Mutation Research, 31 (1975) 87-95 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands

87

M I C R O S O M A L A C T I V A T I O N TO M U T A G E N S O F A N T I S C H I S T O S O M A L METHYL THIOXANTHENONES

A N D I N I T I A L T E S T S ON A P O S S I B L Y

NON-MUTAGENIC ANALOGUE*

PHILIP E. HARTMAN D. TAYLORa, d

a, PETER

B. HULBERTb,

e, ERNEST

BUEDING

b AND

DERRICK

Departments of Biology, Pharmacology and Pathobiology, The Johns Hopkins University, Baltimore, Md. (U.S.A.) (Received September 27th, 1974)

SUMMARY F i v e m e t h y l t h i o x a n t h e n o n e a n d m e t h y l b e n z o t h i o p y r a n o i n d a z o l e analogues, including l u c a n t h o n e (Miracil D), are n o n - m u t a g e n i c for Salmonella typhimurium b u t are a c t i v a t e d to m u t a g e n s b y a r a t liver microsome p r e p a r a t i o n . H y d r o x y m e t h y l analogues, including h y c a n t h o n e (Etrenol), are m u t a g e n i c in the absence of microsomes. I t seems reasonable to assume t h a t t h e h y d r o x y m e t h y l d e r i v a t i v e s are the more p r o x i m a l m u t a g e n s a n d t h a t Salmonella is unable to c a r r y out the h y d r o x y l a t i o n necessary for n m t a g e n a c t i v a t i o n . D u r i n g the p a s t 24 years, several million p a t i e n t s with schistosomiasis have been t r e a t e d with lucanthone, a n d in recent y e a r s a b o u t 700000 persons with h y c a n t h o n e . The possible l o n g - t e r m deleterious effects of these agents for m a n even now r e m a i n to be d e t e r m i n e d . Our studies indicate t h a t p a r t i c u l a r modifications in t h e s t r u c t u r e of t h i o x a n t h e n o n e s d r a s t i c a l l y a l t e r their n m t a g e n i c i t y . One a p p a r e n t l y n o n - m u t a g e n i c t h i o x a n t h e n o n e has been found. A n u m b e r of t h e less m u t a g e n i c c o m p o u n d s also e x h i b i t decreased acute t o x i c i t y in the mouse while r e t a i n i n g a p p r e c i a b l e antischistosomal a c t i v i t y , suggesting t h a t genetic a n d schistosomicidal activities m a y be dissociated from each other.

INTRODUCTION

H y c a n t h o n e (Etrenol), some other h y d r o x y m e t h y l - s u b s t i t u t e d t h i o x a n t h e nones, a n d some s t r u c t u r a l l y r e l a t e d b e n z o t h i o p y r a n o i n d a z o l e s used or u n d e r in* This work was supported in part by Research Grant Roi Aioi65o of the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service (to P.E.H.) and National Science Foundation and Rockefeller Foundation Grants (to E.B.). Contribution No. 795 of the Department of Biology, Johns Hopkins University. a The Department of Biology: address reprint requests. b Department of Pathobiology and Department of Pharmacology. c Present address: Department of Pharmaceutical Chemistry, University of Bradford, Bradford, Yorkshire (Great Britain). d Present address: Boston University School of Medicine, 8o East Concord St., Boston, Mass. o2118 (U.S.A.).

88

P . E . HARTMAN gt

al.

vestigation for use in treatment of schistosomal infections are active frameshift mutagens in Salmonella typhimurium. The methyl-substituted analogues, including !ucanthone (Miracil D), are inactive in this test systemg, 11. In contrast, a number of the methyl-substituted derivatives share mutagenic activity with the hydroxymethyl compounds in, for example, mouse lymphoma cellsS, 6. Both hycanthone and lucanthone transform Rauscher virus-infected rat embryo cell cultures, but the hydroxymethyl derivative (hycanthone) does so at a higher frequency than the methyl derivative (lucanthone) 1~. Here we demonstrate that an NADPH-dependent rat liver microsomal enzyme system is able to metabolize the methyl derivatives into compounds effective in mutagenesis of Salmonella. It seems possible that the hydroxymethyl analogues are more closely related to the proximal mutagens and potential carcinogens than are the methyl derivatives which m a y require metabolic activation. Recent studies have encouraged the view that the genetic activity of the ttlioxanthenones and benzothiopyranoindazoles might be dissociated from antischistosomal activity by appropriate structural modifications in the drug molecules4-6,% 24. Here we demonstrate that N-oxide analogues la are relatively less mutagenic for Salmonella and that this inefficiency is only partially due to lesser penetration. We have been unable to detect mutagenic activity for Salmonella with one Farticular thioxanthenone analogue and discuss the significance of this observation. MATERIALS AND METHODS

