INTERNATIONAL JOURNAL OF LEPROSY^ Volume 51, Number 1

Printed in the U.S.A.

Mutagenic Activity of Antileprosy Drugsand Their Derivatives'

John H. Peters, G. Ross Gordon, John F. Murray, Jr.,and Vincent F. Simmon 2

The first report in 1973 that dapsone(4, 4'-diaminodiphenyl sulfone, DDS) wasweakly carcinogenic in male rats ( 3 ) was sub-sequently confirmed by reports from theNational Cancer Institute ( 4) and the Inter-national Agency for Cancer Research ( 12 ).The principal tumors found in these studieswere mesenchymal tumors of the abdomi-nal organs—mainly in the spleen. These re-ports have caused concern that DDS maypresent a risk to patients receiving antile-prosy therapy with this drug ( 7) even thoughthe doses that induced tumors in the ratsranged from 42 mg to 300 mg per kg perday for the lifetime of the animals; whereasthe usual maximum dose of DDS for manis 100 mg per day or about 1 mg to 2 mgper kg per day.

Stimulated by the original report in 1973and before the confirmatory studies wereavailable, we initiated studies using theshort-term presumptive test for carcinoge-nicity of Ames because this screening sys-tem for mutagenic activity had proved tobe a reliable indicator of carcinogenic ac-tivity in rodents for about 90% of the 300compounds tested ( 19).

We tested DDS and all available metab-olites, potential metabolites, and deriva-tives of DDS and also other drugs employedin leprosy chemotherapy using all five of therecommended test strains of Salmonella ty-phimurium with and without metabolic ac-tivation by rat liver microsomal enzymes.The drugs tested, besides dapsone and itsderivatives, were clofazimine, ethionamide,

' Received for publication on 4 August 1982; ac-cepted for publication on 27 October 1982.

2 J. H. Peters, Ph.D., Director, Biochemical Phar-macology Program; G. R. Gordon, M.S., SeniorBiochemist; J. F. Murray, Jr., B.S., Biochemist (de-ceased); and V. F. Simmon, Ph.D., Director, MicrobialGenetics Department, Life Sciences Division, SRI In-ternational, Menlo Park, California 94025, U.S.A.Current address: V. F. Simmon, Genex Corporation,Rockville, Maryland, U.S.A.

prothionamide, prothionamide-S-oxide [themajor metabolite of prothionamide ( 18)], andrifampin (The Figure).

Furthermore, after finding that 4, 4'-dia-minodiphenyl sulfide (DDSD) and 4, 4'-diaminodiphenyl sulfoxide (DDSO) exhib-ited mutagenic activity in the Ames screenand following the report by the NationalCancer Institute that DDSD was stronglycarcinogenic in mice and rats ( 6), we ex-amined pharmaceutical preparations ofDDS and urine from patients receiving DDStherapeutically for the presence of DDSDand DDSO. Finally, urine concentrates fromvolunteers taking DDS were also examinedfor mutagenic activity using the most sen-sitive S. t yphimuriutn strains TA98 andTA100.

MATERIALS AND METHODSChemicals. The following antileprosy

drugs (The Figure) were assayed for muta-genicity: clofazimine was a gift from Dr. L.Levy, Hadassah Medical School, HebrewUniversity, Jerusalem, Israel; ethionamidewas supplied by the Pasteur Institute, Paris,France; prothionamide was a gift from Mayand Baker, Ltd., Dagenham, England; pro-thionamide-S-oxide was synthesized as re-ported previously ( 18); rifampin was ob-tained from CIBA Pharmaceutical Products,Inc., Summit, New Jersey, U.S.A.; DDS wassupplied by Merck & Co., Rahway, NewJersey, U.S.A., and we purified it by re-crystallization from 95% ethanol. Also testedfor mutagenicity were seven derivatives ofDDS shown in Table 1: MADDS, DADDS,and AHADS were synthesized by Dr. W.Colwell, SRI International, Menlo Park,California, U.S.A.; (HA),DS and (HAAc,).DS were provided by Dr. T. Maren, Uni-versity of Florida, Gainesville, Florida,U.S.A.; DDSO was synthesized by a pub-lished procedure ( 17); and DDSD was sup-plied by K & K Laboratories, Plainview,

