genie effects of niridazole over a 2-log dose range (Chart 6).

At niridazole levels of > 10 jug/plate, a marked increase in Ihenumber of revertanls was noted in bolh mutanls. Similarly, Iheaddilion of S-9 had no effecl on Ihe number of revertanls unlildrug levels of > 10 jug/plate were oblained. Al 100 ftg/plale,Ihe addilion of S-9 increased the number of revertanls in

TA98NR101 and TA100NR3 by 45 and 35%, respectively.Studies wilh Ihe reduclase-deficienl mulanls under anaero

bic condilions were hampered by Ihe extensive bacterial killingevident al niridazole concentralions of > 10 /Â¿g/plaie. InTA98NR101, Ihe addilion of S-9 in Ihe presence of niridazole

al 10/jg/plale (Ihe highesl concenlralion yielding no apparenlkilling) enhanced the number of revertanls by 90%. Similarly,in TA100NR3, Ihe addilion of S-9 in Ihe presence of niridazoleal 3 /ig/plale resulted in a 98% enhancemenl. Thus, al suffi-cienlly high drug concenlrations in the presence of sirici an-

aerobiosis, a conlribulion of Ihe mammalian enzyme may beappreciated.

TA98 TAIOO

40 80 I20 40 80 I20 I60

TIME (MIN)Chart 5. Nitroreductase activity in strains TA98, TA100, and their niridazole-

resistant mutants. Bacterial nitroreductase activity was measured under aerobic(â€¢,â€¢)and anerobic (O, Ãœ)conditions in strain TA98 (â€¢,D) and its niridazole-resistant mutant TA98NR101 (â€¢,D) (left) and strain TA100 (â€¢,O) and itsniridazole-resistant mutant TA100NR3 (â€¢.D) (right). Each 2-ml reaction mixturecontained 1.23 ml of a 5-hr log-phase culture, 20 fig of nitrofurazone, and 14 mgof glucose. Assays were performed at 37Â°, and the rate of nitroreduction was

determined as the rate of decrease in absorbance at 375 nm.

Table 3

Anaerobic nitroreduction of niridazole

Niridazole nitroreductase activity was assessed under anaerobic conditions.Each 2-ml reaction mixture contained 0.5 fimol of niridazole, 14 mg of glucose,and 1.23 ml of 5-hr log-phase culture for bacteria or 200 fimol of potassiumphosphate buffer (pH 7.4), 0.5 fimol of niridazole. 2 (imol of NADPH, and 2 mg ofprotein for S-9 fraction from Aroclor-treated rats. The rate of reduction wasdetermined from the initial rate of decrease in absorbance at 400 nm using anextinction coefficient of 10.5 mM~1cm~'. Results represent the average of tripli

cate determinations.

Source ofenzymeTA98

TA98NR101TA 100TA100NR3S-9ReducÃase

activity(nmol/min)0.16

<0.050.31

<0.050.67a

Activity expressed as nmol/min/mg of protein.

4602 CANCER RESEARCH VOL. 40



DISCUSSION

Niridazole is an effective anthelmintic agent which has beenwidely used during the past 20 years in the clinical treatmentof Infestations due to Schistosoma mansoni, Schistosoma hae-

matobium, and Dracunculus (18). Although it has been largelyreplaced by several newer antiparasitic agents, recent reportsof its profound antiinflammatory and immunosuppressive properties have generated renewed interest in its biodisposition (6,29). Thus, reports of bacterial mutagenicity (11, 21, 22) andmammalian carcinogenicity (27) suggest not only that the several hundred thousand patients treated with the drug might beat increased risk for cancer but that the development of avaluable new immunosuppressive agent might be stifled in itsinfancy.

In this report, we have examined the role of the aromaticnitro group in the biological toxicity of niridazole. In addition,we have reevaluated the effectiveness of mammalian liverenzymes in the metabolic activation of niridazole to a bacterialmutagen.

Previous studies by Watson (28) and Collins (10) have shownthat niridazole is an effective antibacterial agent. This is confirmed here for several gram-negative species (Table 1). The

data in the present study suggest that, as with nitrofurans (20),this nitrothiazole requires the presence of an aromatic nitrogroup (Table 1) and its metabolic activation in situ by thebacteria (Table 3) in order to exert antibacterial effect. Sincethe activated nitro group on niridazole is relatively more oxygenlabile than is the comparable substituent of the nitrofurans (6,15), its greater effectiveness against anaerobic species waspredictable.

