mutagenicity to salmonella of the mono methylamino and n-cyanoethyl analogues of...

Mutation Research, 128 (1984) 11-16 11 Elsevier

MTR03896

Mutagenicity to Salmonella of the monomethylarohno and N-cyanoethyl analogues of 4-dimethylaminoazobenzene (DAB) and

6-dimethylaminophenylazobenzthiazole (6BT)

John Ashby, R.D. Callander, P.A. Lefevre, D. Paton and B. Fishwick Imperial Chemical Industries PLC, Central Toxicology Laboratory, Alderley Park and Organics Division, Blackley, Manchester (Great

Britain)

(Received 7 December 1983) (Revision received 17 April 1984)

(Accepted 18 April 1984)

Summary

Replacement of one of the methyl groups of the carcinogens 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT) with a cyanoethyl (-CHECH2CN) substituent dramatically increases their mutagenic potency to Salmonella (strain TA98). The corresponding monomethylamino derivatives ( -NHCH3) are more mutagenic than the parent dimethylamino [-N(CH3)2] compounds, but substantially less mutagenic than the cyanoethyl derivatives. All of these mutagenic activities are liver-S-9- dependent. The very similar dose response curves observed for the two cyanoethyl compounds argues for the formation of a common electrophilic intermediate from each.

6-Dimethylaminophenylazobenzthiazole (6BT, for structures see Scheme) is a potent bacterial mutagen and exceptionally potent rodent hepato- carcinogen (Brown and Sanchorawala, 1968; Stora, 1976; Ashby et al., 1982 1983a-c; Elliott et al., 1983; Ashby, 1983). For this reason its mechanism of carcinogenic action is under study in this laboratory. Data from other studies with the much weaker parent carcinogen 4-dimethylaminoazobenzene (DAB) suggest that the metabolic activa- tion of that agent proceeds through initial oxida- tive demethylation to yield monomethyl- aminoazobenzene (MAB) followed by N-oxidation and conjugation of the derived hydroxylamine (Lin and Wu, 1974). Consistentwith this suggestion are the facts that MAB is a more powerful carcinogen than is DAB (Arcos and Argus, 1974; Miller and Miller, 1953) and that cyclic amine analogues of DAB are weaker mutagens than DAB itself (Ashby et al., 1983c). It therefore became of interest to

study the corresponding monomethyl derivative of 6BT (MA6BT). In addition, the N-cyanoethyl derivatives of both carcinogens (CN-EtDAB and CN-Et6BT, respectively) were prepared for evalua- tion in vitro as they may rearrange to yield the corresponding monomethylamino compounds via loss of cyanoethylene (acrylonitrile).

The present study compares the mutagenicity to Salmonella of DAB and 6BT with their corresponding monomethylamino and N-cyanoethyl derivatives using both the plate-incorporation and liquid pre-incubation assay protocols.

Materials and methods

Chemicals 4,Dimethylaminoazobenzene (DAB) and 6-di-

methylaminophenylazobenzthiazole (6BT) have been described previously, The samples used in the present study were of similar purity to those de-

0027-5107/84/$03.00 © 1984 Elsevier Science Publishers BN.

12

scribed earlier (Ashby et al., 1982). 6-(4 '-M ethylaminophenylazo)benzothiazole

(MA6BT). Diazotised 6-aminobenzothiazole (Ashby et al., 1983c) in excess dilute hydrochloric acid was added to an aqueous solution of N-methylaniline hydrochloride and stirred at room tem- perature for 30 h. The thick paste which was formed was added to a. solution of an excess of sodium acetate trihydrate in water and the oily precipitate extracted with ether. After being dried the extract was evaporated. The dark residual oil was purified using Whatman PLK5F chromatography plates developed in chloroform. The dark red oil obtained slowly solidified and the product was recrystallised from methanol producing red crystals m.p. 116-117°C. TLC (Whatman LHP- KF silica plate, methanol/chloroform 1:49) revealed no impurities. Theory for C14H12NnS (268): C, 62.7; H, 4.5; N, 20.9. Found: C, 62.9; H, 4.5; N, 21.1%. NMR (90MHz, CDCI3): $ 2.8, s(3H)NCH3; 4.0, s(1H)NH; 6.6, d(2H)aromatic protons adjacent to N-methyl; 7.8-8.4, m(5H) benzothiazole protons H-4, 5 and 7 and aromatic protons adjacent to azo-nitrogen; 8.95, s(1H) benzothiazole H-2; 3.7, trace of impurity. Mass spectrum m + 268, 134 (CH3NHPhN~-), 106 (134-N2).