45

46^ International Journal of Leprosy^ 1983

New York, U.S.A.; Aroclor 1254, which wasused for induction of rodent liver post-mi-tochondrial metabolic activation system(S9), was received from Monsanto Com-pany, St. Louis, Missouri, U.S.A.; chemi-cals used as positive controls in mutagenesistests were sodium azide (Difco Laborato-ries, Detroit, Michigan, U.S.A.); 9-ami-noacridine (Pfaltz & Bauer, Inc., Stamford,Connecticut, U.S.A.); 2-nitrofiuorene and2-anthramine (Aldrich Chemical Co., Mil-waukee, Wisconsin, U.S.A.); and 2-(2-furyI)-3-(5-nitro-2-furyl) acrylamide (also calledAF-2). High-purity reagents used for high-performance, liquid chromatographic pro-cedures (HPLC) were ethylene dichloride(Burdick & Jackson Laboratories, Muske-gon, Michigan, U.S.A.); dimethyl sulfoxide(DMSO) (MCB Manufacturing Chemists,Inc., Gibbstown, New Jersey, U.S.A.); thio-diglycol (Pierce Chemical Co., Rockford, Il-linois, U.S.A.); and water, purified througha Super-Q water purification system by Mil-lipore Corporation, Bedford, Massachu-

setts, U.S.A. All other chemicals were ofreagent grade.

Mutagenesis assay. The full panel of S.typhiinurium strains recommended for mu-tagenesis testing, i.e., TA 1535, TA1537,TA1538, TA98, and TA100, were obtainedfrom Dr. B. N. Ames, University of Cali-fornia, Berkeley, California, U.S.A. Theplate incorporation method of the Saltno-nella/mammalian-m icrosome m utagenici-ty test of Ames, et al. (') was followed toassay all chemical compounds for mutagen-ic activity. All compounds were dissolvedin dimethyl sulfoxide (DMSO). The S9batches were prepared from liver followinginduction with Aroclor 1254 by the methodof Ames, et al. ( 1 ); also S9 preparations fromlivers of hamsters and mice were used inaddition to S9 from rats in some experi-ments. Sterility was confirmed for all S9batches. Where it was required, 0.50 ml ofS9 (10% S9 fraction: 90% cofactors in buff-er) were added per plate. We modified thestandard method in some tests by using a

51, 1^Peters, et al.: Mutagenicity of Antileprosy Drugs^ 47

TABLE 1.^Chemical structures of DDS and derivatives studied.

—(0)— R3

Compound R, R, R3 Abbreviation

4, 4'-Diaminodiphenyl sulfone NH, SO, NH, DDS4-Amino-4'-acetamidodiphenyl sulfone NH, SO, NHAc' MADDS4, 4'-Diacetamidodiphenyl sulfone NHAc SO, NHAc DADDS4-Amino-4'-hydroxyaminodiphcnyl sulfone NH, SO, NHOH AHADS4, 4'-Bis(hydroxyamino)diphenyl sulfone NHOH SO, NHOH (HA),DS4, 4'-Bis(N,0,-diacetylhydroxyarnino)diphenyl sulfone NAcOAc SO, NAcOAc (HAAc2),DS4, 4'-Diaminodiphenyl sulfoxide NH, SO NH, DDSO4, 4'-Diaminodiphenyl sulfide NH, S NH, DDSD

Ac = (—LOCH,).

20 min preincubation of bacteria and S9with the compound being tested before add-ing the top agar overlay and pouring ontothe test plate.

Adult male rats (two strains), hamsters,and mice (Simonsen Labs, Gilroy, Califor-nia, U.S.A.) were used for S9 preparationby pooling livers from the following num-bers of animals for each rodent strain: 15Sprague-Dawley or Fischer 344 rats (200-250 g); 30 Syrian hamsters (100 g); and 100B6C3F, mice (30 g). All animals were housedin quarantine rooms and fed Purina LabChow and water ad libitum until 12 hr be-fore sacrifice.

To ensure the validity of mutagenesis testresults, we routinely confirmed the nutri-tional requirements and biochemical char-acteristics of the S. typhimurium strains asrecommended by Ames, et al. (I). Diagnos-tic chemicals used routinely for confirmingthe reversion properties of the strains with-out metabolic activation were: sodium azide(1.0 µg/plate with strain TA1535); 9-ami-noacridine (100 µg/plate with strainTA 1537); 2-nitrofluorene (10 pg/plate withstrain TA1538); and AF-2 (0.10 pig/platewith strains TA98 and TA 100). To confirmreversion in the presence but not in the ab-sence of metabolic activation, 2.5 pg/plateof 2-anthramine with and without S9 weretested on all five S. typhinntrium strains.The numbers of spontaneous revertants ob-served (Tables 2, 3, and 4) were in agree-ment with the findings of Ames, et al. ( 1 ).In all tests, our criterion for mutagenic ac-tivity was a dose-related increase in numberof revertants to a level of at least twofoldgreater than the number of spontaneousrevertants.