As a bacterial mutagen, niridazole is intriguing in its ability tocause both frame-shift and base substitution mutations (Table

2). Similar observations were made by McCann ef al. (22).McCalla ef al. (21) also demonstrated alkaline-labile lesions in

DNA from cells of strain TA1975 after exposure to niridazole.Whether the molecular events associated with these 2 processes are the same or different has not yet been elucidated;however, the present study indicates that the nitro group isrequired for both frame-shift- and base substitution-type mu

tagenicity. Thus, desnitro niridazole is ineffective in elicitinghistidine-independent revertants over a wide dose range (Chart

4). Moreover, both types of mutation were also found to depend

Table 4Response of niridazo/e-resistant mutants to standard mutagens

Mutant strains derived from Sa/monella tester strains TA98 and TA100 wereplated in the presence of the indicated concentrations of the standard mutagenslisted below. Experiments were performed as described by Ames ef al. (2).

CompoundDose

(jig/plate)

Revertants/plate

S-9 TA98NR101 TA100NR3

None

2-Aminoanthracene

Sodium azide

1.00

1.00

15

21

15170

162195

171466

407

2-NitrofluoreneNiridazole10.00+0.33+783821626154230

TA98NRIOI


TAIOONR3

60O 3600

' O.I I.O IO.O IOO.O " O.I I.O IO.O KDO.O

[NIRIDAZOLE]^jg/pioteChart 6. Dose-dependent effect of niridazole on reversion of niridazole-re-

sistant mutants TA98NR101 (left) and TA100NR3 (right) determined underaerobic conditions in the absence (â€¢)and presence (A) of rat liver S-9.

on bacterial nitroreductase activity. In nitroreductase-deficient

mutants derived from TA98 and TA100, the mutagenic potencyof niridazole was reduced more than 10-fold (Table 2; Chart

6).The nitroreductase-deficient mutants used In the present

study were stable mutations of the parent strains which retainedthe R factor for ampiclllin resistance. They were generatedunder aerobic conditions, and thus, it Is not unreasonable that,in at least one strain (TA100NR3), the aerobic nitroreductaseactivity was more profoundly inhibited than was the anaerobicenzyme activity. Similar resistance has been reported forstrains of EscherichÃ¬acoli lacking one of the soluble nitrore-

ductases (3). As in the case of the E. coli mutants, our strainsshowed decreased sensitivity to other nitro-containing compounds (e.g., nitrofurantoin and metronidazole).4

Several possible mechanisms could account for the observedmutagenicity of higher doses of niridazole in nitroreductase-

deficient mutants. The simplest would be to attribute the effectto residual nitroreductase activity. In this case, our inability todetect anaerobic niridazole nitroreductase activity probablyreflects the fact that the activity in parental strains approachesthe limit of detectability of the assay. However, this alternativeis rendered less probable by the observation that, at 100 jug/plate, both mutant strains show the same number of his*

revertants that were seen in parental strains at 0.33 /Â¿g/plate(Charts 2 and 6) despite the fact that there was more residualreducÃase activity present in TA100NR3 than there was inTA98NR101 (Charts).

Since all of the biochemical determinations in the presentstudy were made using intact bacteria, it is possible thatmutations affecting bacterial permeability might contribute tothe niridazole resistance of the mutant strains. Such a mutationwould have to be rather specific for nitroaromatic compoundssince these tester strains have essentially no cell wall. Also,the mutants are as sensitive as TA98 and TA100 to otherstandard mutagens (Table 2 versus Table 4). Moreover, the

4 J. L. Blumer. A. Friedman, L. W. Meyer, E. Fairchild, L. T. Webster, Jr., and

W. T. Speck, unpublished observations.

DECEMBER 1980 4603



J. L. Blumer et al.

relative resistance of the TA98NR101 strain to 2-nitrofluorene

quantitatively reflects the relative deficiency in nitroreductaseactivity characteristic of this strain (Table 4; Chart 5). This isconsistent with the differences in sensitivity beween the mutants and the parental strains to the mutagenicity of nitroaro-

matics resulting wholly from differences in nitroreductase activity.

