Mutagenicity of nitro- and amino-substituted carbazoles in Salmonella typhimurium. III. Methylated derivatives of 9H-carbazole

Mutation Research 389 Ž1997. 247–260

Mutagenicity of nitro- and amino-substituted carbazoles in Salmonella typhimurium. III. Methylated derivatives of 9H-carbazole V. Andre´ a,b, C. Boissart a,b, F. Sichel a,b, P. Gauduchon a,b,) , J.Y. Le Talaer ¨ J.C. Lancelot c , C. Mercier d , S Chemtob d , E. Raoult e, A. Tallec e

a,b

,

a

d

Laboratoire de Cancerologie Experimentale, Centre F. Baclesse, Route de Lion sur Mer, 14021 Caen cedex, France ´ ´ b EA1772, UniÕersite´ de Caen, 14032 Caen cedex, France c Centre d’Etude et de Recherche sur le Medicament de Normandie, 1 rue Vaubenard, 14032 Caen cedex, France ´ ´ ITODYS de l’UniÕersite´ Paris 7-Denis Diderot, Associe´ au CNRS, URA 34, 1 rue Guy de la Brosse, 75005 Paris, France e Laboratoire d’Electrochimie, Campus Scientifique de Beaulieu, 35042 Rennes-Cedex, France Received 1 May 1996; revised 11 September 1996; accepted 24 October 1996

Abstract The mutagenic potency of nine methylnitrocarbazoles, four methylaminocarbazoles and the methylcarbazole parent compounds was evaluated in Salmonella typhimurium TA98 and TA100, in the absence and presence of S9 isolated from Aroclor-induced rat liver. Nitro derivatives were additionally tested in TA98NR and TA98r1,8DNP6 , and mutagenicity of nitrocarbazoles bearing methyl groups in positions 1 and 4 was also determined in TA1537 and TA1977, with and without S9. The addition of methyl groups on non-mutagenic carbazole can induce a mutagenic response where the intensity and nature of the effect depends on the position of the substitution: base-pair substitutions were only observed for N-methylated carbazoles, whereas 1,4-dimethylated compounds exhibited frameshift mutagenicity. All these activities depended on the presence of S9. From its dependence on classical nitroreductases, direct mutagenicity of methylnitro derivatives should be attributed to bacterial reduction of nitro groups. The influence of the methyl groups Žand other additional substituents. on mutagenicity of these derivatives is discussed through their effects on life-time and reactivity of the intermediates Ži.e., hydroxylamines and nitrenium ions., taking into account the nature, the position and the number of substituted sites. Mutagenic activity of methylnitrocarbazoles was also tentatively correlated with various molecular descriptors. Among them, hydrophobicity was found to be strongly correlated with the mutagenicity of the 1,4diMe3NC isomers. On the other hand, mutagenic potency of the nitrated and aminated methylcarbazoles varied independently of parameters linked to their oxidoreduction properties Ži.e., reduction and oxidation potentials, LUMO and HOMO energies.. Keywords: Methylnitrocarbazole; Salmonella mutagenicity; Hydrophobicity; Oxidoreduction property; Structure–activity relationship

)

Corresponding author. Tel.: q33 02 3145-5070; Fax: q33 02 3145-5053; E-mail: [email protected].

1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 6 . 0 0 1 5 5 - 3

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1. Introduction It is well recognized that nitrated and aminated PAHs and heteroaromatics constitute a group of chemicals potentially hazardous for human health since some of them are able to develop genotoxic andror carcinogenic activities w1,2x. Using convenient short-term tests, mainly the Ames test, many studies have been carried out with the goal: Ž1. to evaluate the genotoxic potential of most common environmental pollutants; and Ž2. to obtain insight into their mechanism of interaction with target DNA. For congeneric compounds, quantitative structure– activity relationships have been searched in order to tentatively identify the structural properties that are closely related to expression or modulation of mutagenicity. In this context, we have undertaken to study the mutagenicity of diversely substituted carbazole derivatives in Salmonella typhimurium w3–5x. According to the general behaviour of the nitro and amino-PAHs, nitro- and aminocarbazoles act mainly as reactive frameshift mutagens and exhibit comparable activity in TA1538 and TA98. After bioactivation into hydroxylamines andror their esters, they give rise to reactive electrophilic nitrenium ions, through N–O heterolysis. In the series of mononitro-, monoamino- and ortho-aminonitrocarbazoles, limited variations in the position and nature of the substituents have profound effects on the mutagenic activity. We have found that, among molecular descriptors chosen to reflect different steps in the expression of mutagenicity, hydrophobicity is an important factor, compared to parameters linked to oxidoreduction properties Ženergies of frontier molecular orbitals, reduction and oxidation potentials. w4,5x. The ability of methyl groups to modulate mutagenic potency has been reported on various aromatic series. This has been best documented for heterocyclic amines from cooked foods w6–8x. In the aminobiphenyl and aminonaphthalene series, substitution by a methyl group ortho to an amino group increases mutagenicity w9x. On the other hand, for nitroarenes and nitroheterocyclic hydrocarbons, the