Collection and concentration of urine formutagenesis assay. Two nonsmoking, malehuman volunteers, known to excrete mu-tagen-free urine in other studies ( 2 '), weregiven 50 mg DDS (Winthrop Laboratories,New York, New York, U.S.A.) orally andurine was collected during 0-12, 12-24, 24-36, and 36-48 hr. Urine aliquots (100 ml)were treated by the XAD-2 resin columnmethod ( 28 ) to yield 200-fold concentratesin DMSO. Aliquots of 5, 10, 30, and 75 Alof each urine concentrate were tested formutagenic activity using strains TA98 andTA I00 with and without rat liver S9. Prein-cubation was not employed in these tests.

Search for DDSO and DDSD in phar-maceutical preparations of DDS and in ur-ine. To examine for the presence of DDSOand DDSD in pharmaceutical preparationsof DDS, we triturated tablets of DDS (25-mg tablets, Winthrop Laboratories; 100-mgtablets, Ayerst Laboratories, New York,New York, U.S.A.) in ethanol. After cen-trifugation, an aliquot of the clear super-natant liqud was evaporated to dryness andthe residue dissolved in the mobile phasefor HPLC.

We also examined 24 hr urine collectionsfrom six volunteers receiving a single oraldose of 50 mg of DDS (Winthrop Labora-tories) and from nine patients receiving dai-ly oral doses of 100 mg of DDS (AyerstLaboratories) for the presence of DDSO andDDSD. Urine specimens (10 ml) were al-kalinized (pH 12-13) and extracted with 25ml of ethylene dichloride. After separationof the organic phase by centrifugation, were-extracted it with 5.0 ml of 2.0 N HC1.The acid phase was separated, made alka-line with 1.1 ml of 10 N NaOH, and ex-

48^ International Journal of Leprosy^ 1 983

TABLE 2. Mutagenicity of antileprosy drugs in Salmonella strains.

Compound^Rat S9 Microgramscompound/plate

Revertants/plate

TA1535 TA1537 TA1538 TA98 TA100

Negative controls 0 16 7 10 13 1160 13 8 23 20 120

Clofazimine 5 to 1000 10 —2 3 9 75 to 1000 1 —1 8 10 17

Ethionamide 5 to 1000 19 0 5 8 195 to 1000 0 3 5 5 21

Prothionamide 5 to 10005 to 1000

143

32

5—1

1010

1822

Prothionamidc-S-oxide 5 to 1000 —1 1 3 3 315 to 1000 6 —2 3 40

Rifampin 0.001 to 1.0 3 1 3 6 160.001 to 1.0 9 —1 7 4 17

a For antileprosy drugs, values are the differences between the highest number of revertants observed at anyconcentration of drug and the negative controls.

tracted with 1.0 ml of ethylene dichloride.This organic extract was evaporated to dry-ness, and the residue was dissolved in themobile phase for HPLC.

This procedure was applied to untreatedurine aliquots and to additional urine spec-imens that were hydrolyzed in 1.9 N HCIcontaining 0.90 mM Na,S,O, at 90-100°Cfor 60 min.

The residues from either extracts of thepharmaceutical preparations or the urinesamples were chromatographed on a 3.2 X250 mm column packed with Lich-rosorb SI-60 silica (Altex Scientific, Inc.,Berkeley, California, U.S.A.) using ethyl-ene dichloride : DM SO : water : thiodiglycol(988: 5:5:2.5, v/v) as the mobile phase. De-tection was by absorption at 280 nm andquantitation was by automatic integrationof the peak areas. Retention times forDDSD, DDS, MADDS (a major metaboliteof DDS), and DDSO were 5, 12, 21, and 26min, respectively, with base-line resolution.Limits of sensitivities were 5 ng for DDSOand 1 ng for DDSD by these methods.

RESULTSMutagenicity of antileprosy compoundsand derivatives

Tests of clofazimine, ethionamide, pro-thionamide, and prothionamide-S-oxideusing 5, 10, 50, 100, 500, and 1000 i.tg perplate using all five test strains with and with-out S9 from Sprague-Dawley rats yielded

no evidence of mutagenicity. Table 2 listsonly the highest net number of revertantsfound in these tests. Because rifampin ex-hibits marked toxicity for S. typhinutrium,we tested this compound at levels rangingfrom 0.001 jig to 1.0 pg per plate. As shownin the last line of Table 2, we also foundrifampin to be nonmutagenic under any testconditions.

