105

Mutation Research, 54 (1978) 105--112 © Elsevier/North-Holland Biomedical Press

Short Communication

COMUTAGENICITY, COMPETITIVE ENZYME SUBSTRATES, AND IN VITRO CARCINOGENICITY ASSAYS

JOHN ASHBY and J.A. STYLES

Imperial Chemical Industries Limited, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire (Great Britain)

(Received 12 December 1977) (Revision received 21 February 1978) (Accepted 27 February 1978)

Sugimura and his colleagues recently reported that the mutagenic potency of 4~limethylaminoazobenzene (DAB; I) in the Ames assay can be increased (by about 40-fold) when tested in the presence of norharman (II) [23,31]. Since that report, Nagao, from the same laboratory, has observed that compounds such as aniline (III) and o-toluidine (IV) produce a positive response in this assay in the presence of norharman [22], despite the. fact that each is negative when tested alone [14,22]. Sugimura et al. [31] have indicated that attempts to devise a new screening method, to be based upon these observations, are in hand, in his laboratory.

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During the last year or so the response given by the Ames assay to several compounds has changed from negative to positive following modifications to the test protocol, examples being the fluorocarbon F22 [7], the fire-retardant Fyrol FR2 [25], ascorbic acid [29], 4-dimethylaminoazobenzene (DAB} [1,5, 23,26,31], aniline (III) [14,22], o-toluidine (IV) [14,22] and azathioprine [27]. Although each of these modifications seems defensible in isolation, the time is approaching when a critical assessment will have to be made of what the test protocol of the Ames and related assays should be, and what limitations should be set on modifications. This is primarily because it is not yet clear whether all such modifications uniquely enable negative in vitro test responses for animal carcinogens to be transformed into positive responses, or whether they will also entail, as seems possible, the co-generation of an unacceptably high proport ion of false positive in vitro predictions of in vivo carcinogenic activity. This could result in these tests reverting to their original and limited role of simply defining microbial mutagens. As the possible addition of nor- harman to the medium of in vitro assays represents the most fundamental change so far suggested, and as its possible implications are so great, it is discussed below using the model carcinogens selected by Sugimura et al. and Nagao, namely, 4-dimethylaminoazobenzene (DAB) (I) and o-toluidine (IV).

Whilst DAB (I) usually gives a negative [1,5,23] response in the Ames assay when tested alone, it is highly mutagenic when tested in the presence of norhar- man [21,23]. Further, norharman has been shown to alter the overall in vitro metabolism of both this and other carcinogens [23]. One of the possible explanations for the above enhanced mutagenic effects is that partial denatura- tion of the bacterial DNA, brought about by norharman [9] , potentiates the reaction of active chemical species with genetic material [ 23 ]. But this explana- tion is not supported by the observation that the in vitro mutagenic potency of some direct-acting mutagens such as 4-nitroquinoline-N-oxide or the direct- acting N-acetoxy derivative of DAB are unaffected by the addition of nor- harman to the assay medium [21,23] , or by the observation that harman is less effective in vitro than norharman despite the fact that it intercalates with DNA more easily than does norharman [9]. An alternative explanation, involving critical norharman-mediated changes to the in vitro metabolism of those muta- gens requiring activation, is therefore more attractive and this explanation implies that substrates other than norharman might achieve similar effects. Direct-acting mutagens can be detoxified either by the S-9 mix [ 6,21] or perhaps even by the bacteria themselves, in the absence of S-9 mix. It is therefore pos- sible that the observed mutagenic potency of some direct-acting mutagens could be increased by the addition of an appropriate competitive detoxification substrate to the assay medium.

