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Genotoxicity Risk Assessment of Diversely Substituted Quinolines Using the SOS Chromotest n,1 Carlos Eduardo Puerto Galvis,2 Leidy Tatiana Dıaz-Duran,1 Nathalia Olivar-Rinco 2 Vladimir V. Kouznetsov, Jorge Luis Fuentes Lorenzo1 nesis Ambiental, Escuela de Biologıa, Facultad de AQ2 AQ3 1Laboratorio de Microbiologıa y Mutage Ciencias, Universidad Industrial de Santander, Bucaramanga, Colombia 2

nica y Biomolecular, Escuela de Quımica, Facultad de Ciencias, Laboratorio de Quımica Orga Universidad Industrial de Santander, Bucaramanga, Colombia

Received 3 March 2013; revised 6 August 2013; accepted 12 August 2013 ABSTRACT: Quinolines are aromatic nitrogen compounds with wide therapeutic potential to treat parasitic and microbial diseases. In this study, the genotoxicity of quinoline, 4-methylquinoline, 4-nitroquinoline-1-oxide (4-NQO), and diversely functionalized quinoline derivatives and the influence of the substituents (functional groups and/or atoms) on their genotoxicity were tested using the SOS chromotest. Quinoline derivatives that induce genotoxicity by the formation of an enamine epoxide structure did not induce the SOS response in Escherichia coli PQ37 cells, with the exception of 4-methylquinoline that was weakly genotoxic. The chemical nature of the substitution (C-5 to C-8: hydroxyl, nitro, methyl, isopropyl, chlorine, fluorine, and iodine atoms; C-2: phenyl and 3,4-methylenedioxyphenyl rings) of quinoline skeleton did not significantly modify compound genotoxicities; however, C-2 substitution with a-, b-, or cpyridinyl groups removed 4-methylquinoline genotoxicity. On the other hand, 4-NQO derivatives whose genotoxic mechanism involves reduction of the C-4 nitro group were strong inducers of the SOS response. Methyl and nitrophenyl substituents at C-2 of 4-NQO core affected the genotoxic potency of this molecule. The relevance of these results is discussed in relation to the potential use of the substituted quinolines. The work showed the sensitivity of SOS chromotest for studying structure–genotoxicity relaC 2013 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, tionships and bioassay-guided quinoline synthesis. V 2013.

Keywords: genotoxicity; quinolines; structure–activity relationship; SOS chromotest

INTRODUCTION

Correspondence to: J. L. F. Lorenzo; e-mail: [email protected] (or) [email protected]

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Contract grant sponsor: Vicerrectorıa de Investigaciones y Extensi on (VIE), Universidad Industrial de Santander, Contract grant number: VIE 5176. Contract grant sponsor: Patrimonio Aut onomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnologıa y la Innovaci on, Francisco Jose de Caldas, Contract grant number: RC-0572–2012 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21905

The quinolines (Qs) and their structural analogs are flat aromatic molecules widely distributed in nature that display a wide spectrum of biological activities (Michael, 1999). Diverse substituted Qs showed therapeutic potential in the treatment of diseases of microbial origin (Urbina et al., 2000; Buller et al., 2002; Hoemann et al., 2002; Jacquemond-Collet et al., 2002; Gomez-Barrio et al., 2006; Gholap et al., 2007; Nandhakumar et al., 2007; Melendez Gomez et al., 2008), as well as antitumor (Rodrıguez-Loaiza et al., 2004; Khan, 2007; Li et al., 2008; Loza-Mejıa et al.,

C 2013 Wiley Periodicals, Inc. V

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DIAZ-DURAN ET AL.

Fig. 1. Synthetic scheme for preparation of the substituted quinolines evaluated in this study.

2008) and neuroprotective (Wright et al., 1996; Di Fabio et al., 2003) agents. Despite the therapeutic potential of Qs, their use as pharmaceuticals is limited because some of these compounds are carcinogenic in the colon and liver of animal models (Hirao et al., 1976; Futakuchi et al., 1996; Saeki et al., 1997a), an effect that has been associated with their mutagenic potency in the corresponding organ (Nagao et al. 1977; Asakura et al., 1997; Suzuki et al., 1998, 2000). Therefore, the development of any therapeutic agents based on a quinoline (Q) skeleton must be focused on a rigorous examination of newly designed bioactive Qs for their genotoxicity and carcinogenicity. The Qs can exert genotoxic effect by different mechanisms. Q genotoxicity is mediated by hepatic enzymes of the cytochrome P450 family that transform this compound to a highly DNA-reactive metabolite, 1,4-dihydro-4-hydroxy2,3-epoxy-Q (Tada et al., 1982; Saeki et al., 1993; Hirano et al., 2002). This metabolite produces guanine and cytosine DNA adducts generating G ! T and G ! C transversions (Suzuki et al., 1998, 2000) that initiate different types of liver carcinomas in mammals (Hirao et al., 1976; Futakuchi et al., 1996; Saeki et al., 1997a). A similar genotoxic mechanism has been reported for halogenated Qs (Takahashi et al., 1988), as well as for 4-methylquinoline (4-MeQ) and its halogenated derivatives (Saeki et al., 1996). On the other hand, 4-nitroquinoline-1-oxide (4-NQO) is a powerful chemical carcinogen (Bailleul et al., 1989). Mutagenic activity of 4-NQO is associated with its reduction product 4-hydroxyaminoquinoline 1-oxide (4-HAQO) (Okabayashi, 1962; Okabayashi and Yoshimoto, 1962). When 4-HAQO is metabolized to an electrophilic reactant, selyl-4HAQO (Tada and Tada, 1975), it reacts with DNA to form