effects of methylation are varied. In the nitrobiphenyl and nitronaphthalene series, ortho-methylation dramatically decreases or inhibits mutagenic activity w10x. In contrast, N-methylation of the heterocyclic nitrogen leads to a strong enhancement of the mutagenicity for 2-nitrobenzimidazole, 2-nitroimidazow4,5-f xquinoline Žnitro-demethyl-IQ. and 2nitro-imidazow4,5-f xnaphthalene Žnitro-demethyl-NI. w11,12x. Finally, mutagenicity of nitroindoles, nitroindolines or nitroindazoles remains unchanged after N-methylation w11x. In the present paper, we have investigated an original series of nitro and amino methylated carbazoles, together with their precursors. Mutagenicity was systematically evaluated in TA98 and TA100, with and without Aroclor-induced rat S9. Compounds displaying significant direct mutagenicity w e re a d d itio n a lly te ste d in T A 9 8 N R Ž nitroreductase-deficient variant . w 13 x and TA98r1,8DNP6 Ž O-acetyltransferase-deficient variant. w14x. Some molecules were also tested in TA1537 and its repair-proficient analogue TA1977. In parallel, hydrophobicity, energies of the frontier molecular orbitals HOMO Ž EHO MO . and LUMO Ž ELUMO ., reduction Ž E red . and oxidation Ž E ox . potentials and hydroxylamine life-time were determined as molecular descriptors w4x, and correlations between these descriptors and mutagenicity in TA98 tentatively established.

2. Materials and methods 2.1. Chemicals Ø 1,4,6-trimethylcarbazole Ž 1,4,6triMeC . and 1,4,9-trimethylcarbazole Ž1,4,9triMeC. have been sythesized according to w15x; Ø 9-methylcarbazole Ž9MeC., 9-methyl-2-nitrocarbazole Ž9Me2NC. and 9-methyl-3-nitrocarbazole Ž9Me3NC. have been obtained from w16x and w17x; Ø 1,4-dimethylcarbazole Ž1,4diMeC., 5,8-dimethyl3-nitrocarbazole Ž5,8diMe3NC., 1,4,6-trimethyl-

Fig. 1. Methylated nitro- and aminocarbazoles: structures and nomenclature.

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

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250

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

3-nitrocarbazole Ž1,4,6triMe3NC., 1,4-dimethyl6-hydroxy-3-nitrocarbazole Ž1,4diMe6OH3NC., 1 ,4 -d im e th y l-6 -a c e ty l-3 -n itro c a rb a z o le Ž1,4diMe6Ac3NC. and 1,4-dimethyl-3,6-dinitrocarbazole Ž1,4diMe3,6diNC. have been obtained from w18x and w19x; Ø 1,4-dimethyl-3-nitrocarbazole Ž1,4diMe3NC. and 1,4,9-trimethyl-3-nitrocarbazole Ž1,4,9triMe3NC. have been prepared from w20x; Ø 1,4-dimethyl-3-aminocarbazole Ž1,4diMe3AC. have been synthesized according to w21x; Ø 1,4-dim ethyl-6-hydroxy-3-am inocarbazole Ž1,4diMe6OH3AC. and its methoxy derivative 1,4-dim ethyl-6-m ethoxy-3-am inocarbazole Ž1,4diMe6OMe3AC. have been prepared from w19x and w18x. See Fig. 1for structures of methylated nitro- and aminocarbazoles.

plied, using a vitreous carbon electrode as working electrode. The relative stability of hydroxylamines were roughly estimated from cyclic voltammograms obtained in buffered medium Žmother solution in DMF q acetic buffer 0.5 M 1:1.. Several sweeps were applied using a Scanning Potensiostat Model 362 ŽE & G Princeton Applied Research. with rates varying from 50 to 500 mVrs. Voltammograms were recorded on a Kipp Zonen XY recorder. The stability Žreported in Table 4. was estimated from the ratio between the magnitude of the reduction cathodic

Table 1 Mutagenicity of methylated carbazoles towards Salmonella typhimurium TA98 and TA100 without Žy. and with Žq. S9 mix Žrevrnmol a . Molecule

TA100 yS9

qS9

yS9

9MeC 1,4diMeC 1,4,6triMeC 1,4,9triMeC 2NC

- 0.2 - 0.3 - 0.3 - 0.3 4.9"0.2

20.1"0.9 - 0.3 - 0.3 Ž7.3. 5.1"0.6

- 0.04 - 0.04 - 0.03 - 0.03 47"0.6

3NC

4.7"0.2 3"0.2

9Me2NC 9Me3NC 1,4,9triMe3NC 1,4diMe3NC 5,8diMe3NC 1,4,6triMe3NC

Ž8.1. 15.8"1 Ž7.4. -1.1 - 0.2 - 0.2

Ž103. Ž15.7. Ž7.7. -1.4 Ž9.1. - 0.3

1,4diMe6OH3NC 1,4diMe6Ac3NC 1,4diMe3,6diNC 3AC 9Me3AC 1,4diMe3AC 1,4diMe6OH3AC 1,4diMe6OMe3AC