Tile isolation of the Salmonella typhirnurium strain LT-2 frameshift mutations used in our reversion tests have been described elsewhere ~°. Sub-strains defective in cell wall biosynthesis and in DNA repair were obtained front Dr. B. N. AMES (strains TAI534, TAI535, TAI537, TAI538, TAI952, TAI978) 3 or by P22-mediated transduction into strain No. 712-1, that is, strain SB849, defective in DNA-repair (hcr-z) (refs. 22, 28). Mutagenic responses to chemical agents were tested b y two methods. The "direct plating test" consisted of plating together the agent and about 2 .lO 8 bacteria from a saturated Difco-nutrient broth culture on to VOGEL-BONNER J~ medium ~5 containing 0.2% glucose, 1.25% (by volume) liquid Difeo-nutrient broth and, in cases where uvrBA mutants were used, 4 #g/ml biotin. The "agar-layer test" was as described by AMES et al. 1 except that histidine was omitted from the agar overlay which was poured on to plates containing the broth-supplemented medium just described. In the agar layer test, "S-9" microsomes from phenobarbital-treated male SpragueDawley rats were prepared as described by AMES et al. 1 and incorporated along with NADP and glucose-6-phosphate as an efficient regenerating system for NADPH, the substrate for microsomal mixed-function oxidation of the test chemicals 1. The above media allow several rounds of DNA synthesis and cell division in the presence of chemical agents at sub-inhibitory concentrations, allowing detection of mutations that m a y only occur during DNA replication (cf. ref. 18). Experimental data are average values in each case for two or more plates and, for controls, 4 or more plates. RESULTS

Fig. I shows the structures of the methyl- and hydroxymethyl-substituted

89

MUTAGENIC ANTISCHISTOSOMAL AGENTS 0

R3

N--N--R3

R2

R2

Ring A (thioxan thenones)

Agent

Ring B (benzothiopyranoindazoles)

Ring

RI

R2 -CH3

Luconthone

A

-H

Lucanthone N-oxide

"

*'

Chlorolucanthone N-oxide

"

-CI

Hycanthone Hycont hone

-H

_NHCH2 CH2IN(C2 HS)2 O® -CHaOH-NHCH2CH2N(C2 HS)2 _NHCHzCH2~I (C2 HS)2 OE~

"

-CJ

Piperazine #ION-oxide

-NHCH2CHEN(C2 HS} 2

E~

N-oxide

P i p e r a z i n e # I0

R3

-CH 3

-N

"

-N

"

N-CH 3

/--Xo

N-CH 3

k---/\o o IA-5 IA-5

-CH2CH2N (C2H5) 2

N-oxide

-CH2CH2~. (C2H5) 2

60

IA-4

-CH20H - CH2CH2 N(C2 HS)2

I A - 4 N-oxide

-CH2CH2I~(C2H5)2 o®

Fig. I. S t r u c t u r e s of t h i o x a n t h e n o n e s a n d b e n z o t h i o p y r a n o i n d a z o l e s t e s t e d for m u t a g e n i c i t y . P i p e r a z i n e N o . i o is 6 - c h l o r o - 4 - m e t h y l - i - ( 4 - m e t h y l - i - p i p e r a z i n y l ) - t h i o x a n t h e n - 9 - o n e .

TABLE I ACTIVATION OF LUCANTHONE BY RAT LIVER MICROSOMES plus NADPH-REGENERATING SYSTEM IN THE AGAR LAYER TEST A v e r a g e n u m b e r of His + r e v e r t a n t s p e r p l a t e a b o v e s p o n t a n e o u s (control s u b t r a c t e d ) . Agent

Concentration ofagent (~moles/plate)

Bacterial strain T A 1538 = hisD3o52 uvrBzJ rfa T A 1537 = hisC3o67 uvrBz] rfa Additions Additions None MicroMicroN one MicroMicrosomes somes 4somes somes + NA DPH a NA DPH a

Hycanthone 0.0055 m e t h a n e s u ] f o n a t e 0.022 o.o55 0.22

21 76 179 615

8 71 149 437

Lucanthone hydrochloride

o 3 2 Inhibition

o o 13 Inhibition

22

32

Spontaneous

0.007 0.028 0.07 0.28

6 41 133 588 4 20 29 36 20

35 127 305 lO47

5 36 lO5 493

38 38 87 497

4 6 8 Inhibition

i o 9 21

6 I9 25 47

8

7

6

a N A D P H i n d i c a t e s a d d i t i o n of g l u c o s e - 6 - p h o s p h a t e a n d N A D P (TPN).