DNA adducts (Galiegue-Zouitina et al., 1985, 1986; Kohda et al., 1991) that are considered responsible for its carcinogenicity (Kawazoe et al., 1967, 1969). Different studies on the structure–genotoxicity relationship of Qs have shown that the incorporation of certain substituents in the Q core change their clastogenicity, mutagenicity, and carcinogenicity. For example, as indicated in Figure 1, a fluorine moiety incorporated at positions C-5 and C-7 of Q increases significantly its genotoxic activity; however, the incorporation at position C-3 produces a complete reduction of its genotoxicity in different experimental models (LaVoie et al., 1991; Saeki et al., 1997a,b; Miyata et al., 1998; Kato et al., 1999; Hakura et al., 2007; Suzuki et al., 2007). It has been also demonstrated that chlorine and fluorine incorporation at positions C-2 and C-3 of Q, respectively, result in the reduction of its carcinogenicity (Hirao et al., 1976; Saeki et al., 1997a). The 4-MeQ also showed loss of mutagenicity with fluorine and chlorine incorporation at C-2 and C-3, respectively (Saeki et al., 1996; Kato et al., 2000). Nevertheless, there are no reports on the mutagenicity of Qs containing strong electron-withdrawing substituents as halogen atoms (F and Cl) and nitro group (NO2) and poor donating substituents like alkyl groups (methyl and isopropyl) mainly at positions C-6 and C-8 of the Q skeleton or aromatic electron-withdrawing substituents (phenyl and pyridinyl) and strong donating substituents like 3,4-methylenedioxyphenyl moiety at position C-2. The diversely substituted Qs selected for this study demonstrated in vitro antifungal and antiprotozoal properties (Vargas et al., 2003; Kouznetsov et al., 2005, 2007; Melendez Gomez et al., 2008). As nothing was known about the genotoxicity of these Q derivatives, we have studied the

Environmental Toxicology DOI 10.1002/tox


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GENOTOXICITY RISK ASSESSMENT OF DIVERSELY SUBSTITUTED QUINOLINES

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Fig. 2. Synthetic scheme of new 4-nitroquinoline-N-oxide derivatives.

genotoxicity of these compounds by using the SOS chromotest (Quillardet et al., 1982). This assay is known to be sensitive in the detection of DNA damage caused by Qs with different genotoxic mechanisms (Cenci et al., 2008). This study had the following objectives: (i) to assess the genotoxicity of Qs derivatives by testing SOS chromotest; (ii) to evaluate whether the introduction of various substituents (phenyl, 3,4-methylenedioxyphenyl, pyridinyl, and 4-nitrophenyl) in the C-2 position of the Q, 4-MeQ, or 4-NQO skeletons could modify their genotoxicity; and (iii) to estimate whether the incorporation of halogen atoms or alkyl groups into the benzene ring of 2-phenylquinolines, 2-(3,4-methylenedioxyphenyl)quinolines, and 4-methyl-2-pyridinylquinolines modify their genotoxicity.

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phenyl-b-D-galactopyranoside or ONPG) and alkaline phosphatase (p-nitrophenylphosphate [PNPP]) and the compound nicotinamide adenine dinucleotide phosphate were purchased from AMRESCO (Solon, Ohio, USA). The lyophilized S9 fraction of male rat liver was purchased from MOLTOX (Molecular Toxicology, Boone, NC, USA). The remaining chemicals were obtained from J.T. BAKER (Phillipsburg, NJ, USA).

Bacterial Strain and Culture E. coli PQ37 strain [F2 thr leu his-4 pyrD thi galE galK or galT lacDU169 srl300::Tn10 rpoB rpsL uvrA rfa trp::Muc1 sulA::Mud(Ap,lac)ts] that has been recommended for the detection of genotoxic carcinogens (Quillardet et al., 1982) was used in this work. This strain carries the sulA::lacZ fusion gene as a reporter gene for primary DNA damage induced during the SOS response. The cells were grown overnight at 37 C and shaken at 100 rpm in LB medium (10 g tryptone/L, 5 g yeast extract/L, 10 g sodium chloride/L, pH 7.4) supplemented with ampicillin (50 lg/mL) and tetracycline (17 lg/mL).