- 0.2 - 0.2 Ž0.14. - 0.3 Ž0.15. - 0.2 - 0.2 - 0.23

- 0.3 - 0.3 Ž0.14. - 0.3 Ž9.6. - 0.2 - 0.3 - 0.3

2.2. Mutagenicity testing All derivatives were tested up to 250 mgrplate, according to the Ames standard plate incorporation test w22x, as detailed in our first paper w3x. S9 fraction ŽIffa-Credo, l’Arbresle, France., was isolated from male Sprague–Dawley rats pretreated with Aroclor 1254. The tests in TA98NR and TA98r1,8DNP6 were only performed in the absence of S9, since we could not rule out the presence of nitroreductases or O-acetyltransferases in the microsomal fraction. 2.3. Electrochemistry A mother solution 2 mM in dimethylformamide ŽDMF. was prepared for each compound. The electrolyte used in DMF was tetrabutylammonium tetrafluoroborate NBF4 Bu 4 Ž0.2 M in DMF, first recrystallized in methanol.. Redox potentials were determined by cyclic voltammetry, in a mixture of mother solutionrelectrolyte solution Žratio 1:1 vrv. Žsweep rate applied at the stationary mercury electrode: 100 mVrs.. Reduction potentials of nitro groups were determined as the average between cathodic and anodic peak potentials recorded when negative potentials were applied. Oxidation potentials of amino groups were derived from anodic peaks obtained when positive potentials were ap-

TA98 qS9

- 0.1 Ž0.62. Ž15.4. 1.9"0.2 36"3.4 b 86"8.6 b 45"0.8 40 w1x c 58 w10x c 199"11 173"8 Ž279. Ž92. Ž170. 19"2.5 6.5"0.7 3"0.1 - 0.09 14.4"1 37"1 11.3 w2.5 mgx c 45.8 w10 mgx c Ž1.9. - 0.2 - 0.2 - 0.2 Ž2.4. Ž4.4. - 0.03 Ž4.9. Ž0.2. Ž115. - 0.04 Ž0.35. Ž0.8. - 0.2 Ž55. - 0.2

Parentheses indicate non-linear response. Mutagenicity is calculated from the dose giving the highest response. -, not mutagenic at the highest dose tested. The limit value is calculated from this dose and the minimal detectable response Žtwice the background.. a Calculated from the slope Ž"standard error. of a least square regression of the linear portion of the dose–response curve. b Biphasic response Ženhanced slope at higher dose.. c Non-linear response, enhanced for the highest doses. Activity is calculated from the wmgx doses.

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

peak and the magnitude of the oxidation anodic peak. In every case, the reference electrode was a saturated calomel electrode ŽSCE. in aqueous solution. 2.4. RP-HPLC Methylcarbazole derivatives were dissolved in a buffered mixture Žammonium acetate Rectapur Pro-

251

labo 0.02 MrHPLC-grade acetonitrile Scharlau, 1:1. then filtrated on DynaGard 0.2 mm. Samples were injected on a reverse-phase column Ž250 = 4 mm, Lichrospher 100RP-18, 5 mm, Merck. using a 20-ml Rheodyne loop injector and were isocratically eluted with ammonium acetate 0.02 Mracetonitrile 1:1 Žflow rate 1 mlrmin, Pharmacia LKB HPLC pump 2248.. Absorbance was continuously recorded at 230 and 320 nm Žband characteristic of the presence of

Fig. 2. Mutagenic activity of N-methylated carbazoles in TA100 in the absence ŽA, C. and presence ŽB, D. of S9. A and B: precursor N-methylated carbazoles 9 MeC and 1,4,9triMeC. C and D: N-methylated nitrocarbazoles 9Me2NC, 9Me3NC and 1,4,9triMe3NC.

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V. Andre´ et al.r Mutation Research 389 (1997) 247–260

the nitro group. using a Pharmacia LKB spectrophotometer VWM-2141. 2.5. Quantum data The energies of the highest occupied molecular orbitals Ž E HO MO . and of the lowest unoccupied molecular orbitals Ž ELUMO . were calculated by means

of the semi-empirical quantum mechanical method AM1 w23x from the MOPAC 6 package. 2.6. Statistical methods Correlation between mutagenicity in Salmonella typhimurium TA98 strain and molecular descriptors of methylcarbazoles was assessed using linear regression analysis. Two-tailed tests were used. Corre-

Fig. 3. Mutagenic activity in TA98 in the absence ŽA, C. and presence ŽB, D. of S9. A and B: N-methylated nitrocarbazoles 9Me2NC, 9Me3NC and 1,4,9triMeC. C and D: 1,4-dimethylated nitrocarbazoles 1,4diMe3NC; 1,4,6triMe6NC; 1,4diMe6OH3NC; 1,4diMe3NC and 5,8diMe3NC.

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

lation coefficient r was considered significant at the 0.05 level.