6-[p-(N-~-Cyanoethyl-N-methylamino)phenyl- azo]benzothiazole (CN-Et6BT). 6-Aminoben- zothiazole (Ashby et al., 1983c) was dissolved in dilute aqueous hydrochloric acid and diazotised using a slight excess of sodium nitrite. The di- azonium solution was treated with sulphamic acid and added to a solution of 1 equivalent of N- cyanoethyl-N-methylaniline (courtesy of ICI PLC, Organics Division, Manchester, U.K.) in acetone at < 10 °. The mixture was neutralised im-

~ , _ N =N . . . ~ N/cH3 N, .~N = N..,~N ~'CH3 k = / - - ~CH3 ~ / ~ J k = / - - ~CH3 DAB S 6B T

/ H

MAB Ls )-~ M~,B'~=' --c.,

~ /CHzCH2CN ~ ~ /CH2CH2CN N"~ ')"- N = N "-~ / \)--N~ ~ ~(~'N----" N ~ N ~ c H 3 ~,S/~_~J ~ ~-CH l

CN-Et DAB CN~Et 6BT

Scheme 1. Chemical structure and abbreviated name of chemicals discussed in the text.

mediately by addition of an excess of an aqueous solution of sodium acetate, After stirring for 6 h in an ice-bath the product was collected and washed with water and allowed to dry. The crude product was purified by chromatography on florisil eluting with methanol/chloroform (1:4) and recrystallised from methanol, m.p. 141-141.5°C. TLC (Merck GF254, methanol/chloroform 1 :49) showed the produce to be pure. Theory for C17HasNsS (321):C, 63.6; H, 4.7; N, 21.8. Found: C, 63.7; H, 4.7; N, 22.1%. NMR (60 MHz, d 6 DMSO): 82.86, t(2H) methylene; 3.15, s(3H)N- methyl; 3.9, t(2H)methylene; 7.05, d(2H)aromatic protons adjacent to -NR2; 7.9-8.4, m(4H)benzothiazole H-4 and 5 and aromatic protons adjacent to azo-nitrogen; 8.68, m-split s(1H)benzothiazole H-7; 9.6, s(1H)benzothiazole H-2. Mass spectrum: m + 321, 281 (M+-CHzCN), 187 (NCCH2CH 2- NCH3PhN~- ), 159 (187-N2), 134 (benzthiazyl ring), l19(159-CH2CN).

4-Methylaminoazobenzene. (MAB) was prepared according to the method published by Miller and Baumann (1945) and was recrystallised from cyclohexane, m.p. 88-89 ° C (Miller and Baumann

3S0O- TA98 Plate j/-~_, =._~-~_,/cH,c.,c, Liver $9 ~ - m ~--/ ~-c.,

(CN Et DAB)

2800-

~ 2100-

~ 1400,

z

700 N = N"~N(~H,

~ , M A B , ~ _ .=N _ . ~ .(Col:

DMSO 0.5 5 50 500

Dose (pg/plate) Fig. 1. The plate incorporation assay was conducted as described in the Methods section. Plate counts for DAB and MAB are shown in Table 1.

3s0o- TA98 Plate Liver S9

I~S--~- N N I NI/CMzCMICN 2800" = " ~ ~(H 1

2100- ~ iCNet68rJ

~ 1400- N N--N N/Pl - - C ~ - -

7 0 0 - r / ! ~ j ~ / / ~

0 ~'-- ' '= . . . . DMSO 0.5 5 50 500

Dose (pg/plate)

Fig. 2. The plate incorporation assay was conducted as described in the Methods section.

report 88-88,5 o ). C, H and N contents were within 0.1% of the theoretical values. The proton N M R spectrum (90 MHz, CDC13) and mass spectrum were consistent with this structure and revealed no impurities.