Tests of DDS and its N-acetylated andN-hydroxylated derivatives of up to 1000Aig or 5000 i.tg per plate for the five strainswith and without S9 from Sprague-Dawleyrats also yielded negative results as shownin Table 3. Again, only the highest net num-ber of revertants observed at any of the levelsof compound are shown. These tests wererepeated using a 20 min preincubation withand without S9 from Sprague-Dawley rats.Again, no mutagenic activity of these com-pounds with any of the five strains of bac-teria was detected (results not shown). Sub-sequently, 10, 50, 100, 500, 1000, and 5000

DDS were tested using 20 min preincu-bation, all five test strains, and S9 fromSprague-Dawley and Fischer 344 rats, Syr-ian hamsters, and B6C3F, mice. Again, nomutagenic activity was detected under anytest condition (results not shown).

In contrast with the above negative re-sults, Table 4 shows that the sulfoxide an-alog of DDS, DDSO, was mutagenic forstrains TA1538 (at 100 lig) and TA100(at 50 pg) in the presence of S9. It wasinactive under all conditions without met-

51, 1^Peters, et al.: Mutagenicity of Antileprosy Drugs^49

TABLE 3. Mutagenicity of DDS and derivatives in Salmonella strains.

Compound^Rat S9 Microgramscompound/plate

Revenants/plates

TA1535 TAI537 TA1538 TA98 TA100

Negative controls 0 22 6 11 26 1400 10 5 15 28 123

DDS 10 to 5000 3 1 —4 —12 —4710 to 5000 3 3 8 —5 —16

MADDS 5 to 5000 6 7 5 2 115 to 5000 5 5 16 9 8

DADDS 5 to 5000 10 8 6 —' 65 to 5000 5 8 16 13 13

AHADS 5 to 1000 0 6 10 —9 —95 to 1000 3 6 15 5 47

(HA) W DS 5 to 1000 2 1 18 —8 315 to 1000 5 3 14 3

(HAAc 2 ) 2 DS 5 to 1000 l 3 4 —1 —85 to 1000 3 7 6 11 12

a For DDS and derivatives, values are the differences between the highest number of revertants observed atany concentration of drug and the negative controls.

abolic activation. Clearly, DDSO exhibitedmutagenic activity. As shown, the sulfideanalog of DDS, DDSD, was weakly activein the absence of metabolic activation forstrain TA 100 ( 100 pg) but inactive for theother strains. However, DDSD exhibitedsubstantial activities with added S9 for strainTA1538 ( ^ 100 pg), for strain TA98 ( 10pg), and for strain TA 100 ( ^ 5 pg). The max-imum increases over the negative controlvalues in the presence of S9 were 1 100% forDDSO (at 1000 pg) and for DDSD (at 500pg) with strain TA 100. These comparativeincreases suggest that DDSD was about twiceas active as was DDSO.

Studies of pharmaceutical preparations ofDDS and urine collections fromvolunteers and patients

Examination of ethanolic extracts of re-crystallized DDS or DDS tablets from Ay-erst or Winthrop Laboratories yielded no de-tectable DDSO or DDSD. From the limitsof sensitivity for detection and the amountsanalyzed, we can calculate that these DDSpreparations contained <0.01% contami-nation by these compounds. Thus, patientsreceiving 100 mg DDS per day of these DDSpreparations would receive <10 pg of eitherDDSO or DDSD.

Application of the same detection meth-od to extracts of aliquots of urine from vol-unteers receiving a single oral dose of 50 mg

DDS or from patients receiving daily oraldoses of 100 mg DDS yielded no detectableDDSO or DDSD in any urine specimen (re-sults not shown). These results indicate that<0.01% of the daily dose of 100 mg DDSwas excreted as either DDSO or DDSD.Because major quantities of urinary metab-olites of DDS and MADDS in man are acid-hydrolyzable ('°) and because DDSO orDDSD, if formed, would probably be ex-creted as such conjugates, we re-examinedurine samples from the patients after acidhydrolysis for the presence of DDSO andDDSD. As shown in Table 5, these urinesamples contained less than 10 ng DDSOor DDSD per ml. Thus, the sum of thesecompounds and their potential conjugatesdid not exceed 0.01% to 0.02% of the dailydose of DDS administered.