Genotoxicity Assay MATERIALS AND METHODS Tested Quinoline Molecules In total, 24 Qs were evaluated for genotoxicity (Fig. 1). Commercial Q, 4-MeQ, 8-hydroxyquinoline (8-HQ), 4,7dichloroquinoline (4,7-DCQ), and 4-NQO were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Syntheses of noncommercial polyfunctionalized 2-phenylquinolines, 2(3,4-methylenedioxyphenyl)quinolines and 2-aryl-4methylquinolines tested in this work, were carried out using diverse aldehydes and anilines as starting materials following known synthetic procedures (Kouznetsov et al., 2007; Melendez G omez et al., 2008). Spectral data for known Qs were identical to those published. New quinoline-N-oxide derivatives were prepared using established synthetic protocols (Sasaki et al., 1998). Briefly, Q was mixed with hydrogen peroxide in the presence of acetic acid at 70 C yielding quinoline N-oxide. After separating the resulting solid, it was treated with potassium nitrate and sulfuric acid at room temperature to give the desired compound with the nitro group selectively at position C-4 F2 (Fig. 2).

Culture Media and Chemicals Luria–Bertani (LB) medium, antibiotics (ampicillin and tetracycline), mutagens such as aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and 4-NQO, and the compounds D-glucose6-phosphate and dimethylsulfoside were obtained from Sigma-Aldrich. The substrates for b-galactosidase (o-nitro-

Genotoxicity was evaluated using the SOS chromotest as described by Quillardet and Hofnung (1985). Briefly, overnight cultures were grown in fresh LB medium to an optical density (OD600 nm) of 0.4. Then, 0.1 mL of the culture is diluted in either 0.9 mL of fresh LB medium for assay without metabolic activation or 0.9 mL of S9 mix for assay with metabolic activation. Fractions of 150 lL are distributed into a series of Eppendorf tubes containing 5 lL of samples of the compounds to be tested. The mixtures are incubated first for 30 min at 8 C and after 2 h at 37 C with shaking in an Eppendorf Termomixer apparatus (Sao Paulo, Brazil). Negative (distilled water) and positive (B[a]P and/or 4NQO) controls were always included in each assay. A minimum of three independent experiments per treatment with four replicate each were conducted. b-Galactosidase and alkaline phosphatase were assayed in 96-well plates (Brand GMBH, Germany) as follows: for b-galactosidase activity, cell membranes were disrupted by mixing 135 lL of Z buffer (110 mM mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM Mg2SO4, 0.1% SDS, and 40 mM b-mercaptoethanol, pH 7.0) with 15 lL of cell culture for 20 min at 37 C. The reaction was started by adding 30 lL of ONPG (4 mg/mL in phosphate buffer: 61 mL Na2HPO4 0.1 M and 39 mL NaH2PO4H2O 0.1 M, pH 7). After 40 min, the enzymatic reaction was stopped by adding 100 lL of 1 M Na2CO3. For alkaline phosphatase activity, cell membranes were disrupted by adding 135 lL of T buffer (1 M Tris and 0.1% SDS, pH 8.8) to 15 lL of cell culture followed by mixing vigorously and incubation for 20 min at 37 C. The enzyme reaction was started by adding 30




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lL of PNPP solution (4 mg/mL in T buffer). After 40 min, the reaction was stopped by adding 50 lL of 2 M HCl. After 5 min, 50 lL of 2 M Tris was added to restore the color. The final absorbances of the b-galactosidase and alkaline phosphatase assays were measured at 420 nm using a Multiskan Go microplate reader (Thermo Scientific, Milford, MA, USA). b-Galactosidase and alkaline phosphatase activities were calculated using the relationship: 420 (Quillardet et al., 1982), where Enzyme units 5 10003A t A420 is the OD at 420 nm and t is the length of incubation (min) with substrate (ONPG or PNPP). The ratio of b-galactosidase units to alkaline phosphatase units b -Galactosidase units (R5 Alkaline phosphatase units ) reflects the induction of the sulA gene relative to protein synthesis, thus accounting for any effect of the test substance on protein synthesis. The genotoxicity criterion used was the SOS induction factor (I) that represents the normalized induction data of the sulA gene in each treatment and was therefore considered to be an indirect measure of the primary DNA damage (genotoxicity) induced by the treatments. This parameter was calculated as follows: I5 RRntt , where t and nt are the treated and nontreated cells, respectively. A substance was classified as not genotoxic if I was 2.0, and a clear concentration–response relationship was observed. The SOS-inducing potency (SOSIP) or the slope of the linear region of the dose–response curves (Quillardet et al., 1982) was calculated for genotoxic compounds. This parameter represents the induction factor per mass unit or per nanomole of compound tested and this was computed as DI , where DI is the difference follows: SOSIP 5 DC between two points in the linear region of the dose response curve, and DC is the difference between the corresponding concentrations.