3soo. TA98 Pre-inc. Liver S9

N N N ~cH2CHzcN

(CN-Et DAB)

1 ~ 2100- I '~ 1400- 0

700 ~ ~ f'CH3

/ e/e..-o-o ~oAsJ

0 I L P ~ : DMSO 0.5 i 50 500

Dose (pg/plato) Fig. 3. The pre-incubation assay was conducted as described in the Methods section. Plate counts for DAB are shown in Table 1.

13

3500

2800-

e 21oo

o • 1400

o Z

TA98 Pre-inc. Liver S9

~CHzCH~CN T

0 = ~ r U ~ e DMSO 0.5 5 5() 500

Dose (pg/plate)

Fig. 4. The pre-incubation assay was conducted as described in the Methods section.

4-[N-(fl-cyanoethyl)-N-methylamino] azobenzene. (CN-EtDAB) was prepared by ICI PLC Organics Division, Manchester, U.K.; m.p. 79-79.5°C. Theory for C16H16N4(264): C 72.7; H, 6.1; N, 21.2. Found: C, 72.7; H, 6.1; N, 21.3%. The N M R and mass spectra were consistent with the structure shown and revealed no impurities.

Salmonella assay All chemicals were evaluated in strain TA98 of

S. typhimurium in the plate assay according to our previously described protocol (Ashby et al., 1982). Aroclor-induced rat-liver $9 mix was prepared as described previously (Ashby et al., 1982). The dose-levels employed are shown in Figs. 1-4. 3 Plates per dose-level were used for the test chemicals, 5 for the DMSO controls and 2 for the strain-specific positive control chemical (2AA). In a separate study, 6BT, CN-Et6BT, DAB and CN- EtDAB were evaluated in the same tester strain using a 1-h period of pre-incubation before plating (Ashby et al., 1982, Lefevre and Ashby, 1981). These experiments were conducted in the presence of liver $9 mix and employed 5 plates per dose- level. 2-Aminoanthracene (2AA) was used as an $9

14

TABLE 1

PLATE AND PRE-INCUBATION ASSAY DATA FOR DAB AND MAB

Dose level 0 10 20 25 50 100 200 250 500 ( # g/plate)

DMSO (plate) 30.0 + 4.9 DAB(plate) 42.0_+8.0 43.7_+10.2 73.0_+6.1 65.0___13 61.3_+2.5 MAB(plate) 44.7+15.5 47.3_+7.6 97.7_+12.4 150.7_+29.9 164.0_+14 DMSO (pre-inc.) 41.0_+4.1 DAB(pre-inc.) 73.0+5.6 204.0_+i8.1 276.8_+17.7 276.2_+18.9

144.7_+11.4 56.3_+15

These data are plotted in Figs. 1 and 3 but due to the scales adopted these weak responses are difficult to discern.

control . Al l exper iments were repea ted at least once with fresh samples of liver $9 mix.

Results

Data f rom a single de te rmina t ion of the muta- genici ty of all of the test chemicals in the pla te assay are shown in Figs. 1 -2 , and using the prein- cuba t ion assay in Figs. 3 -4 . Al l responses were cons idered posit ive. The d o s e - r e s p o n s e curves shown for D A B and M A B in the p la te assay and for DAB in the p re - incuba t ion assay are weak, therefore the p r imary da ta for these tests are shown in Table 1. Al l exper iments have been rep roduced and representa t ive da ta are shown. In the case of

TABLE 2

SILOLUBILITY OF THE CHEMICALS SHOWN IN WET OCTANOL (Smith et al., 1975) TOGETHER WITH THEIR EST~IMATED log P VALUES

Chemical Solubility Rel. solubility Estimated (mg/ml; wet (CN-Et6BT = 1) log P value octanol, 20 o C, (octanol/ 24 h stirring water)

DAB 12.4 26 4.58 MAB 63.6 133 3.98 CN-EtDAB 15.1 32 3.85 6BT 4.2 .9 4.29 MA6BT 20.4 42 3.69 CN-Et6BT 0.5 1 3.56

The latter were calculated by the method of Hansch and Leo (1979) and based on the experimentally derived value of 4.58 for DAB (Kurihara and Fujita, 1973). The relative solubilities were calculated to illustrate the wide range of values evident. These differences are reduced in in vitro tests due to the common employment of DMSO as a solvent, but they may become critical to the expression of genotoxicity in rodents dosed in vivo.

er ror bars ( + SD) missing f rom the Figures these are conta ined by the da t a -po in t symbol . A single de te rmina t ion (da ta not shown) made using the p la te assay ind ica ted the total dependence of the mutagenic i ty of the two cyanoethyl compounds on $9 mix, a fact previous ly es tabl ished for D A B and 6BT (Ashby et al., 1982).