9°

p.E. HARTMAN el al.

thioxanthenones and benzothiopyranoindazoles (hereafter termed indazoles) used in our tests for mutagenicity. The upper left quadrant of Table I shows that the hydroxymethyl compound, hycanthone, is active in reverting Salmonella frameshift strain TAI538 in the absence of microsomes. Strain TAI538 contains a repeating CGCGCGCG/GCGCGCGC sequence and reverts only by intragenic frameshift mutations 14. The number ot revertants is proportional to hycanthone dosage, as portrayed in more detail elsewhere 9. There m a y be a slight quenching of mutagenicity upon the addition ot microsome% and this is relatively independent of the presence of N A D P H in the microsomal extract. The upper right quadrant of Table I presents similar data for reversion of strain TAI537 which contains a CCCC/GGGG sequence susceptible to frameshiit mutagenesis (reviewed in ref. 3). The lower portion of Table I shows the relative inactivity of the methylsubstituted analogue, lucanthone, in eliciting reversion of these two bacterial strains in the absence of microsomes. The small numbers of colonies found without microsome addition ("None" colunm) are not significantly above the background spontaneous frequencies (listed at the bottom of the Table and subtracted from data in the body TABLE

II

ACTIVATION OF I A - 3 BY RAT LIVER MICROSOMES AGAR LAYER TEST

plus

N A D P I ~ - R E G E N E R A T 1 N G SYSTEM IN THE

Average number of His + revertants per plate above spontaneous.

Agent

Concentration Bacterial strain of agent T A I 5 3 8 =hisD3o52 uvrBA rfa TAz537 =hisC3o76 uvrBA rfa (Itmoles /p late) Additions Additions None Microsomes + None Microsomes + NA DPH NA DPH

IA- 4

0.005 o.o21 0.05 o.21

6 12 38 74

o 28 64 59

0.005 o.o21 0.05 o.21 -

8 o o Inhibition 34

7 4 12 35 38

nlethanesulfonate

IA 3

methanesulfonate Spontaneous TABLE

75 143 346 Inhibition

17 36 76 386

o o o

o o 60 399 14

Inhibition 9

III

LOWERED MUTAGENICITY OF HYCANTHONE N - O X I D E AS OPPOSED TO HYCANTHONE IN THE DIRECT PLATING TEST

Average number

of H i s +

revertants per plate above spontaneous.

Bacterial strain SB8o52 TAI534 TAI538 TAI978 SB8o76 TAI952 TAI537

= ~ ~ = ~ ~ ~

D3o52 D3o52 uvrB D3o52 uvrB rfa D3o52 rfa C3o76 C3o76 uvrB C3o76 uvrB rfa

Hycanthone (free base) (0.28 #mole/plate)

Hycanthone N-oxide (j~'ee base) (2. 7 izmole/plate) (0.27 #mole/plate)