RESULTS

scopic data. Spectral data for quinoline-N-oxide derivatives are as follows: Data of 2-methylquinoline N-oxide: 79% yield, mp: 109– 111 C; FTIR (KBr disk, cm21): 3033 m(@CHAr), 2946 m(ACH3), 1589 m(CAr@CAr), 1541 m(CAN). 1H NMR (400 MHz, CDCl3), d(ppm): 8.27 (1H, d, J 5 7.3 Hz, 8-HAr), 7.87– 8.08 (3H, m, 5-, 6-, and 7-HAr), 7.62 (1H, dd, J 5 7.7, 1.4 Hz, 4-HAr), 7.21 (1H, d, J 5 7.7 Hz, 3-HAr), 2.67 (3H, s, HCH3). 13C NMR (101 MHz, CDCl3), d(ppm): 137.1, 133.7, 130.1, 129.6, 126.9, 125.2, 123.1, 121.6, 120.3, 19.2. Data of 2-methyl-4-nitroquinoline N-oxide: 45% yield, mp: 127–129 C; FTIR (KBr disk, cm21): 3033 m(@CHAr), 2946 m(ACH3), 1589 m(CAr@CAr), 1542 m(CANO2), 1357 m(CANO2). 1H NMR (400 MHz, CDCl3), d(ppm): 8.27 (1H, d, J 5 7.4 Hz, 8-HAr), 7.88–8.08 (3H, m, 5-, 6-, and 7-HAr), 7.65 (1H, s, 3-HAr), 2.72 (3H, s, CH3). 13C NMR (101 MHz, CDCl3), d(ppm): 148.5, 140.8, 132.1, 131.9, 127.2, 124.7, 122.6, 120.9, 120.1, 20.0. Data for new 2-(4-nitrophenyl)quinoline: 62% yield, Rf 5 0.63 (5:1 hexane/EtOAc); mp: 134–136 C; FTIR (KBr disk, cm21): 3043 m(@CHAr), 2946 m(@CHAr), 1589 m(CAr@CAr), 1541 m(CANO2), 1342 m(CANO2). 1H NMR (400 MHz, CDCl3), d(ppm): 8.28 (2H, d, J 5 8.6 Hz, 30 - and 50 -HAr), 8.22 (1H, m, 3-HAr), 8.14 (1H, d, J 5 7.7 Hz, 8-HAr), 7.88 (1H, d, J 5 8.6 Hz, 4-HAr), 7.83 (1H, d, J 5 8.1 Hz, 5-HAr), 7.73 (1H, ddd, J 5 8.2, 7.2, 0.7 Hz, 6-HAr), 7.55–7.52 (1H, m, 7-HAr), 7.45 (2H, d, J 5 8.6 Hz, 20 - and 60 -HAr). 13C NMR (101 MHz, CDCl3), d(ppm): 154.4, 148.9, 148.8, 141.3, 138.2, 137.4, 133.3, 129.9, 129.7, 127.6, 127.5, 127.1, 123.9, 122.4, 118.3. GC: tR 5 24.12 min.; MS (EI): 250 (M1). Data of new 2-(4-nitrophenyl)quinoline N-oxide: 81% yield, mp: 139–141 C; FTIR (KBr disk, cm21): 2946 m(@CHAr), 1604 m(CAr@CAr), 1542 m(CANO2), 1357 m(CANO2). 1 H NMR (400 MHz, CDCl3), d(ppm): 8.38 (2H, d, J 5 8.7 Hz, 30 - and 50 -HAr), 8.19 (1H, d, J 5 7.5 Hz, 8-HAr), 8.02 (1H, d, J 5 7.8 Hz, 3-HAr), 7.98 (2H, d, J 5 8.7 Hz, 20 - and 60 -HAr), 7.81 (1H, d, J 5 7.8 Hz, 4-HAr), 7.63 (1H, d, J 5 7.4 Hz, 5HAr), 7.54–7.60 (2H, m, 6- and 7-HAr). 13C NMR (101 MHz, CDCl3), d(ppm): 148.9, 137.5, 134.9, 132.5, 129.8, 129.2 (2C), 128.3, 127.0, 126.8 (2C), 123.4, 122.5, 121.6, 120.3. Data of new 4-nitro-2-(4-nitrophenyl)quinoline N-oxide: 56% yield, mp: 149–151 C; FTIR (KBr disk, cm21): 2946 m(@CHAr), 1604 m(CAr@CAr), 1542 m(CANO2), 1357 m(CANO2). 1 H NMR (400 MHz, CDCl3), d(ppm): 8.41 (2H, d, J 5 8.6 Hz, 30 - and 50 -HAr), 8.27 (1H, s, 3-HAr), 8.16 (1H, d, J 5 7.2 Hz, 8-HAr), 8.00 (2H, d, J 5 8.7 Hz, 20 - and 60 -HAr), 7.77 (1H, d, J 5 6.8 Hz, 5-HAr), 7.37–7.48 (2H, m, 6- and 7-HAr). 13 C NMR (101 MHz, CDCl3), d(ppm): 148.9, 148.2, 136.4, 135.1, 133.6, 130.8 (2C), 129.0, 126.4, 125.3 (2C), 123.1, 121.3, 120.9, 120.2.

Synthesis of Quinoline-N-Oxide Derivatives

Induction Kinetics of the sulA Gene by Standard Mutagens

Five new quinoline-N-oxide compounds were obtained, and their structures were elucidated through 1H and 13C spectro-

To select the optimal genotoxic dose for standard mutagens used as positive controls in genotoxicity assays, the

Statistical Analysis The I values were expressed as the mean 6 SEM. The data normality was tested using the Kolmogorov-Smirnov test, and the homogeneity of variances was assessed by analysis of variance. Statistical comparisons were done using Dunnet’s test. Product–moment (Pearson) correlation analysis was used to examine the concentration–response relationship in the genotoxicity experiments. A value of p < 0.05 indicated significance in all cases. All data analyses were done using the STATISTICA software package v.6 (StatSoft, Tulsa, OK, USA).