4-Dimethylaminoazobenzene (DAB) (I) was first defined as a rat-liver car- cinogen by Kinosita [12], in Japan. However, initial at tempts to repeat these in vivo experiments in America were unsuccessful [2]. This divergence of results was finally at tr ibuted to test animal diet variation between the two countries -- in particular, the Japanese animals had low levels of liver azoreduc- tase enzymes, a deficiency eventually associated with their low riboflavin intake [2]. It is therefore possible that the variations in the carcinogenic potency of DAB observed in vivo might be reflected as variations in the muta-

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genic potency of DAB in an in vitro assay if variations in the levels or activities of these enzymes in the microsomal S-9 mix occurred.

The scheme shows a simplified picture of the metabolic pathways available to DAB in the presence of rat-liver enzymes, either in vivo or in vitro. Oxidative N-demethylat ion and N-hydroxylat ion of DAB is a plausible and suggested mutagenic and carcinogenic pathway (Scheme, pathway a) [2,13]. Acting in competi t ion to this is molecular cleavage of DAB at the azo-linkage by azo- reductase enzymes [11,13,15,19,20] (pathway b) and ring hydroxylat ion of the intact molecule or its monodemethyla t ion product followed by conjugation (pathway c) [13,32]. It is possible that in the in vitro experiments discussed above, norharman is acting as an alternative substrate for C-hydroxylation enzymes (pathway c), thereby increasing the relative contr ibution made by the mutagenic pathway in the overall metabolism of DAB by the S-9 mix. This could lead to the observed increase in mutagenic in vitro potency.

We had independently evaluated the above possibility by determining the cell-transforming ability of DAB in both the presence and absence of an alter- native substrate for the azoreductase enzymes. We have previously shown that DAB is consistently positive in the cell transformation assay of Styles [ 3 ] when using the standard, induced rat-liver S-9 mix and normal test protocol [30]. Fig. 1 represents the composite response produced by this chemical over 7 separate experiments. The negative test response given by this assay for DAB either in the absence of S-9 mix or when using uninduced S-9 mix confirms that the metabolic activation of DAB is not only necessary but critically dependent upon the type of S-9 mix employed. In our experiment the non-carcinogen azobenzene (V) [28] was selected to act as a competitive substrate to DAB for both the C-hydroxylase and azoreductase enzymes of the S-9 mix. If azoben- zene were to act in this way an increase in the test response for a given dose of DAB would be expected. 3 experiments were conducted simultaneously.

In the first, the carcinogen DAB (I) and the structurally related, putative non-carcinogen, 3-methyl-DAB (VI) [16--18] were tested in the presence of

108

induced rat-liver S-9 mix, giving the expected positive (Fig. 2) and negative response (Fig. 3) respectively. The non-carcinogen azobenzene (V) was evaluated separately and gave a negative response (Fig. 4). In the third experiment a one- to-one mixture of DAB (I) and azobenzene (V) was evaluated at each dose level (e.g., 83 pg DAB + 83 pg azobenzene at the 83 pg dose-level). The increased positive response obtained is shown in Fig. 5, an approximately 25-fold increase in cell-transforming potency being apparent at the 83-pg dose-level. In these experiments, whose results parallel closely those produced in vitro by testing DAB in the presence of norharman, and those produced in vivo by blocking 4 '-hydroxylat ion of DAB with an ethyl group [18], azobenzene has most probably simply potent iated the mutagenic efficacy of the S-9 mix for DAB. With other azo compounds the reverse effect might be observed. For example, Garner has shown that the in vitro reductive splitting by azoreductase enzymes of the azo dye chrysoidine (VII) favourably contributes to the ob- served mutagenic potency of this compound [8]. In this case, therefore, the presence of azobenzene in the test medium might well reduce the observed mutagenic potency.

The term comutagenicity was used by Sugimura et al. [23,31] to describe

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Fig. 1- -5 . The g e n e r a t i o n , p r e s e n t a t i o n and i n t e r p r e t a t i o n of these resul t s is as p rev ious ly desc r ibed [ 3 0 ] .