In a further study, urine was collected at12 hr intervals for a total of 48 hr from twovolunteers following a single 50 mg oraldose of DDS. After concentration by theXAD-2 resin column method, each samplewas tested for mutagenicity with TA98 andTA 100 by the standard procedure with andwithout S9 from Sprague-Dawley rats. Nomutagenic activity was detected when ali-quots ranging from 5 ill to 75 ill of DMSOextracts (equivalent to 1 ml to 15 ml ofurine) were tested. This study supports ourearlier observations that DDSO and DDSD,the mutagenic analogs of DDS, were absent

Rat S9^Microgramscompound/plate TAI535 TA1537 TA1538^TA98^TA 100

CompoundRevertants/plate'

ofPatient

no.dose

ng/mlof

urine

50^ International Journal of Leprosy^ 1 983

TABLE 4. Mutagenicity of DDSO and DDSD in Salmonella strains.

Negative controls^ 0^

23^5^16^18^115^0

^16^5^23^32^128

DDSO^

5 to 1000b^—1^6^

3^—1^515 to 10b^—5

^1^0^0^0

^

50^—3^

7^—2^255

^

100^

5^

40^—2^371

^

500^

8^

83^12^664

^

1000^1^76^25^1404

DDSD 5 to 50b100500

10005

1050

100500

1000

—6—7

—12—13

—386

141011

^

4^0^2 ^0

^

—1^—3—1^2

^

I^0^3 ^4^2 ^19

^

1^29^2 ^47

—2^26

13126

11183792

204227177

53131180201304475891

107413871316

Values represent averages of two experiments. For DDSO and DDSD, values arc the differences betweenthe numbers of revertants observed and the negative controls.

"The number of revertants listed arc the differences between the highest number of revertants observed atany concentration of drug and the negative controls.

from the urine of subjects receiving DDS;it further suggests the absence of any mu-tagenic metabolites of DDS in the urine ofhumans receiving this drug.

DISCUSSIONThe similarity of chemical structure be-

tween DDS and such strong rodent aro-matic amine carcinogens as benzidine, 4, 4'-

TABLE 5. Search for DDSO and DDSDin acid-Thdrolj ,zed urine .from patients."

DDSO^DDSD

ng/mlof

urine

1 <10 <0.01 <10 <0.012 <10 <0.01 <1 0 <0.013 <10 <0.01 <1 0 <0.014b <10 <0.01 <1 0 <0.015 <10 <0.02 <10 <0.026 <10 <0.01 <1 0 <0.017 <10 <0.01 <10 <0.018' <10 <0.02 <1 0 <0.029 _d <1 0 <0.01

° Samples were aliquots of 24 hr urine collectionsfrom patients receiving 100 mg DDS daily.

b 300 mg rifampin/day was also given.DDS dose was 50 mg/day.

° Not determined.

oxydianiline, 4, 4'-methylene-dianiline, and2, 7-fluorenediamine ( 13 ) makes DDS un-usual in its very weak carcinogenic activityfor rats. Most arylamines are activated toproximate carcinogens by N-hydroxylation( 20) but our studies have shown that neitherAHADS, the mono-N-hydroxy derivative,(HA),DS, the di-N-hydroxy derivative, nor(HAAc,),DS, a di-N-hydroxy derivativewherein the N-OH groups are protected byacetylation, are mutagenic under any con-ditions tested using the S. typhimuratinstrains (Table 3). Thus, these derivatives,along with DDS and its mono- and di-ace-tylated analogs, were not mutagenic underany test conditions we employed (Tables 3and 4).

Evidence of N-hydroxylation of DDS andthe possible formation of nitroso and azoxyanalogs of DDS was found in vitro with ratmicrosomes (s), and observations in manalso reported the formation of N-hydroxymetabolites of DDS ("). Using relativelynon-specific colorimetric methods, Uehlekeand Tabarelli ( 27) reported that N-hydroxymetabolites can comprise as much as 50%of the oral dose of 200 mg DDS to man.However, we found, using highly specificliquid chromatographic techniques, thatAHADS in 24 hr urines accounted for only

To ofdose

51, 1^Peters, et al.: Mutagenicity of Antileprosy Drugs^51

4.2% (mean of 6; S.D. = 2.3) of the dailydose of 100 mg of DDS taken by leprosypatients ("). Substantially more AHADSwas found after weak acid hydolysis of urinealiquots and reassay for AHADS. This mildtreatment yielded a mean increase corre-sponding to 27.6% (S.D. = 13.6%) of thedose. Thus, N-hydroxylated DDS metabo-lites are important urinary metabolites ofDDS in man although, in contrast with suchderivatives of other arylamines, these de-rived from DDS exhibit no mutagenic ac-tivity in the Ames screen. It would appearthat N-hydroxy compounds may not be thecause of tumors in rats receiving DDS.