4 2o6 192o 816

15 65 3Ol 14

16 15 ioo 4

o

o

o

84 1919

3° 845

o o

MUTAGENIC ANTISCHISTOSOMAL AGENTS

91

of the Table). The data presented are averages of colonies on duplicate plates in but one of a series of experiments, and other tests have shown lucanthone to be inactive. There may be a slight induction of mutations by higher concentrations of lucanthone upon the addition of microsomes alone; however, this mutagenicity is decidedly enhanced in the presence of NADPH. There is some quenching of the inhibition of strain TAI537 by the addition of microsomes. This could either be due to binding to microsomes of lucanthone or to its metabolism to a form less active in mutagenesis. Data in Table II show that the hydroxymethyl-substituted indazole compound, IA- 4, actively reverts the same two Salmonella strains while the methylsubstituted analogue, IA-3, is inactive unless microsomes and NADPH are present. The inability of methyl-substituted thioxanthenoI~es and indazoles to revert the Salmonella mutants while the hydroxymethyl-substituted derivatives are active indicates that the latter may be more closely related to the actual mutagens. Salmonella appears unable either to hydroxylate the methyl derivatives or to metabolize them in other ways to the actual mutagens. This is not merely due to the presence in the strains of uvrB deletion mutations, for tests with uvr+ strains and with a repairdefective point mutation (e.g. strain SB2294 ---- hisF3o3~ hisoI242 hcr-z) show the same differential effects of lucanthone and hycanthone in eliciting frameshift mutations (data not shown). The varying mutagenic activities of the above compounds encouraged a search for further analogues with still less mutagenicity. The data in Table III show that hycanthone N-oxide (P. HULBERT AND E. BUEOING, in preparation) exercises lower mutagenic activity in reverting the histidine frameshift mutations, hisD3o52 and hisC3o76, in a variety of genetic backgrounds 3 than does hycanthone. However, the N-oxide also is less toxic to bacterial growth, and an increase in N-oxide level results in increased mutagenesis (Table III). Therefore, in this case mutagenesis largely may reflect decreased penetration of the drug. Similar data have been obtained by D. S. STRAUS (personal communication). Screening of fftrther analogues was carried out by STRAUS et al. 24 who demonstrated greatly decreased mutagenesis in Salmonella of two 4-hydroxymethyl-Ipiperazinyl analogues of hycanthone. Methyl-substituted analogues ot these two compounds were inactive in their tests carried out in the absence of microsomes. Table IV shows the barely detectable mutagenic activity of one of the methylpiperazyl derivatives, compound No.Io of STRAUS et al. 24 (Fig. I), as tested here in the presence of microsomes. Comparative data in tests with other compounds in the same experiments or in similar expeliments (Table IV, footnote b) also are presented in Table IV. Included are newly synthesized N-oxide derivatives (P. B. HULBERT AND E. BUEDING, in preparation), some ot which show enhanced antischistosomal activity 13. The data in Table IV demonstrate the lessened mutagenicity for Salmonella strain TAI538 of compounds containing the following structural features: indazole ring system (also compare Tables I and II), methyl group, piperazinyl ring, and N-oxide function. Combination of the latter three modifications (piperazinyl No.Io N-oxide) eliminates detectable mutagenesis although penetration appears nearly as effective as penetration of hycanthone as judged by the close end-points at which bacterial growth inhibition is obtained. A total of 13.4/,moles of the piperazinyl N-oxide failed to induce detectable mutation when tested at sub-inhibitory concentrations on a total of 20 plates including microsomes and NADPH. This amount

92 TABLE

P.E.

H A R T M A N et

al.

IV

COMPARATIVE MUTAGENIC ACTIVITIES BY THE AGAR LAYER TEST OF HYCANTHONE AND SOME ANALOGUE~ STRAIN T A I 5 3 8 (hisD3o52 uvrB/] rfa) IN THE ABSENCE AND IN THE PRESENCE OF RAT LIVER MICROSOMES NADPH A v e r a g e n u m b e r of H i s + r e v e r t a n t s p e r p l a t e a b o v e s p o n t a n e o u s ( c o n t r o l s u b t r a c t e d ) .

Concentration of agent (#moles/plate)

o.I O.5 I.O 2.o

Agent a in presence ( + ) or absence (0) of microsomes plus N A D P H Hycanthone Hycanthone Lucanthone Lucanthone (--CH20H) N-oxide (--CHs) N-oxide ( -- CH20H ) ( -- CH3)

Chlorolucanthone N-oxide (--CH3)

0

+

0

+

0

+

0

+

0

248 1562 Inh a Inh

212 1262 Inh Inh

13 54 NT e 193

IO 56 NT 198

°b Inhb NT Inh

3 ob 7 ob NT Inh

4 3 NT Inh

14 22 NT Inh

o o o Inh

of drug similarly tested would have produced over IO ooo revertants were hycanthone used and over IOOO revertants were lucanthone used. Previous experiments have demonstrated that hydroxymethyl-substituted thioxanthenones and structurally related benzothiopyranoindazoles only revert Salmonella strains carrying certain frameshift mutations and do not revert strains carrying base-substitution mutationsg,U, 24. Similarly, no mutagenicity in reversion ot the base-substitution mutation in strain TAI535 (hisG46 uvrBA rfa) was detected for lucanthone or for piperazine No. IO and piperazine No. IO N-oxide when o.5/,mole of the test compound was plated on each of two plates in the presence of microsomes plus substrate. DISCUSSION