The t r a n s f o r m a t i o n f r equenc i e s ( co r r ec t ed to a t h e o r e t i c a l L D 0 ) and the cell survxvals o b t a i n e d a f t e r t reat- m e n t o f the cells w i t h 4 - d i m e t h y l a m i n o a z o b e n z e n e (I) [as an average o f 7 e x p e r i m e n t s ] (Fig. 1),

4 - d i m e t h y l a m i n o a z o b e n z e n e (1) as t e s ted in the p r e sen t e x p e r i m e n t (Fig. 2), 3 - m e t h y l - 4 - d i m e t h y l a m i n o - a z o b e n z e n e (VI ) (Fig . 3) , a z o b e n z e n e (V) (Fig . 4) and a 1 : 1 m i x t u r e of 4 - d i m e ~ h y l a m i n o a z o b e n z e n e (I)

and a z o b e n z e n e (V) (Fig . 5) are shown . All e x p e r i m e n t s were c o n d u c t e d us ing dup l i ca t e p la tes at each dose level. Ful l deta i ls o f these and re la ted e x p e r i m e n t s will be r e p o r t e d e l sewhere [ 5 ] .

The B H K 2 1 / C 1 3 cells used in these e x p e r i m e n t s were a new sub-clone of c lone 13. The s p o n t a n e o u s t r a n s f o r m a t i o n f r e q u e n c y o f th i s new clone is 10 in con t ra s t to the p rev ious ly e m p l o y e d c lone (as used for the e x p e r i m e n t s s h o w n in Fig. 1) w h i c h had a s p o n t a n e o u s t r a n s f o r m a t i o n f r e q u e n c y o f 50 [ 3 0 ] . This is r e f l ec t ed as a change in the pos i t i on o f the h o r i z o n t a l d o t t e d line in Figs. 2--5 f r o m those p rev ious ly desc r ibed [ 3 0 ] and as s h o w n in Fig. 1. Pos i t ive resul t s w e r e d e f i n e d b y a 5-fold increase in t r a n s f o r m a t i o n f r e q u e n c y (ho r i zon t a l d o t t e d l ine) at the L D s 0 dose level (ver t ical d o t t e d l ine) over c on t ro l values.

T h e t r a n s f o r m a t i o n f r e q u e n c y seen in the m i x e d 4 - d i m e t h y l a m i n o a z o b e n z e n e / a z o b e n z e n e e x p e r i m e n t (Fig. 5) was the h ighes t ye t obse rved wi th th i s assay (10 4 per 10 5 surv ivors , i.e. 1% t r a n s f o r m a t i o n ) .

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the effects produced on DAB by norharman, but this could not be used to describe a reduction in mutagenic potency. We have therefore not adopted it to describe the effects produced by azobenzene. Azobenzene is probably no more nor less a comutagen under these circumstances than is the S-9 mix itself. The term metabolic potentiation is suggested and this effect may well be compound-, or at least chemical class-, specific.

Following the observations with DAB, Nagao [22] has demonstrated that the rat carcinogen o-toluidine (IV), which had previously been found to be negative in the Ames assay [14], is positive when tested in the presence of Dorharman. Again it could be argued that the balance between the N-oxidase enzymes {putative activation enzymes) and the C-hydroxylation enzymes {puta- tive deactivation enzymes) of rats in vivo is such as to produce tumours, yet in

110

the process of preparing the S-9 mix from rat liver the relative contribution made by the activation enzymes is lowered. The presence of norharman in the assay medium may effectively restore this balance to that encountered by o-toluidine in vivo, thereby enabling a positive response in the Ames assay to be obtained. In a related study Garner has shown that whilst he too finds o-toluim dine negative in the Ames assay, 2,4-dimethylaniline (VIII), is positive. Garner had similarly anticipated that the introduction of a methyl group into the 4-position of o-toluidine [giving (VIII)] would reduce the effective contribu- tion made by C-hydroxylase enzymes to the overall metabolism of the toluidine nucleus, thereby potentiating N-oxidase effects and in vitro muta- genicity (R.C. Garner, private communication and permission to refer to this unpublished result). A similar argument could be used to explain the increased carcinogenic potency observed for 4'-ethyl-DAB [18].