3. Results 3.1. Mutagenicity None of the methylated precursor compounds displayed direct mutagenic activity in TA100 and TA98 ŽTable 1 and Fig. 2A.. Upon addition of S9, 1,4diMeC, 1,4,6triMeC and 1,4,9triMeC developed a weak to moderate activity in TA98 ŽTable 1.. In TA100, only N-methylcarbazoles Ž9MeC and 1,4,9triMeC. were active, with higher mutagenic potency for the monomethylated isomer 9MeC ŽTable 1 and Fig. 2B.. Most of methylnitrocarbazoles showed direct activity in TA98, with large variations depending on the position and the number of substituents.The most active compounds in this series were again the Nmethyl substituted ones, and were classed as follows: 9Me3NC) 9Me2NCf 1,4,9triMe3NC ŽTable 1 and Fig. 3A.. The other trimethylated derivative 1,4,6triMe3NC was about 4.5-fold less active than its N -m eth y lated iso m er. 1 ,4 d iM e3 N C , 1,4diMe3,6diNC and 1,4diMe6OH3NC were weakly active, whereas 5,8diMe3NC did not display any direct mutagenicity ŽTable 1 and Fig. 3C..

253

Addition of S9 induced various effects, depending on the molecule considered ŽTable 1 and Fig. 3B, D.. Among N-methylated compounds, a clear-cut decrease of activity was observed for 1,4,9triMe3NC Žalmost 9-fold decrease. and, in to a lesser extent, for 9Me3NC Ž3-fold decrease., whereas mutagenicity of 9Me2NC remained unchanged. In the series 1,4-dimethylated nitrocarbazoles, mutagenic activity of 1,4diMe3,6diNC was not modified after addition of S9, whereas mutagenicity of 1,4diMe3NC was only slightly decreased. In contrast, 1,4diMe6OH3NC became inactive. For 1,4,6triMe3NC at the lowest doses, addition of S9 decreased the activity. On the contrary, at the highest ones, it tended to activate the molecule ŽTable 1.. 5,8diMe3NC was the sole molecule for which activity was entirely dependent on the presence of S9. 1,4diMe6Ac3NC was inactive whatever the conditions used. In TA100, the most active direct mutagens were again N-methylnitrocarbazoles that could be classed in the same order as in TA98 ŽTable 1 and Fig. 2C.. We have noted that 9Me3NC became toxic beyond 1 0 m g r p la te . A m o n g 9 H -c o m p o u n d s, 1,4diMe3,6diNC was the sole chemical to develop a weak mutagenic activity. Upon addition of S9 ŽTable 1 and Fig. 2D., activities of 9Me3NC, 1,4,9triMe3NC and 1,4diMe3,6diNC, although weak, were not modified. In contrast, mutagenic potency of 9Me2NC

Fig. 4. Mutagenic activity of methylated aminocarbazoles in TA98 in absence ŽA. and in presence ŽB. of S9.

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

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Table 2 Direct mutagenicity of methylnitrocarbazoles towards Salmonella typhimurium TA98NR and TA98r1,8DNP6 Žexpressed as % of direct mutagenicity towards the parent strain TA98.

Table 3 Mutagenicities of 1,4diMe3NC and substituted isomers towards Salmonella typhimurium TA1537 and TA1977, without Žy. and with S9 mix Žrevrnmol. a

Molecule

TA98NR

TA98r1,8DNP6

Molecule

2NC 3NC 9Me2NC 9Me3NC 1,4,9triMe3NC 1,4diMe3NC 1,4,6triMe3NC 1,4diMe6OH3NC 1,4diMe3,6diNC

17% 14% 46% 37.5% 38% 12.5% 0% 0% 12.5%

20% 14% 31% 47% 47% 18.6% 57% 33% 37.5%

1,4diMe3NC 1,4,6triMe3NC 1,4diMe6OH3NC 1,4diMe6Ac3NC 1,4diMe3,6diNC

TA1537 yS9

qS9

TA1977 yS9

qS9

- 0.01 Ž0.04. Ž1.3. - 0.03 Ž0.04.

Ž0.2. Ž1. Ž0.6. - 0.04 Ž2.1.

Ž0.2. - 0.07 Ž1.4. - 0.02 Ž0.4.

Ž0.2. Ž0.3. Ž0.6. Ž0.6. Ž0.1.

For legend, see Table 1.

underwent a 14-fold increase and this compound became the most active one. Again, 5,8diMe3NC gained moderate mutagenicity. None of the aminomethylcarbazoles was directly mutagenic in TA98, except 9Me3AC for which the response became significant only for the highest dose tested Ž100 mgrplate. ŽTable 1 and Fig. 4A.. After metabolism by the S9 fraction ŽTable 1 and Fig. 4B., 9Me3AC and, to a lesser extent,

1,4diMe6OMe3AC became active, the mutagenicities of the two other compounds remaining close to the detection threshold. Lastly, only 9Me3AC was mutagenic in TA100, mainly in the presence of S9 ŽTable 1.. Methylnitrocarbazoles displaying significant mutagenicity in TA98 were also tested in TA98NR and TA98r1,8DNP6 ŽTable 2.. Mutagenic activities of 1,4-dimethylated derivatives were dramatically decreased Žfor 1,4diMe3NC and 1,4diMe3,6diNC. or

Table 4 Physicochemical properties of methylnitro- and methylaminocarbazoles Žreduction and oxidation potentials, retention volumes, LUMO and HOMO energies, stability of hydroxylamine intermediates and molecular weight. Molecules

E red ŽV.