A more likely suggestion is that the rat iscapable of converting small amounts of thehigh DDS doses used in the carcinogenesisstudies to the mutagenic DDSO or to thestrongly mutagenic and carcinogenic DDSD(Table 4). Recently, other authors (H) havealso reported that DDS is non-mutagenicand that DDSD is a strong mutagen of S.typhimurium strains TA98 and TA 100.However, we are not aware of any reportsat this time that DDS can be metabolicallyreduced to either DDSO or DDSD in therat or any other species. Many years agowhen DDSO was being considered as a ther-apeutic agent for leprosy chemotherapy, acomparative study of the urinary excretionof metabolites by man receiving DDS andDDSO was performed. The author ( 9 ) foundthat DDSO was converted to DDS but thatDDS was not excreted as DDSO. It appearsfrom this information that the fully oxi-dized sulfone form may be the end productof metabolism; whereas the reduced formssuch as DDSD and DDSO may be prefer-entially oxidized to the sulfone.

Our search for the presence of DDSD andDDSO in pharmaceutical preparations ofDDS and in urine, both in conjugated andunconjugated forms, yielded the conclusionthat these compounds were not present inpharmaceutical preparations of DDS nor inurine from patients receiving 100 mg DDSdaily. Also, using a technique that detectsmutagens readily in urine concentrates fromcigarette smokers (21 28 ), we could not detectmutagenic activity in urine from volunteerstaking DDS. Other workers ( 2) found thatDDS caused chromosome damage in hu-man lymphocytes at 4.0 Ag/m1 but not at0.4 pg/ml.

A number of epidemiologic studies haveexamined cancer incidence in leprosy pa-tients (22, 24, 26 , ,) and all concluded that lep-rosy patients do not exhibit statisticallyhigher cancer incidence than do controlpopulations. In only one study was drugtherapy considered. These authors ( 15 ) ana-lyzed their data in two time periods: priorto 1950, i.e., prior to sulfone or DDS ther-apy; and after 1950, when DDS therapy wasused extensively. In the lepromatous andthe tuberculoid groups of patients they foundrisk ratios of 1.6 and 1.4, respectively, forthe period before 1950. After 1950, theseratios were 1.5 and 0.6 for the two types ofdisease. No ratio was significantly differentfrom unity, however. Such results do notsuggest patients receiving DDS routinely areat undue risk.

Of the other antileprosy drugs we foundto be non-mutagenic in the Ames screen(Table 2), only rifampin and ethionamidehave been tested previously for mutagenicor carcinogenic activity. Rifampin was non-mutagenic in a number of test systems, car-cinogenic only in female mice of one strain,and not carcinogenic in rats. Rifampin incombination with other drugs for the che-motherapy of tuberculosis patients did notincrease the frequency of chromosomedamage in leukocytes ( 25 ). Ethionamide wasnot carcinogenic in mice and rats in thestandard bioassay of the National CancerInstitute ( 5 ).

SUMMARY'We tested the mutagenic activity of an-

tileprosy drugs (clofazimine, ethionamide,prothionamide, prothionamide-S-oxide,rifampin, and dapsone and many of its de-rivatives) using the Ames Salmonella/mi-crosome assay system. None of these, in-cluding N-acetylated and N-hydroxylatedderivatives of dapsone, were found to bepositive with or without metabolic activa-tion in this test. However, the sulfoxide andsulfide analogs of dapsone were found to bemutagenic with metabolic activation. Thesetwo analogs could not be detected in phar-maceutical preparations of dapsone(<0.01%), nor could they be found (in eitherunconjugated or conjugated form) in urinefrom volunteers taking a single oral dose of50 mg of dapsone or from patients receivingdaily oral doses of 100 mg of dapsone. Also,

52^ International Journal of Leprosy^ 1983

urine concentrates from volunteers taking50 mg of dapsone did not exhibit mutagenicactivity in the Ames screen. These resultsindicate that patients receiving antileprosytherapy with clofazimine, dapsone, ethion-amide, prothionamide, and/or rifampin arenot being exposed to mutagenic (and there-by possible carcinogenic) drugs.

RESUMENSc probO Ia actividad mutagenica de las drogas anti-

leprosas clofazimina, etionamida, protionamida, sul-foxido de protionamida, rifampina, dapsona y variosde sus derivados, usando el sistema de cnsayo de Amescon microsomas y Salmonella. Ninguna de ellas, in-cluycndo a los derivados N-acetilados y N-hidroxic-tilados de Ia dapsona, result() positiva en la prueba demutagenicidad ann despuOs de su activaciOn metab()-lica. Sin embargo, los derivados sulfOxido y sulfuro dela dapsona resultaron mutagênicos despuás de su ac-tivaciOn metabOlica. Estos dos andlogos no se pudierondemostrar en las preparaciones farmaceuticas dc dap-sona (<0.01%) ni en la orina de voluntarios que to-maron una dosis (mica de 50 mg de dapsona, ni en Iaorina de pacientes tratados con dosis orales diarias de100 mg de dapsona. Los concentrados de orina de losvoluntarios que tomaron 50 mg de dapsona tampocomostraron actividad mutagánica en el ensayo de Ames.Estos resultados indican que los pacientes en trata-miento con clofazimina, dapsona, etionamida, protio-namida, o rifampina, no estan expuestos a drogas mu-tagónicas, potencialmente carcinogênicas.