Our interest in the compounds tested here (Fig. I) stems from the widespread use of lucanthone and, now, of hycanthone in treatment of human schistosomiasisL the clearly demonstrated mut agenicity of hycanthone in several test systems 5,6,9,1i, 15,17 and the lessened mutagenicity6,~, 17 yet high antischistosomal activity of some indazole derivatives closely related in structure to lueanthone and hycanthone 4. These indications that particular structural modifications might dissociate mutagenicity from antischistosolnal activity have encouraged a search for still more beneficial modifications. In the series of compounds tested to date, it appears that the hydroxymethyl substituent is requisite for effective mutagenesis in Salmonellag,n, ~4 and important for antisehistosomal activity in the monkey and in man 21. The hydroxymethyl substituent also m a y be requisite for earcinogenesisS, 12 since the frequency of transformation of Rauscher virus-infected cell cultures is approximately Io-fold lower following treatment with lucanthone than it is with hycanthone 1~. On the other hand, the latter compound shows lower cytogenetic activity 19, and the mutational responses of tissue culture cells to various analogues are as yet somewhat unpredictable 6. We show here that methyl-substituted thioxanthenones and a methyl-substituted benzothiopyranoindazole which are inactive in mutagenesis ot Salmonella, can be metabolized to nmtagens by an NADPH-dependent rat microsomal enzyme system. A similar activation to proximal mutagens has been demonstrated for a variety of

: I~ 8: Ii

93

MUTAGENIC ANTISCHISTOSOMAL AGENTS

Piperazine No. Io (--CH3)

Piperazine No. zo N-oxide ( -- CH3)

IA -3 (-- CH3)

IA-3N-oxide ( -- CH3)

IA-4 (--CH2OH)

IX-4 N-oxide (--CH2OH)

O

+

O

+

O

+

0

+

O

+

O

+

9 3 Inh Inh

o io Inh Inh

o o o Inh

o o o Inh

o Inh NT Inh

6 7° NT Inh

oe o o Inh

o 5 8 Inh

i5 b I 5 ob NT NT

4 ob Ioob NT NT

9 2o

15 18 NT 29

a All compounds

are the methanesulfonate

NT 19

salts.

b Extrapolated from data in Tables I and II. c Low numbers of excess colonies on occasional plate. o Inh, inhibition of bacterial growth. e NT, not tested. other compounds1,16. A distinct possibility, susceptible to future examination is the simple conversion of the methyl compounds to the more directly mutagenic hydroxymethyl derivatives. It is possible that Salmonella is deficient in this hydroxylation reaction, just as hydroxylation in man and the monkey appears to be less than in hamsters and in hamsters less than in mice 21. One class of possible metabolites still containing methyl groups, the sulfoxide derivative "i, for two reasons appears less likely as a candidate for mutagenesis of Salmonella. First, while hycanthone sulfoxide is mutagenic (T. M. Cook AND C. A. KING, cited in ref. 9), it is quantitatively less active than hycanthone itself (D. S. S T R A U S ; T . M . C O O K A N D C . K . G O L D M A N , personal communications). Secondly, in tests with a variety of analogues, only the hydroxymethyl derivatives are mutagenic ~4. Differential rates of metabolism to these various derivatives could account for variations in genetic activities of hycanthone and its analogues found in mammalian systems. An additional variation could arise from polyamine content of various tissues and fluids since polyamines partially reverse the inhibitory effects of lucanthone in E . coli ~7 and of growth inhibition and mutagenesis in Salmonella (P. E. HARTMAN, unpublished). Although other possibilities exist 9, it seems likely that the mutagenic activity of hycanthone and its analogues is due to metabolism to (a) derivative(s) able to react covalently with DNA while still retaining ability to intercalate, as indicated for other potent mutagens and carcinogens". This would account for the increased level of mutagenesis detected in DNA repair-defective Salmonella mutants (Table I I I and ref. 9). Clearly, the DNA of repair-defective treated cultures should be examined for its content of covalently bound agents and repair-efficient strains for release of a deoxynucleotide-bound agent. Knowledge of the ultimate mutagen might assist in the design of a completely non-mutagenic substitute for hycanthone. Elsewhere"-l~,~ we have reviewed the evidence indicating that hycanthone is exclusively a frameshift mutagen in Salmonella and, like irradiation and most frame-