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induction kinetics of the sulA gene in E. coli PQ37 following treatment with mutagens B[a]P (in the presence of metabolic activation) and 4-NQO (in the absence of metabolic activation) were studied for ranges between 0.1 and 400 lM and 0.13 and 282 lM, respectively (data not shown). The I values increased with B[a]P and 4-NQO dose and were significant beginning at 12.5 and 0.13 lM, respectively. Based on these results, B[a]P and 4-NQO dose of 50.0 and 0.27 lM, respectively, which yields I maximum values and nonreduction of protein synthesis as indicated by the constitutive b-galactosidase and alkaline phosphatase activities (data not shown), were used as positive controls in each Qs genotoxicity assay.

Quinoline Derivatives Genotoxicity The genotoxic effect of Q derivatives was investigated by T1 means of the SOS chromotest (Table I). The Q and 8-HQ did not significantly increase the I values in E. coli PQ37. The incorporation of chlorine at positions C-4 and C-7, phenyl group at position C-2, and halogens (chlorine, fluorine, and iodine) at position C-6 of Q core did not modify molecule genotoxicity. Last, incorporation of the 3,4-methylenedioxyphenyl at position C-2 of Q core alone or combined with groups methyl at positions C-5, C-6, and C-7 or methoxy at position C-6 neither modify genotoxicity. The results showed that these compounds do not induce the SOS response in E. coli.

4-Methylquinoline Derivatives Genotoxicity

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The genotoxic effect of 4-MeQ derivatives was also investiT2 gated (Table II). The 4-MeQ in the presence of metabolic activation, but not in the absence, significantly increased the I values from a dose of 8.8 lM. A moderate but significant correlation between concentration and DNA damage was observed in product–moment correlation analysis (R 5 0.5; p < 0.05). This indicates that 4-MeQ is weakly genotoxic in E. coli cells. The 4-MeQ SOSIP value (0.37) was consistently lower than B[a]P (0.63) and AFB1 (50.1), two standard mutagens that generate epoxides reactive with DNA by metabolic activation. SOSIP values depend on compound size as follows: AFB1 > B[a]P > 4-MeQ, suggesting that the size of DNA adduct produced by epoxides may be a factor in induction of SOS response. Incorporation of (a, b, and c) isomeric pyridinyl rings at position C-2, nitro group at position C-5, chlorine, fluorine, and methyl groups at position C-6, or fluorine and isopropyl group at position C-8 of 4-MeQ core did not significantly increase the I values in E. coli PQ37. In addition, change of nitrogen position inside pyridinyl groups (i.e., pyridin-2-yl, pyridin-4-yl, and pyridin-5-yl) of 4-MeQ derivatives either modified the genotoxicity patterns of these molecules. Thus, the vast majority of these derivatives showed significant

5

reduction for I values when compared with 4-MeQ genotoxic concentration (Table II).

4-Nitroquinoline 1-Oxide Derivatives Genotoxicity Genotoxicity data of 4-NQO derivatives are shown in Table III. 4-NQO molecule induced sulA gene coming from a dose of 0.13 lM reaching the I maximum value (34 6 3) at a dose of 0.27 lM. The incorporation of methyl and 4-nitrophenyl groups at position C-2 of 4-NQO ring (i.e., 2-methyl-4-NQO and 2-(4-nitrophenyl)24-NQO derivatives) significantly reduces its genotoxic effect. These two 4-NQO derivatives show I maximum values (42.6 6 1.2 and 12.2 6 1.2) at higher dose of 141 lM and 35.2 lM, respectively. The SOSIP values for these three compounds were as follows: 4-NQO (836.95) > 2-(4-nitrophenyl)24-NQO (3.79) > 2methyl-4-NQO (1.77). The incorporation of 4-nitrophenyl and methyl groups at position C-2 of 4-NQO ring reduced 221 and 472 times the SOSIP values of this compound, respectively. Quinoline-N-oxidized derivatives such as 2-(4-nitrophenyl)quinoline-1-oxide and 2-methylquinoline-1-oxide), where NO2 group at position C-4 is absent, did not induce genetic damage in the SOS chromotest, confirming the importance of the nitro group at position C-4 for genotoxicity of 4-NQO derivatives.

DISCUSSION This work provides new information about genotoxicity of polyfunctionalized Qs with therapeutic potential as antifungal and antiprotozoal agents (Vargas et al., 2003; Kouznetsov et al., 2005, 2007; Melendez Gomez et al., 2008). In total, five new quinoline-N-oxide compounds were obtained, and their structures were elucidated through 1H and 13C spectroscopic data. As an example, we highlight the absence of the signal near to 7.81 ppm in the 1H NMR spectrum of the 4-nitro-2-(4-nitrophenyl)quinoline N-oxide that would correspond to proton 4-H, indicating that this position was substituted by the nitro group in the final step. The concordance of the remaining signals with the proposed structure prove that the nitro group was not inserted in other position and were in agreement with the signals depicted by the 2-(4-nitrophenyl)quinoline N-oxide and the 2-(4nitrophenyl)quinoline. The Q and their derivatives 8-HQ and 4,7-DCQ did not induce genetic damage in the SOS chromotest, whereas 4MeQ resulted in a weak genotoxic response in accordance with previous findings showing quinolines I maximum values between 0.95 to 6.74 with a mean of 1.23 (He et al., 2005). According to these findings, the Qs appear to be poor to moderate genotoxic compounds. However, mutagenic properties of the Q and 4-MeQ have been widely demonstrated by means of the Salmonella/microsome assay (Nagao