Alternatively, the results could be interpreted as consistentwith the appearance of a new nitroreductase in the mutantswhich has a marked increase in the Km for nitroaromatic substrates. Finally, a new mechanism of mutagenesis not involvingthe nitro group cannot be ruled out. Further work is in progressto distinguish among these possibilities.

The lack of effect of rat liver enzyme on bacterial mutagenesis by niridazole has been reported previously (11 ). However,since these studies were performed under aerobic conditionsat low niridazole concentrations, it was felt that the role ofmammalian enzymes should be reevaluated in light of ourpresent understanding of niridazole metabolism. Under aerobicconditions, niridazole can be converted to at least 4 polarmetabolites (5). One of these, the 4,5-glycol, indicates that anepoxide intermediate is formed during aerobic metabolism (23).However, in neither TA98 nor TA100 did the addition of ratliver S-9 significantly increase the yield of his* revertants

(Chart 3). This lack of effect may be explained by the differencebetween the observed Km for niridazole in liver microsomalpreparations (=25 JUM)and the maximum nonbactericidal concentration used in the plate incorporation assay (=0.8 UM).However, if this were the only explanation, a marked enhancement would then be expected at the levels of drug used withthe nitroreductase-deficient mutants (=230 fiM). Since no real

enhancement was noted (Chart 6), we conclude that, underaerobic conditions, either mammalian liver enzymes cannotform a bacterial mutagen or the mutagen once formed cannotenter the bacteria. Further studies are necessary to distinguishbetween these possibilities.

Under anaerobic conditions, mammalian nitroreductase canform an oxygen-labile hydroxylamine from niridazole (15). WithTA98 and TA100 tester strains, this pathway could not significantly enhance the mutagenicity of niridazole (Chart 3). In fact,only under the extreme conditions typified by anaerobiosis plushigh drug concentration plus the absence of bacterial nitroreductase could any effect of S-9 be demonstrated.

From the above discussion, one might question the idea thatniridazole poses a risk to those individuals taking the drug.Nevertheless, at least one group of investigators has demonstrated niridazole carcinogenicity in mice and hamsters (8, 27).Their study involved continuous feeding of the drug to theanimals for 40 to 70 weeks. It is not clear from their data,however, that the 5- to 7-day course of therapy (the usual dose

is 25 mg/kg/day) generally used to treat schistosomiasisposes a similar threat. Since the drug has a relatively shorthalf-life in animals (14) and in humans5 and since the DNA

lesions caused by the drug in bacteria are readily repairedupon removal of the agent (21 ), the true carcinogenic potentialof the drug remains unknown. More intensive investigationsunder physiologically relevant and clinically pertinent conditions must be performed to assess the true risk of niridazoleexposure.

One possible area of interest for future investigations relates5 J. W. Tracy, J. L. Blumer, and L. T. Webster, Jr.. unpublished observations.

4604

to the possible involvement of intestinal microorganisms in themetabolic activation of niridazole to eukaryotic mutagens and/or carcinogens. Such a mechanism has been reported recentlyto account for the presence of mutagenic activity in urine ofanimals receiving the antiparasitic agent 4-isothiocyano-4'-ni-

trodiphenylamine (4) despite its lack of mutagenic activity invitro in the presence or absence of rat liver enzymes. Similarly,reactive niridazole metabolites formed by intestinal microorganisms may be absorbed and result in genetic damage indistant tissues and organs. The intestinal flora have been foundto contribute significantly to the formation of the immunosup-pressive metabolite of niridazole found in the urine of nirida-zole-treated animals.6

The present study demonstrated that the mutagenicity of anitro-containing compound, niridazole, for tester microorga

nisms was dependent upon specific bacterial nitroreductaseactivity that was not present in the mammalian activation mixture. These results suggest that mutagenicity testing with standard tester microorganisms overestimates the potential long-term hazards of nitro-containing compounds. More specifically,

many of these compounds may be mutagenic for bacteria yetincapable of being converted to DMA-modifying agents in eu-

karoytic cells under physiological conditions.

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DECEMBER 1980 4605



1980;40:4599-4605. Cancer Res Jeffrey L. Blumer, Allen Friedman, LeRoy W. Meyer, et al. Nitroreductases for Niridazole MutagenesisRelative Importance of Bacterial and Mammalian

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Relative Importance of Bacterial and Mammalian Nitroreductases … · aerobic mutagenicity testing was performed by placing the inoculated agar plates into Gas Pak jars (BBL Microbiology