E ox ŽV.

V ret Žml.

E LU MO ŽeV.

EHOMO ŽeV.

Stability

MW Žgrmol.

9MeC 1,4diMeC 1,4,6triMeC 1,4,9triMeC 2NC 3NC 9Me2NC 9Me3NC 1,4,9triMe3NC 1,4diMe3NC 5,8diMe3NC 1,4,6triMe3NC 1,4diMe6OH3NC 1,4diMe6Ac3NC 1,4diMe3,6diNC 3AC 9Me3AC 1,4diMe3AC 1,4diMe6OH3AC 1,4diMe6OMe3AC

y y y y y1.16 y1.27 y1.10 y1.23 y1.30 y1.36 y1.23 y1.39 y1.37 y1.29 y1.12 y y y y y

y y y y y y y y y y y y y y y q0.47 q0.50 q0.39 q0.35 q0.40

42.1 36.9 18.7 83.5 21.2 16.7 43.4 35.2 65.2 34.5 42.5 53.0 8.4 17.6 32.4 5.9 10.0 8.1 3.2 6.4

y0.126 y0.140 y0.118 y0.816 y1.219 y0.936 y1.209 y0.911 y0.880 y0.849 y0.908 y0.833 y0.858 y1.121 y1.489 y0.144 y0.115 y0.134 y0.246 y0.194

y8.317 y8.313 y8.227 y8.720 y9.931 y9.024 y8.843 y8.895 y8.099 y8.863 y8.820 y8.741 y8.662 y9.097 y9.468 y8.002 y7.918 y7.901 y7.870 y7.845

y y y y 0.36 0.22 0.20 0.15 0.34 0.10 0.26 0.04 0.04 0.48 0.26 y y y y y

181 195 209 209 212 212 226 226 254 240 240 254 256 282 285 182 196 210 226 240

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

completely abolished Žfor 1,4,6triMe3NC and 1,4diMe6OH3NC. in TA98NR compared to those observed in TA98. In contrast, mutagenicity of Nmethylated compounds seemed to be only partly dependent upon the ‘classical’ nitroreductases, since 37–46% of the initial activity was retained in the nitroreductase-deficient strain. Mutagenic activity of 1,4,6triMe3NC in TA98r1,8DNP6 was almost 60% of its initial activity. At the opposite end, 1,4diMe3NC exhibited the strongest dependence upon bacterial O-acetyltransferases. For the remaining compounds, direct mutagenicity was estimated between 30 and 50% of that in TA98. Finally, some molecules were tested in parallel in TA1537 and TA1977 ŽTable 3.. They were either inactive or displayed a weak direct mutagenicity in T A 1 5 3 7 , th e m o st a c tiv e o n e b e in g 1,4diMe6OH3NC. Upon addition of S9, the derivatives tended to be more active or became active, except for 1,4diMe6OH3NC and 1,4diMe6Ac3NC. In the UVr-proficient strain TA1977, only 1,4diMe6OH3NC clearly developed a mutagenic activity. The four other isomers were either inactive or close to the detection limit. The influence of S9 was similar to that described previously in TA1537, except for 1,4diMe6Ac3NC, which became active, and for 1,4diMe3,6diNC, which was less mutagenic than in the absence of S9. 3.2. Physicochemical and quantum properties Reduction potentials of nitro groups and oxidation potentials of amino groups determined in DMF are displayed in Table 4. The addition of a methyl group on the nitrogen heteroatom or the addition of two methyl groups on positions 5 and 8 of the carbazole ring of 2NC or 3NC led to 9Me2NC, 9Me3NC and 5,8diMe3NC which were more easily reduced than their parent compounds. The opposite effect was observed when the dimethyl moiety was located on positions 1 and 4. As compared with 3AC, N-methylation rendered oxidation of the amine more difficult. Conversely, oxidation was greatly facilitated by 1,4-dimethylation. According to the isomer considered, RP-HPLC retention volumes varied over a large range. NMethylated derivatives Žprecursors or nitrated ones.

255

Fig. 5. Voltammograms of 1,4diMe6OH3NC ŽA., 1,4,6triMe3NC ŽB. and 1,4diMe3,6diNC ŽC. obtained in DMFrammoniacal buffer. Sweep rate, 100 mVrs; sensibility, 0.5.

globally displayed a marked hydrophobicity, whereas aminomethylcarbazoles were the most hydrophillic compounds of the series ŽTable 4.. Stability of hydroxylamine intermediates Žobtained from voltammograms shown in Fig. 5. are presented in Table 4, together with frontier orbital energies E LUMO and EHOMO calculated with the semi-empirical method AM1.