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TABLE I. SOS induction factor (I) in E. coli PQ37 cells treated with Q derivatives using the SOS chromotest Compounds

Treatments

With S9 Fraction

Without S9 Fraction

Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141.0 lM 282.0 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM

1.0 6 0.1 – 3.1 6 0.4a 1.1 6 0.2b 0.9 6 0.1b 0.9 6 0.1b 0.8 6 0.2b 0.9 6 0.2b 0.9 6 0.1b 1.0 6 0.1b 1.1 6 0.2b 1.2 6 0.2b 1.0 6 0.2b 1.0 6 0.2b 0.8 6 0.1b 1.0 6 0.0 – 3.3 6 0.5a 1.0 6 0.1b 1.0 6 0.2b 1.1 6 0.1b 1.1 6 0.1b 1.1 6 0.1b 1.0 6 0.0b 1.2 6 0.1b 1.2 6 0.1b 1.3 6 0.1b 1.2 6 0.1b 0.7 6 0.1b 0.5 6 0.0b 1.0 6 0.0 – 3.4 6 0.3a 0.9 6 0.1b 1.1 6 0.1b 1.1 6 0.1b 1.0 6 0.2b 1.0 6 0.1b 1.0 6 0.1b 1.3 6 0.4b 1.1 6 0.1b 1.1 6 0.2b 1.1 6 0.1b 0.9 6 0.1b 0.6 6 0.1b 1.0 6 0.1 – 3.3 6 0.3a 1.1 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 1.1 6 0.0b 1.2 6 0.2b 1.0 6 0.1b

1.0 6 0.1 11.1 6 1.3a – 0.9 6 0.2b 0.9 6 0.2b 0.9 6 0.1b 1.0 6 0.2b 1.1 6 0.2b 1.0 6 0.2b 1.1 6 0.1b 0.8 6 0.2b 0.6 6 0.1b 0.2 6 0.1b 0.2 6 0.0b 0.3 6 0.1b 1.0 6 0.1 4.8 6 0.9a – 0.7 6 0.1b 0.8 6 0.1b 0.7 6 0.2b 0.6 6 0.1b 0.7 6 0.1b 0.7 6 0.2b 0.7 6 0.1b 0.7 6 0.1b 0.5 6 0.1b 0.5 6 0.1b 0.4 6 0.0b 0.2 6 0.1b 1.0 6 0.1 12.7 6 1.4a – 1.2 6 0.1b 1.2 6 0.1b 1.3 6 0.1b 0.6 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 1.2 6 0.4b 1.0 6 0.3b 0.8 6 0.1b 0.9 6 0.2b 0.9 6 0.2b 0.5 6 0.1b 1.0 6 0.1 14.2 6 3.3a – 1.1 6 0.3b 1.0 6 0.1b 1.0 6 0.2b 1.1 6 0.1b 1.2 6 0.2b 1.2 6 0.2b




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TABLE I. Continued Compounds

Treatments 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM

With S9 Fraction b

Without S9 Fraction 1.2 6 0.2b 1.2 6 0.1b 1.2 6 0.1b 1.3 6 0.2b 1.5 6 0.1b 1.3 6 0.1b 1.0 6 0, 13.6 6 3.7a – 0.9 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 1.1 6 0.1b 0.9 6 0.1b 1.0 6 0.1b 0.8 6 0.0b 1.1 6 0.1b 0.9 6 0.1b 0.9 6 0.0b 0.9 6 0.1b 1.0 6 0.1 15.3 6 3.0a – 1.0 6 0.1b 0.9 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 0.9 6 0.2b 1.0 6 0.3 11.1 6 1.4a – 1.1 6 0.1b 1.1 6 0.0b 1.1 6 0.1b 0.8 6 0.2b 0.8 6 0.2b 0.8 6 0.1b 1.0 6 0.2b 0.8 6 0.1b 0.9 6 0.1b 0.7 6 0.1b 0.8 6 0.1b 0.5 6 0.1b 1.0 6 0.0 12.4 6 2.5a – 0.9 6 0.0b

1.0 6 0.1 1.0 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.7 6 0.0b 1.0 6 0.1 – 3.3 6 0.3a 0.9 6 0.1b 0.9 6 0.0b 1.0 6 0.1b 1.0 6 0.0b 1.1 6 0.1b 1.2 6 0.1b 0.7 6 0.2b 0.8 6 0.1b 0.7 6 0.1b 0.8 6 0.2b 0.8 6 0.2b 0.8 6 0.2b 1.0 6 0.1 – 3.3 6 0.4a 1.0 6 0.2b 0.9 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 1.0 6 0.2b 1.0 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.9 6 0.2b 0.8 6 0.2b 1.0 6 0.0 – 3.0 6 0.2a 1.0 6 0.0b 0.7 6 0.1b 1.0 6 0.1b 1.1 6 0.0b 1.1 6 0.1b 0.9 6 0.1b 1.0 6 0.0b 0.9 6 0.1b 0.9 6 0.0b 0.8 6 0.0b 0.9 6 0.0b 0.8 6 0.1b 1.0 6 0.0 – 3.6 6 0.3a 1.2 6 0.1b