The solubil! ty of the test chemicals in wet octanol (4.4% H 2 0 ) is shown in Table 2 together with their es t imated log P values based on the f igure of 4.58 es tabl ished exper imenta l ly for D A B (Kur iha ra and Fuj i ta , 1973). The use of wet octanol as solvent enables compar i sons to be made with the es t imated log P values (Smith et al., 1975) which were ca lcula ted by the methods of Hansch and Leo (1979).

Discussion

Earl ier studies f rom this L a bo ra to ry have indi- ca ted that the po ten t carc inogenic i ty of 6BT (E1- l iot t et al., 1983) is unl ikely to be related to the two amines formed upon cleavage of its centra l azo group (Ashby et al., 1983b; El l iot t and Ashby, 1983). Fur ther , the absolu te requi rement observed for induced liver $9 mix when evaluat ing 6BT for mutagen ic i ty in vi tro (Ashby et al., 1982) coupled with the reduced mutagenic i ty of two cyclic amine analogues (Ashby et al., 1983c) suggests that metabo l ic ac t iva t ion of the d ime thy l amino group of 6BT presents the p r ima ry source of its mutagenic and carc inogenic activity. These conclusions para l le l closely the requi rements for carc inogenic act ivi ty suggested for DAB, namely, an ini t ial N-deme thy l a t i on fol lowed by N-ox ida t ion and conjuga t ion of the der ived hydroxy lamine to yield the u l t imate D N A - r e a c t i v e species (Lin and Wu,

1974). Consistent with this proposed mechanism, the initial demethylation product of DAB (MAB) is a more potent carcinogen than is DAB (Arcos and Argus, 1974) and azo reductive cleavage re- duces the carcinogenic potency of DAB (Arcos and Argus, 1974). It therefore became of interest to examine the role of the first N-methyl group of 6BT in the expression of its mutagenicity in vitro.

The data shown in Figs. 1 and 2 show that the monomethylamino derivatives of 6BT (MA6BT) and DAB (MAB) are approxiamtely twice as mutagenic as the parent dimethylamines. These Figures also show that the corresponding N- cyanoethyl derivatives (CN-EtDAB and CN- Et6BT) possess much higher and approximately equal mutagenic activity. The latter derivatives were evaluated as a possible alternative way of presenting the tester bacteria with the monomethylamino derivatives via S9-independent retro- Michael reaction (R2N-CH2-CH2CN ~ R2NH + C H 2 = CHCN).

The reason for the potent mutagenicity of the two cyanoethylderivatives remains unclear, but several factors seem relevant. First is the fact that they each possess an almost identical profile of mutagenic activity in both the plate and pre-incubation assays (Figs. 1-4). This suggests that a mutagenic intermediate is formed from each that is not directly influenced by the fundamental aromatic substructure, the marked difference between the mutagenicities of DAB and 6BT them- selves being evident in these Figures. The possibil- ity that the mutagenicity of the cyanoethyl derivatives is due to acrylonitrile per se, possibly liberated via a retro-Michael reaction, is weakened by the high mutagenic potency and absolute requirement of these agents for $9 mix, acrylonitrile being a weak and S9-independent mutagen (Milvy and Wolff, 1977; Venitt et al., 1977).

It is concluded that the monodemethylation of 6BT, as with DAB, may represent the initial step in its bioactivation as a mutagen and carcinogen. The potent mutagenicity of the two cyanoethyl compounds remains to be explained; their genotoxic activity in rodents in vivo will be discussed in a future publication.

The widely differing solubilities of the present 6 chemicals (Table 2) suggests that the expression in vivo of the mutagenicity observed for each in vitro

15

may be significantly modulated by this factor. In particular, the high solubility of MA6BT may lead to it appearing more genotoxic in vivo than the relatively insoluble cyanoethyl analogue, despite the fact that the latter compound is considerably more mutagenic in vitro.