Whilst the effects produced in vitro by norharman and azobenzene are possibly acceptable when dealing with potentiation (or initiation) of the formation of mutagenic species from established carcinogens, there may be very real dangers hidden in this practice. For example, Nagao has also shown that aniline (III) is positive in the Ames assay when tested in the presence of norharman [22], while both she and others [14] have shown that it is negative when tested alone. The IARC analysis of the possible carcinogenicity of this compound con- cluded [10] that none of the many animal studies conducted on this chemical could be taken as indicating that it is carcinogenic.

Further, several epidemiological studies have indicated that aniline is not car- cinogenic to man [10]. In this case, therefore, norharman may have poten- tiated a minor component of the overall metabolism of aniline to the point of unreality in vivo. Many more such false positive in vitro predictions of single- compound in vivo carcinogenicity could follow in the wake of aniline if nor- harman or other individually effective chemical adjuncts are added indiscrimi- nately to the medium of the Ames assay. The dramatic effects of S-9 variations on the observed mutagenic potency of benzo[a]pyrene in this assay [4,24] add further weight to this concern.

It is possible that individual batches of S-9 mix could be titrated with nor- harman (or other individually appropriate substrates) to produce a new balance of enzyme activities which would relate more closely than the origin~ mix to in vivo conditions. For example, it may be useful to find the dose of norhar- man which would initiate mutagenicity for the carcinogen o-toluidine in the Ames assay yet leave the putative non-carcinogen aniline as negative. Such a balanced activation system might also potentiate the mutagenicity of related carcinogens such as 4-aminobiphenyl without incurring an increased incidence of false positive results. This controlled method of using chemical adjuncts would be time-consuming and would necessitate further research, nonetheless, in this role, such adjuncts may provide a valuable means of finely adjusting the activity of the in vitro S-9 mix, to that encountered by a single test chemical in vivo.

The most disturbing question raised by the above experiments is that of the possible potentiation of carcinogens by non-carcinogens or even carcinogenesis produced synergistically by two or more individually non-carcinogenic mate- rials. For example, it is interesting to consider the likely outcome of testing the

111

above two adjuncts, norharman and azobenzene, together. Whilst both have been shown to be individually inactive in separate in vitro assays, when tested together the aniline produced by azoreductase cleavage of the azobenzene might be potentiated as a mutagen by the norharman, leading to a possible overall positive in vitro response. Whilst this prediction might accurately fortell the outcome of testing azobenzene together with norharman in vivo (assuming that each chemical was at the right place at the right time with respect to each other), the extra load that the generation of such data would put upon environ- mental legislative machinery may be too great for it to withstand at the moment . This leads us to suggest that for the immediate future such chemical adjuncts should be retained as research tools and only become involved with environmental screening methods after careful thought and further experi- ments, and the production of an exact definition of the purpose and scope of such screening programmes.

An exciting aspect of these findings is that they should enable the mode of action of mutagens and/or carcinogens to be studied using in vitro assays by testing compounds in the presence of alternative chemical substances for each of the postulated enzymic steps in their metabolism. A decreased response in the presence of an alternative activation substrate and an increased response in the presence of an alternative deactivation substrate should define the existence of a given pathway and enable its relative importance to be assessed.

References

1 Ames , B.N., W.E. Durs ton , E. Yamasak i and F.D. Lee, Carcinogens are mutagens : A single tes t sys tem com bi n i ng liver h o m o g e n a t e s for ac t iva t ion and bac te r ia for de t ec t i on , Proc. Natl . Acad. Sci. (U.S.A.) , 70 (1973) 2 2 8 1 - - 2 2 8 5 .

2 Arcos , J.C., and M.F. Argus, Che mma l I n d u c t i o n of Cancer , Vol. II B, A c a d e m i c Press, New York , 1974 .