4. Discussion We show here that substitution of non-mutagenic carbazole by methyl groups can induce a mutagenic activity that requires the presence of S9. Clearly, position and number of methyl groups determine the type of mutagenic response. Base-pair substitutions

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were only observed for N-methylated carbazoles, according to the previous results presented by Lavoie et al. w24x. These authors reported a decreased activity of N-methylcarbazole upon addition of a second methyl group, and we also observed this unfavourable effect in the case of 1,4,9diMeC. Gorrod and Temple w25x have proposed that N-methylcarbazoles could be first metabolized into N-hydroxymethylcarbazoles. Through a N-dealkylation reaction, which can occur spontaneously, but is catalysed by the S9 fraction, these intermediates would release formaldehyde which would finally be responsible for DNA lesions. The mutagenicity of purified N-hydroxymethylcarbazole in TA100 is in keeping with this mechanism w24,26x. Furthermore, a recent re-evaluation of the mutagenic properties of formaldehyde itself has shown an activity mainly in TA100, the intensity of response varying strongly according to protocol w27x. The four methyl-9H-carbazole isomers tested by Lavoie et al. w24x were inactive in TA98. We demonstrate here that 1,4-dimethylated compounds are able to develop frameshift mutagenic activity. With regard to 1,4diMeC itself, the addition of a third methyl group on position 6 or 9 enhanced this response, with a more pronounced effect for position 6. Since methyl groups do not per se induce any direct activity in TA98 and TA100, the bacterial reduction of the nitro group must be responsible for the mutagenicity of methylnitro derivatives. This is further confirmed by the strong dependence of their activity towards ‘classical’ bacterial nitroreductases, which is, however, less pronounced for 9-CH 3 compounds. In this case, it might be assumed that, particularly when the ‘classical’ enzyme is absent, other nitroreductases are involved for which 9-CH 3 compounds may be better substrates. The presence in Salmonella of at least two pathways for bioactivation of nitroarenes has been proposed w28x. Beside the ‘classical’ one-electron nitroreduction, a two-electron transfer pathway, tightly coupled to the esterification step w29x, was postulated to be responsible for the mutagenic activation of some nitropyrenes. In our case, the dependence toward esterification was less pronounced for 9-methylated nitrocarbazoles than for their 9H congeners. Furthermore, the number of electrons transferred in the first step of ni-

Fig. 6. Mechanism of spontaneous loss of electrophilic character for the nitrenium ions derived from 2NC and 3 NC.

troreduction was not significantly modified by Nmethyl substitution Ždata not shown.. Thus, the implication of the two-electron pathway in the remaining mutagenicity of 9-methylated nitrocarbazoles in TA98NR is unlikely, and additional ‘non-classical’ pathways may contribute to their initial reduction step. The increased activity of N-methylnitro derivatives in TA98 and TA100 as compared with their 9H homologues, may partly result from the higher efficiency of these ‘non-classical’ pathways. In addition, the N-methyl group may influence the properties of the proximate or ultimate mutagen ŽFigs. 6 and 7.. Ž1. The weaker dependence of the activity of N-methylated derivatives upon the presence of Oacetyltransferase might suggest that the hydroxylamine species itself may be more readily converted into the ultimate electrophile through heterolysis of the N–O bond, the esterification step becoming less crucial. Ž2. In benzimidazole series, Vance et al. w11x have reported an increased direct mutagenicity for Nmethylnitrobenzimidazoles as compared to unmethylated compound. They hypothesized that N-methyl on the imidazole ring might extend the life-time of nitrenium ions by avoiding loss of their electrophilic character through spontaneous dissociation of the hydrogen ion from the heteroatom. This mechanism might also explain the influence of N-methylation on mutagenic activity of 2NC and 3NC. As shown in Fig. 6, the nitrenium ions derived from these compounds are both able to lose their electrophilic character through spontaneous dissociation of the 9H proton. Whereas the addition of S9 had a limited effect on the mutagenicity of 3NC in TA98 and induces only a

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

2-fold decrease of the activity of 1,4diMe3NC, a clear-cut decrease of activity is observed for their 9-CH 3 derivatives. This may be related to the biotransformation of N-methyl into a less favourable N-CH 2 OH group or to partial restoration of a free N–H moiety. In TA100, this unfavourable effect could be balanced by the specific action of the reactive species formed from the N-hydroxymethyl group towards the target sequences of this strain. In contrast, the adverse effect of addition of S9 on mutagenicity in TA98 is no longer observed for 9Me2NC. In this case, the 12-fold increase of activity in TA100 may fully reflect the influence of N-methyl hydroxylation. The addition of 1,4-dimethyl moiety on 3NC is

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systematically associated with a decrease of direct mutagenicity in TA98. Steric hindrance in the vicinity of the nitro group might inhibit bonding to nitroreduction enzymes. In addition, electrodonating effects of methyl groups might also contribute to a decrease in density of positive charge on the nitrenium ion, leading to a less reactive species ŽFig. 7.. The mutagenicity of 1,4,9triMe3NC undergoes is due to the conflicting influence of methyl groups located on positions 1,4 and on the nitrogen heteroatom. Four derivatives with the 1,4diMe3NC moiety bear an additional substituent on position 6. When the influence of the nature of substituent is evaluated by the ratio between the mutagenicity of these com-

Fig. 7. Mechanistic interpretation of the effects of methyl substitution on the mutagenic activity of methylnitro- and methylaminocarbazoles compared to their mononitro- or monoamino-parent compounds.