Acknowledgements

We thank G. Morris of ICI PLC (Pharmaceuti- cals Division) for estimating the partition coeffi- cients and Sue Rodger for secretarial assistance.

References

Arcos, J.C., and M.F. Argus (1974) Chemical Induction of Cancer, Vol. liB, Academic Press, New York, 1974.

Ashby, J. (1983) The unique role of rodents in the detection of possible human carcinogens and mutagens, Mutation Res., 115, 177-213.

Ashby, J., P.A. Lefevre, J.A. Styles, J. Charlesworth and D. Paton (1982) Comparisons between carcinogenic potency and mutagenic potency to Salmonella in a series of derivatives of 4-dimethylaminoazoazobenzene (DAB), Mutation Res., 93, 67-81.

Ashby, J., B.M. Elliott, P.A. Lefevre, J.A. Styles and E. Longs- taff (1983a) Initiation/promotion versus complete carcino- genicity in the rodent liver, Environ. Hlth. Perspect., 50, 339-346.

Ashby, J., P.A. Lefevre and R.D. Callander (1983b) The possible role of azoreduction in the bacterial mutagenicity of 4-dimethylaminoazobenzene (DAB) and two of its analogues (6BT and 5I), Mutation Res., 116, 271-279.

Ashby, J., D. Paton and P.A. Lefevre (1983c) Cyclic amines as less mutagenic replacements for dimethylamino substituents on aromatic organic compounds: Implications for carcino- genieity and toxicity, Cancer Lett., 17, 263-271.

Brown, E.V., and C.J. Sanchorawala (1968) Carcinogenic activities of analogues of p-dimethylaminoazobenzene VI, J. Med. Chem., 11, 1074-1075.

Elliott, B.M., and J. Ashby (1983) Correlation between azo reductase activity and carcianogenicity of analogues of 4-di- methylaminophenylazobenzene (DAB) in the rat, European Environmental Mutagen Society Meeting, 1983, Poster II 2B7.

Elliott, B.M., M. Robinson and J. Ashby (1983) 6-p-Dimethyl- amino-phenylazobenzothiazole (6BT): A potent hepatocar- cinogen in the rat, Cancer Lett., 21, 69-76.

Hansch, C., and A. Leo (1979) Substituent constants for correlation analysis in chemistry and biology, Wiley, New York.

Kurihara, N., and T. Fujita (1973) Studies on BHC isomers and related compounds, V Biochem. Physiol., 2, 383-390.

Lefevre, P.A., and J. Ashby (1981) The effects of pre-incubation period and norharman on the mutagenic potency of 4-dimethylaminoazobenzene and 3'-methyl-4-dimethylaminoazobenzene, Carcinogenesis, 2, 927-931.

16

Lin, J.K., and J.R. Wu (1974) Synthesis, toxicities and carcino- genicities of carcinogenic bifunctional amino azo dyes, Cancer Res., 34, 2274-2282.

Miller, J.A., and C.A. Baumann (1945) The determination of p-dimethylaminoazobenzene in tissue, Cancer Res., 5, 157-161.

Miller) J.A., and E.C. Miller (1953) The carcinogenic aminoazo dyes, Adv. Cancer Res., 1,339-365.

Milvy, P., and M. Wolff (1977) Mutagenic studies with acrylonitrile, Mutation Res., 48, 271-278.

Smith, R.N., C. Hansch and M.M. Ames (1975) Selection of a reference partitioning system for drug design work, J. Pharm. Sci., 64, 599-606.

Stora, C. (1976) t:,tude comparative dans les deux sexes du pouvoir canc6rog6ne du N,N-dimethyl-p-(6-benzothiazo- lylazo)aniline (6BT), Bull. Cancer, 63, 87-94.

Venitt, S., C.T. Bushell and M. Osborne (1977) Mutagenicity of acrylonitrile (cyanoethylene) in Escherichia coil, Mutation Res., 45, 283-288 see also, discussion between authors of the above paper and those of Milvy and Wolff herein, Mutation Res., 57 (1978) 107-113.

mutagenicity to salmonella of the mono methylamino and n-cyanoethyl analogues of...

Documents