3 A s h b y , J. , and I .F .H. Purchase , The select ion of app rop r i a t e chemica l class con t ro l s for u s e w i t h short- t e r m tests for po ten t i a l ca rc inogen ic i ty , Ann . Occup. Hyg. , 20 (1977) 2 9 7 - -3 0 1 .

4 A s h b y , J., and J .A. Styles, Does carc inogenic p o t e n c y corre la te wi th mu tagen ic p o t e n c y in the Ames assay? Na tu re ( L o n d o n ) , 271 ( 1 9 7 8 ) 452 - -455 .

5 Ashby , J., J .A. Styles and D. Pa ton , The in vi t ro eva lua t ion of some der ivat ives of the ca rc inogen b u t t e r yel low: Impl ica t ions for e n v i r o n m e n t a l screening, Brit. J. Cancer , in press.

6 De Flora , S., Metabol ic deac t iva t ion of m u t a g e n s in the Sa lmone l l a -mic rosome test , Na tu re ( L o n d o n ) , 271 (1978) 4 5 5 - - 4 5 6 .

7 Food and Chemica l News, N o v e m b e r 22 (1976) 64. 8 Garner , R.C., and C.A. N u t m a n , Test ing of some azo dyes and their r educ t ion p r o d u c t s for mu ta -

genic i ty using Salmonella typhimurium TA 1538 , Muta t ion Res., 44 (1977) 9- -19 . 9 Hayash i , K., M. Nagao and T. Sug imura , In t e rac t ions of n o r h a r m a n and h a r m a n wi th D N A , Nucleic

Acid Res. , 4 (1977) 3 6 7 9 - - 3 6 8 5 . 10 In t e rna t iona l Assoc ia t ion for Research on Cancer ( IARC) , Mo n o g rap h on the Eva lua t ion of Carcino-

genic Risk of Chemicals to Man, Vol. 4, 1974 , pp. 27 - -39 . 11 Kensler , C.J., The inf luence of diet on the r ibof lavin c o n t e n t and the abi l i ty of ra t liver slices to

d e s t roy the ca rc inogen N,N-dimethyl-p-aminoazobenzene, J. Biol. Chem. , 179 (1949) 1 0 7 9 - - 1 0 8 4 . 12 Kinosi ta , R., Studies on the carc inogenic subs tances , Jpn . Pathol . Soc. Trans . , 27 (1937) 6 6 5 - - 7 2 7 . 13 Lin, J .K. , J .A . Miller, and E.C. Miller, S t ruc tures of hepa t ic nucleic ac id -bound d yes in rats given the car-

c inogen N - m e t h y l - 4 - a m i n o a z o b e n z e n e , Cancer Res., 35 (1975) 8 4 4 - - 8 5 0 . 14 McCann , J. , E. Choi, E. Yamasak i and B.N. Ame s , De tec t ion of carc inogens as m u t a g e n s in the SaN

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Br i t . J . C a n c e r , 36 ( 1 9 7 7 ) 558-- -563. 31 S u g i m u r a , T. , T. K a w a c h i , T. M a t s u s h i m a , M. N a g a o , S. Sa to a n d T. Yahag i , in : D. S c o t t , B.A. Br idges

a n d F .H. Sobe l s (Eds . ) , P rogress in G e n e t i c T o x i c o l o g y , E l s e v i e r / N o r t h H o l l a n d B i o m e d i c a l Press, A m s t e r d a m , 1 9 7 7 , pp . 1 2 6 - - 1 5 4 .

3 2 T o p h a m , J .C . , a n d J .W. W e s t r o p , T h i n - l a y e r c h r o m a t o g r a p h y of 4 - d i m e t h y l a m i n o a z o b e n z e n e a n d s o m e o f i ts m e t a b o l i t e s , J . C h r o m a t o g . , 16 ( 1 9 6 4 ) 2 3 3 - - 2 3 4 .