RÉSUMÉ

On a êtudie l'activitê mutagénique d'une sarie demedicaments contre Ia lepre (clofazimine, ethiona-mide, prothionamide, prothionamide-S-oxyde, rifam-picine, dapsone et plusieurs de ses dárivás), en ayantrecours au systême d'evaluation de Ames qui fait appelaux Salmonella et aux microsomes. Aucun de ces me-dicaments, y compris les derivês N-acetylês et N-hy-droxyles de la dapsone, n'ont revelê une activite mu-tagênique, que ce soit avec ou sans activationmatabolique au tours de l'êpreuve. Neanmoins, lesanalogues sulfoxydês et sulfidês de la dapsone se sontrêválês mutagêniques lorsqu'on avait recours a uneactivation matabolique. 11 n'a pas etc possible de de-teeter Ia presence de ces deux produits analogues, dansdes preparations pharmaceutiques de dapsone( <0.01%); it n'a pas átá davantage possible de les de-celer, tant sous la forme conjuguêe que non conjuguee,dans l'urine de volontaires auxquels on avait admi-nistrê une dose orale unique de 50 mg de dapsone, oudans l'urine de malades recevant des doses quoti-diennes de 100 mg de dapsone par voie buccale. Dememo, des concentrês d'urine de volontaires recevant50 mg de dapsone n'ont pas têmoigne d'activite mu-tagênique dans l'epreuve de Ames. Ces rêsultats in-diquent que les malades qui recoivent une thêrapeu-

tique antil6preuse avec Ia clofazimine, Ia dapsone,Páthionamide, la prothionamide, et/ou Ia rifamicine,ne sont pas exposés a des medicaments mutageniques,et des lors a des carcinogênes éventuels.

Acknowledgments. We thank Drs. R. H. Gelber (PHSHospital, San Francisco, California, U.S.A.) and R. R.Jacobson (National Hansen's Disease Center, Carville,Louisiana, U.S.A.) for providing urine collections fromtheir patients, and W. Tanaka and D. Kashiwase fortechnical assistance.

This study was supported in part by NIH Grant AI-08214.

REFERENCESI. AMES, B. N., MCCANN, J. and YAMASAKI, E.

Methods for detecting carcinogens and mutagenswith the Salmonella/mammalian-microsome mu-tagenicity test. Mutat. Res. 31 (1975) 347-364.BEIGUELMAN, B., PISANI, R. C. B. and EL GUINDY,

M. M. In vitro effect of dapsone on human chro-mosomes. Int. J. Lepr. 43 (1975) 41-44.

3. BERGEL, M. Actividad cancerigena de Ia diami-nodifenisulfulfona (DDS). Pub. Cent. Estud. Le-prol. 13 (1973) 30-40.

4. Bioassay of dapsone for possible carcinogenicity.Nat. Cancer Inst. CAS No. 80-08-0, NCI-CG-TR-20 (1977).

5. Bioassay of ethionamide for possible carcinoge-nicity. Nat. Cancer Inst. CAS No. 536-33-4, NCI-CG-TR-46 (1978).

6. Bioassay of 4, 4'-thiodianiline for possible carcin-ogenicity. Nat. Cancer Inst. CAS No. 139-65-I,NCI-CG-TR-47 (1978).

7. BLOOM, B. R. News about carcinogens: What's fitto print? Hastings Ctr. Rpt., August 1979,5-7.

8. CUCINELL, S. A., ISRAILI, Z. H. and DAYTON, P.G. Microsomal N-oxidation of dapsone as a causeof methemoglobin formation in human red cells.Am. J. Trop. Med. Hyg. 21 (1972) 322-331.

9. ELLARD, G. A. Absorption, metabolism and ex-cretion of di (p-aminophenyl) sulphone (dapsone)and di (p-aminophenyl) sulfoxide in man. Br. J.Pharmacol. Chemother. 26 (1966) 212-217.