94

e . E . HARTMANgt al.

shift mutagens, elicits m a i n l y or exclusively base-pair deletions (in contrast to I C R compounds which also elicit base-pair additions2°,24). Originally we t h o u g h t t h a t h y c a n t h o n e - i n d u c e d base-pair deletions m i g h t be restricted to G/C-rich nueleotide sequences (cf. 14) a n d t h a t this m e c h a n i s m might solely underlie the carcinogenic a c t i v i t y of this a n d other agentsl-a, 16. However, these ideas are open to question because several n m t a g e n s including an i n t e r c a l a t i n g a n d a l k y l a t i n g chemical, ICR372, a d d i t i o n a l l y induce t a n d e m gene duplications in E. Coli ~3. Also, at least in yeastlL p r e l i m i n a r y results indicate t h a t h y c a n t h o n e causes a v a r i e t y of genetic changes, including base substitutions, in contrast to its a p p a r e n t specificity for base-pair deletion in Salmonella. I n this report we show t h a t a n N-oxide derivative of a previously tested piperazine 24 is not detectably m u t a g e n i c in Salmonella, yet this c o m p o u n d retains a b o u t 3o% of the antisehistosomal a c t i v i t y of h y c a n t h o n e in the mouse la. The c o m p o u n d also has a chlorine s u b s t i t u t i o n (Fig. I), a n d of the two chloro-substituted benzothiopyranoindazoles la tested, each is seven times less toxic for the mouse t h a n is its deschloro analogue. Thus, while only relatively small samples of a restricted n u m b e r of analogues have been examined, the observations reported here further encourage the view t h a t genetic a n d schistosomicidal activities m a y be dissociated from each other. Clearly, further testing is required in other n o n - h u m a n systems in order to discern if, in fact, a n effective antischistosomal c o m p o u n d w i t h o u t genetic a c t i v i t y can be a d m i n i s t e r e d to the m a n y million persons 7 infected with schistosome species susceptible to the t h i o x a n t h e n o n e class. ACKNOWLEDGEMENTS

We t h a n k Dr. S. ARCHER for supplying h y c a n t h o n e , Drs. E. ELSLAGER a n d F. J . DE GERRES for providing IA-3 a n d IA-4, a n d Drs. E. ELSLAGER a n d D. WORTH for samples of the two piperazil analogues. REFERENCES I AMES,B. N., ~vV.DURSTON, E. YAMASAKIAND F. D. LEE, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.), 7° (1973) 2281-2285. 2 AMES, B. N., E. G. GURNEY, J. A. MILLER AND H. BARTSCH,Carcinogens as frameshift mutagens; metabolites and derivatives of 2-acetyl-aminofluorene and other aromatic amine carcinogens, Proc. Natl. Acad. Sci. (U.S.), 69 (1972) 3128-3132. 3 AMES, B. N., F. D. LEE AND W. E. I)URSTON, An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.), 7° (1973) 782 786. 4 BUEDING, E., J. FISHER AND H. BRUCE, The antischistosomal activity of a chloroindazole analog of hycanthone in mice infected with Schistosoma mansoni, J. Pharmaeol. Exptl. Therap., 186 (1973) 4o2 4o7 . 5 CLIVE, D., Recent developments with the L5178Y heterozygote mutagen assay system, in F. J. DE SERRES AND W. SHERIDAN(Eds.), The Evaluation of Chemical Mutagenicity Data in Relation to Population Risk, Environmental Health Perspectives No. 6, U.S. Department of Health, Education and Welfare, 1973, pp- 119-125. 6 CLIVE, D., Mutagenicity of thioxanthenes (hycanthone, lucanthone and four indazole derivatives) at the TK locus in cultured mammalian cells, Mutation Res., 26 (1974) 307--318. 7 FRIEBEL, H., Drug safety in theory and practice, WHO Chronicle, 27 (1973) 59-65. 8 HAESE, W. H., D. L. SMITH AND E. BUEDING, Hycanthone-induced hepatic changes in mice infected with Schistosoma mansoni, J. Pharmacol. Exptl. Therap., I8, (1973) 430-44 °. 9 HARTMAN, P. E., H. BERGER AND Z. HARTMAN, Comparison of hycanthone ("Etrenol"), some

MUTAGENIC ANTISCHISTOSOMAL AGENTS

IO

II

12 13

14 15

16 17 18

19 20

21 22

23 24

25 26 27

28

95

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