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TABLE I. Continued Compounds

Treatments 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM

With S9 Fraction b

0.9 6 0.1 1.1 6 0.2b 0.9 6 0.2b 1.0 6 0.1b 0.8 6 0.1b 0.9 6 0.1b 0.8 6 0.0b 0.9 6 0.1b 0.8 6 0.1b 0.8 6 0.1b 0.8 6 0.1b 1.0 6 0.1 – 3.0 6 0.4a 1.1 6 0.1b 1.1 6 0.1b 0.9 6 0.1b 1.1 6 0.1b 1.1 6 0.1b 1.1 6 0.1b 1.1 6 0.1b 1.2 6 0.1b 1.1 6 0.1b 1.2 6 0.1b 1.3 6 0.3b 0.8 6 0.1b 1.0 6 0.16 – 3.0 6 0.4a 0.7 6 0.2b 0.8 6 0.2b 0.8 6 0.2b 0.8 6 0.1b 0.9 6 0.2b 0.9 6 0.2b 1.0 6 0.1b 1.0 6 0.1b 1.0 6 0.0b 0.9 6 0.1b 0.9 6 0.1b 0.9 6 0.1b

Without S9 Fraction 0.9 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 0.8 6 0.1b 0.8 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 0.7 6 0.1b 1.0 6 0.1 11.3 6 1.5a – 1.1 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 1.0 6 0.1 10.6 6 1.0a – 1.3 6 0.2b 1.2 6 0.2b 1.1 6 0.1b 0.9 6 0.2b 1.0 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 0.7 6 0.1b 0.8 6 0.1b 0.8 6 0.1b 0.7 6 0.1b

The values are the mean 6 SEM of minimal three independent experiments with four replicates each. Negative control was distilled water. Positive control for assay with (B[a]P) and without (4-NQO) metabolic activation was used at concentrations of 50.0 lM and 0.3 lM, respectively, which were the I maximum values to each compounds. a Significant differences for I values when compared with the negative control (p < 0.05, Dunnet’s test). b No significant difference was found.

et al., 1977; Takahashi et al. 1988; LaVoie et al., 1991; Saeki et al., 1996, b; Kato et al., 1999, 2000). Contrasting results with SOS chromotest and Salmonella/microsome assay could be explained because these two assays respond to different genotoxic stimuli (Rosenkranz et al., 1999). Salmonella/microsome assay detects point mutations in Salmonella

histidine gene, whereas the SOS chromotest responds to DNA damage that induces SOS response in E. coli. Suzuki et al. (2000) have postulated that exposure to Q produce quinoline-N2-guanine DNA adducts, whose small size may make conformational change that results in misparing with guanine. However, it is improbable that DNA




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TABLE II. SOS induction factor (I) in E. coli PQ37 cells treated with 4-MeQ derivatives using the SOS chromotest Compounds

Treatments

With S9 Fraction

Without S9 Fraction

Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141.0 lM 282.0 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM

1.0 6 0.1 – 4.6 6 1.3a 1.0 6 0.1b 0.9 6 0.1b 1.1 6 0.2b 1.0 6 0.1b 1.1 6 0.1b 1.3 6 0.2b 1.3 6 0.2a 1.5 6 0.4a 1.6 6 0.3a 1.5 6 0.3a 1.5 6 0.3a 1.3 6 0.1a 1.0 6 0.0 – 4.0 6 1.2a 1.0 6 0.1b 1.0 6 0.2b 1.0 6 0.2b 1.0 6 0.2b 1.1 6 0.2b 1.0 6 0.2b 1.1 6 0.1b,c 1.1 6 0.2b,c 1.1 6 0.1b,c 0.9 6 0.1b,c 0.7 6 0.1b,c 0.5 6 0.0b,c 1.0 6 0.1 9.2 6 1.6a – 1.2 6 0.2b 0.8 6 0.2b 0.8 6 0.1b 1.1 6 0.1b 1.2 6 0.1b 1.3 6 0.1b 1.1 6 0.1b,c 1.2 6 0.1b,c 0.9 6 0.2b,c 1.1 6 0.1b,c 0.9 6 0.1b,c 0.6 6 0.1b,c 1.0 6 0.1 9.3 6 0.9a – 0.9 6 0.1b 0.5 6 0.0b 0.8 6 0.1b 0.8 6 0.1b 0.9 6 0.1b 0.9 6 0.1b