10. GELBER, R., PETERS, J. H., GORDON, G. R., GLAZKO,

A. J. and LEVY, L. The polymorphic acetylationof dapsone in man. Clin. Pharmacol. Ther. 12(1971) 225-238.

11. GORDON, G. R., MURRAY, J. F., JR., PETERS, J. H.,GELBER, R. H. and JACOBSON, R. R. Studies onthe urinary metabolites of dapsone in man. Int. J.Lepr. 47 (1979) 681-682.

12. GRICIUTE, L. and TOMATIS, L. Carcinogenicity ofdapsone in mice and rats. Int. J. Cancer 25 (1980)123-129.

13. GfilswoLD, D. P., JR., CASEY, A. E., WEISBURGER,

E. K. and WEISBURGER, J. H. The carcinogenicityof multiple intragastric doses of aromatic and het-erocyclic nitro or amino derivatives in young fe-

51, 1^Peters, et al.: Alutugenicity of Antileprosy Drugs^53

male Sprague-Dawley rats. Cancer Res. 28 (1968)924-933.

14. ISRAILI, Z. H., CUCINELL, S. A., VAUGIIT, J., DAVIS,

E., LESSER, J. M. and DAYTON, P. G. Studies onthe metabolism of dapsone in man and experi-mental animals: Formation of N-hydroxy metab-olites. J. Pharmacol. Exp. Ther. 187 (1973) 135-151.

15. KOLONEL, N. L. and HIROHATA, T. Leprosy andcancer: A retrospective cohort study in Hawaii. J.Natl. Cancer Inst. 58 (1977) 1577-1581.

16. LAVOIE, E., TULLEY, L., Fox, E. and HOHMANN,

D. Mutagenicity of aminophenyl and nitrophenylethers, sulfides, and disulfides. Mutat. Res. 67(1979) 123-131.

17. LEONARD, N. J. and JOHNSON, C. R. Periodateoxidation of sulfides to sulfoxides. Scope of thereaction. J. Org . Chem. 27 (1962) 282-284.

18. MATSUO, Y., TATSUKAWA, H., MURRAY, J. F., JR.

and PETERS, J. H. Prothionamide and prothion-amide-S-oxide in experimental leprosy. Int. J. Lepr.49 (1981) 302-306.

19. MCCANN, J., CHOI, E., YAMASAKI, E. and AMES,

B. N. Detection of carcinogens as mutagens in theSa/monella/microsome test: Assay of 300 chem-icals. Proc. Natl. Acad. Sci. U.S.A. 71 (1975) 5135-5139.

20. MILLER, E. C. and MILLER, J. A. Mechanisms ofchemical carcinogenesis. Cancer 47 (1981) 1055-1064.

21. MORTELMANS, K., SIIAN, G., HAWK-PFIATHER, K.and PETERS, J. H. Application of mutagenicity

testing to detect mutagens in human urine. En-viron. Mutagen. 2 (1980) 305-306.

22. OLEINICK, A. Altered immunity and cancer risk:A review of the problem and analysis of the cancermortality experience of leprosy patients. J. Natl.Cancer Inst. 43 (1969) 775-781.

23. PETERS, J. H., GORDON, G. R., SININION, V. F. andTANAKA, W. Mutagenesis-carcinogenesis of an-tileprosy drugs in animal systems: Pertinence toman. Proc. XI Intl. Lepr. Cong., Mexico City,Mexico, 13-18 November 1978. F. Latapi, et al.,eds. Princeton, New Jersey, U.S.A.: ExcerptaMedica, 1980, p. 230.

24. PURTILLO, D. T. and PANGI, C. Incidence of can-cer in patients with leprosy. Cancer 35 (1975) 1259-1261.

25. RIFAMPIN. IARC Sci. Publ. Monographs on theEvaluation of the Carcinogenic Risk of Chemicalsto Humans 24 (1980) 243-258.

26. TOKUDOME, S., KONG, S., IKEDA, M., KURATSUNE,

M. and KUMAMARU, S. Cancer and other causesof death among leprosy patients. J. Natl. CancerInst. 67 (1981) 285-289.

27. UEHLEKE, H. and TAIIARELLI, S. N-hydroxylationof 4,4'-diaminodiphenyl sulfone (dapsone) by liv-er microsomes, and in dogs and humans. Naunyn-Schmied. Arch. Pharmacol. 278 (1973) 55-68.

28. YAMASAKI, E. and AMES, B. N. Concentration ofmutagens from urine by absorption with the non-polar resin XAD-2: Cigarette smokers have mu-tagenic urine. Proc. Natl. Acad. Sci. U.S.A. 74(1977) 3555-3559.