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pounds in TA98 and that of their common precursor 1,4diMe3NC, compounds can be ordered as follows: 6 y CH 3 Ž =5.7 . ) 6 y H Ž =1 . ) 6 y NO 2 Ž =0.4 . f 6 y OH Ž =0.3 . ) 6 y COCH 3 Ž =0.03 . The hydrophobicity of the molecules widely varies with the kind of substituent, and probably contributes to modulation of mutagenicity Žsee below.. However, comparative analysis of mutagenic activities in TA98NR and TA98r1,8DNP6 and data on the stability of hydroxylamines suggest that additional factors are implicated. Whereas positions 3 and 6 are not resonance-conjugated, clear variations of electrochemically determined hydroxylamine stability suggest that long-distance electronic effects occur, perhaps relayed by the heteroatom located para to positions 3 and 6. The presence of an OH or CH 3 group on position 6 decreases the stability of the hydroxylamine ŽFig. 5.. These electrodonating groups could reinforce the polarization of the N–O bond, facilitating the heterolysis reaction. Further supported by the lower dependence of mutagenicity of these two isomers towards O-acetyltransferase enzymes, this mechanism could especially account for the unexpected activity of the most hydrophillic compound, 1,4diMe6OH3NC. The complete loss of activity for 1,4diMe6Ac3NC could result from its low hydrophobicity and from the extended hydroxylamine stabilisation by the electro-withdrawing acetyl group ŽFig. 5.. The effect of 6-nitro substitution is more difficult to assess, since both nitro groups may undergo metabolic activation. This makes the comparison with the two possible precursors 1,4diMe3NC and 5,8diMe3NC risky, the latter of which is inactive. 1,4diMe3NC compounds are tricyclic analogues of the antitumour drug ellipticine Ž5,11-dimethyl6 H-pyridow4,3-b xcarbazole., and in fact some of them, in particular 1,4diMe6OH3NC and its amino equivalent 1,4diMe6OH3AC, exhibit some cytotoxic activity against murine leukaemia L1210 w21x. The frameshift mutagenicity of ellipticine in TA98 probably involves covalent binding to DNA, while its activity in TA1537 results from intercalation between DNA base pair w30x. Thus, in order to assess the contribution of a non-covalent DNA-binding mechanism to mutagenicity of 1,4diMe3NC deriva-

tives, additional experiments were performed with strains TA1537 and TA1977. The behaviour of the 6-OH derivative is noteworthy since its mutagenicity is very similar in the two strains, and thus does not depend on an efficient UVr repair system. Conversely, its weak activity in TA98 is fully dependent on the formation of hydroxylamine. Hence 1,4diMe6OH3NC frameshift mutagenesis might occur both through the formation of DNA adducts or through non-covalent binding to DNA. In the presence of S9, 1,4diMe3NC exhibits a similar weak activity towards both HisC3076 strains: it might be speculated that this is due to the formation of 1,4diMe6OH3NC through the activity of arylhydroxylases present in the S9 fraction. For the remaining 1,4diMe compounds, non-covalent binding to DNA does not seem to be the preponderant mechanism involved in the mutagenic response ŽFig. 7.. The absence of activity for 5,8diMe3NC is difficult to explain since methyl and nitro groups are located on opposite rings. Electrochemically determined stability indices indicate that the hydroxylamine obtained from 5,8diMe3NC is clearly more stable than that obtained from its 1,4diMe3NC. Stabilisation of the N–O bond could account for the loss of activity of 5,8diMe3NC compared to weakly mutagenic 1,4diMe3NC. Note that this is the only isomer to be activated upon addition of S9 Žfor TA98 as well as TA100., which suggests an activation through methyl groupŽs. metabolism. The reason why this metabolism would not occur for other 1,4diMeC derivatives remains unclear. All aminomethylcarbazoles studied were derived from 3AC. Similar to their precursor, they displayed no direct mutagenicity in TA98 and TA100, except 9Me3AC which developed a limited activity. Upon addition of S9, all the compounds become active in TA98, with important variations between isomers. The most active compound bears a N-methyl Ž9Me3AC., and addition of a 1,4-dimethyl moiety or hydroxy substituent decreases activity. This is similar to what is observed with nitro compounds, and suggests that the substituents mainly influence the processing of the hydroxylamine proximate mutagen, regardless of its formation pathway ŽFig. 7.. In other respects, whereas direct mutagenicity of 3NC and 1,4diMe3NC is notably higher than that of their S9-activating corresponding amines, it is note-

V. Andre´ et al.r Mutation Research 389 (1997) 247–260

worthy that the activities of 9Me3AC and 9Me3NC become equivalent. Considering the longer random walk from extracellular metabolic activation site to DNA target for aminated compounds, it is conceivable that N-methyl substitution, which has been proposed to extend the lifetime of the ultimate electrophile by impeding the loss of electrophilic character, could contribute to the increase of activity in greater proportion for amino compound ŽFig. 7.. Correlations between mutagenic potency of nitro compounds and various molecular descriptors have also been investigated and are discussed below. The aminomethylcarbazole series was too restricted to perform a systematic correlation study. RP-HPLC retention volume V ret is a reflection of hydrophobicity. In the series of 11 nitrated carbazoles, no significant correlation could be found between the logarithm of the activity in TA98 ŽlogTA98 -. and log V ret. However, the correlation became highly significant Ž r s 0.99; n s 5; p s 0.0006. among 1,4diMe3NC isomers, after exclusion of 1,4diMe6OH3NC for which marginal behaviour has been previously discussed. For this limited, but very homogenous series, hydrophobicities appear as a strong determinant of mutagenic potency. The capacity of a molecule to accept or to lose an electron, respectively, can be experimentally determined Ž E red , E ox ., or theoretically estimated by calculating the energies of the frontier molecular orbitals LUMO Ž ELUMO . and HOMO Ž E HOMO .. In the nitrocarbazole series considered as a whole, no significant correlation could be establish between logŽTA98 -. and E red , E LUMO or E HOMO . Experimental E red and calculated E LUMO were found tightly and negatively correlated Ž r s y0.8; n s 9; p s 0.0048.. This had been observed previously for the aminonitrocarbazoles series w4x as well as in other series of nitroaromatic compounds w31x and validates the methods selected for the determination of the molecular descriptors. These results confirm our previous statement that in series of congeneric nitro compounds, hydrophobicity emerges as the determinant parameter in linear correlation studies between mutagenicity in TA98 and molecular descriptors w4x. However, modulation of mutagenic activity by methyl groups is not restricted to their influence on hydrophobicity and depends on the site of methylation ŽFig. 7.. These

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groups as well as other substituents are also likely to influence mutagenicity through their effect on the lability of the hydroxylamine intermediates andror on the life-time of the ultimate electrophilic mutagen. Microsomal metabolization of methyl groups can also lead to substantial activity in TA100. Lastly, 1,4diMe6OH3NC is able to cause frameshift mutations by non-covalent binding to DNA. Acknowledgements We thank Dr. J.F. Heron, Director of the Centre ´ F. Baclesse, for his constant support. The investigation was supported by grants from the Ligue Nationale de Lutte contre le Cancer ŽFederation Na´ ´ tionale des Centres de Lutte contre le Cancer, comite´ de la Manche et du Calvados. and from the University of Caen. References w1x Tokiwa, H. and Ohnishi, Y. Ž1986. Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol., 17, 23–60. w2x Clayson, D.B. and Garner, R.C. Ž1976. Carcinogenic aromatic amines and related compounds, in: C.E. Searle ŽEd.., Chemical Carcinogens, American Chemical Society Monograph 173, Washington, DC. w3x Andre, ´ V., Boissart, C., Lechevrel, M., Gauduchon, P., Le Talaer, ¨ J.Y., Lancelot, J.C., Letois, B., Saturnino, C., Rault, S. and Robba, M. Ž1993. Mutagenicity of nitro- and aminosubstituted carbazoles in Salmonella typhimurium. I. Monosubstituted derivatives of 9H-carbazole, Mutation Res., 299, 63–73. w4x Andre, ´ V., Boissart, C., Sichel, F., Gauduchon, P., Le Talaer, J.Y., Lancelot, J.C., Robba, M., Mercier, C., Chemtob, S., Raoult, E. and Tallec, A. Ž1995. Mutagenicity of nitro- and amino-substituted carbazoles in Salmonella typhimurium. II. Ortho-aminonitro derivatives of 9H-carbazole, Mutation Res., 345, 11–25. w5x Mercier, C., Chemtob, S., Vizet, P., Andre, ´ V. and Gauduchon, P. Ž1995. Modeling carbazole mutagenicity with the DARCrCALPHI system, Toxicol. Model., 3, 191–206. w6x Nagao, M., Wakabayashi, K., Kasai, H., Nishimura, S. and Sugimura, T. Ž1981. Effect of methyl substitution on mutagenicity of 2-amino-3-methylimidazow4,5-fxquinoline, isolated from boiled sardine, Carcinogenesis, 2, 1147–1149. w7x Kaiser, G., Harnasch, D., King, M.T. and Wild, D. Ž1986. Chemical structure and mutagenic activity of aminoimidazoquinolines and aminonaphthimidazoles related to 2-amino-3methylimidazow4,5-f xquinoline, Chem. Biol. Interact., 57, 97–106.

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