1.0 6 0.0 10.0 6 2.5a – 0.9 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 0.8 6 0.1b 0.6 6 0.1b 0.6 6 0.0b 0.4 6 0.0b 0.4 6 0.0b 0.2 6 0.0b 0.3 6 0.0b 1.0 6 0.0 12.5 6 1.6a – 1.1 6 0.1b 1.1 6 0.1b 1.1 6 0.1b 1.1 6 0.0b 1.1 6 0.1b 1.1 6 0.3b 1.1 6 0.0b 1.0 6 0.1b 1.2 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 0.8 6 0.2b 1.0 6 0.1 – 4.2 6 0.5a 0.9 6 0.1b 0.9 6 0.1b 1.1 6 0.1b 1.0 6 0.2b 0.9 6 0.3b 0.9 6 0.2b 1.1 6 0.1b 1.0 6 0.2b 1.1 6 0.2b 1.1 6 0.1b 0.9 6 0.1b 0.7 6 0.1b 1.0 6 0.1 – 3.4 6 1.2a 1.3 6 0.1b 1.0 6 0.1b 1.1 6 0.2b 0.9 6 0.1b 0.9 6 0.1b 0.8 6 0.2b




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TABLE II. Continued Compounds

Treatments 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM

With S9 Fraction b,c

1.0 6 0.1 0.8 6 0.2b,c 1.0 6 0.1b,c 0.9 6 0.1b,c 0.9 6 0.1b,c 0.6 6 0.1b,c 1.0 6 0.0 16.6 6 0.9a – 1.1 6 0.1b 1.0 6 0.1b 1.0 6 0.1b 1.1 6 0.2b 1.2 6 0.2b 1.1 6 0.1b 1.1 6 0.2b,c 1.0 6 0.1b,c 1.2 6 0.2b,c 1.0 6 0.2b,c 0.7 6 0.1b,c 0.45 6 0.1b,c 1.0 6 0.0 9.0 6 1.2a – 1.1 6 0.1b 0.9 6 0.1b 0.9 6 0.1b 1.2 6 0.2b 1.3 6 0.2b 1.3 6 0.2b 1.3 6 0.1b,c 1.2 6 0.1b,c 1.3 6 0.2b,c 1.3 6 0.2b,c 1.0 6 0.1b,c 0.8 6 0.2b,c 1.0 6 0.1 13.9 6 1.5a – 1.1 6 0.2b 1.1 6 0.2b 1.1 6 0.2b 0.9 6 0.1b 1.0 6 0.1b 0.9 6 0.1b 0.9 6 0.1b,c 1.1 6 0.1b,c 1.1 6 0.1b,c 1.1 6 0.0b,c 1.0 6 0.2b,c 1.1 6 0.2b,c 1.0 6 0.1 10.4 6 1.4a – 1.2 6 0.2b



Without S9 Fraction 0.7 6 0.1b 0.7 6 0.1b 0.6 6 0.1b 0.6 6 0.0b 0.5 6 0.0b 0.3 6 0.0b 1.0 6 0.1 – 3.5 6 0.8a 1.1 6 0.3b 1.2 6 0.1b 0.9 6 0.1b 1.0 6 0.1b 1.2 6 0.1b 0.9 6 0.1b 1.1 6 0.0b 1.0 6 0.2b 1.1 6 0.1b 1.0 6 0.1b 1.0 6 0.2b 1.0 6 0.2b 1.0 6 0.1 – 2.8 6 0.4a 1.1 6 0.3b 0.9 6 0.2b 0.9 6 0.2b 1.0 6 0.2b 1.0 6 0.2b 0.9 6 0.1b 1.1 6 0.2b 1.0 6 0.1b 1.2 6 0.2b 0.7 6 0.1b 0.9 6 0.2b 0.7 6 0.2b 1.0 6 0.2 – 3.2 6 0.8a 0.7 6 0.2b 0.7 6 0.1b 0.7 6 0.1b 0.7 6 0.2b 0.8 6 0.2b 0.9 6 0.2b 1.0 6 0.2b 0.6 6 0.1b 0.6 6 0.3b 0.6 6 0.2b 0.6 6 0.2b 0.5 6 0.1b 1.0 6 0.2 – 4.5 6 1.3a 0.8 6 0.1b


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TABLE II. Continued Compounds

Treatments 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM Negative control Positive control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141 lM 282 lM

With S9 Fraction b

1.4 6 0.3 1.2 6 0.2b 1.0 6 0.2b 0.9 6 0.2b 0.7 6 0.1b 0.7 6 0.1b,c 0.9 6 0.1b,c 0.7 6 0.1b,c 1.0 6 0.2b,c 1.0 6 0.3b,c 0.9 6 0.2b,c 1.0 6 0.2 13.0 6 2.0a – 1.0 6 0.2b 1.1 6 0.1b 1.0 6 0.2b 0.9 6 0.1b 0.9 6 0.2b 0.9 6 0.2b 0.8 6 0.1b,c 0.9 6 0.1b,c 0.9 6 0.2b,c 0.9 6 0.1b,c 0.8 6 0.1b,c 0.8 6 0.0b,c

Without S9 Fraction 0.9 6 0.1b 0.9 6 0.1b 1.0 6 0.2b 1.2 6 0.2b 1.0 6 0.2b 0.9 6 0.2b 1.0 6 0.2b 0.8 6 0.1b 1.1 6 0.3b 0.7 6 0.1b 1.1 6 0.3b 1.0 6 0.1 – 3.4 6 0.9a 0.7 6 0.1b 0.8 6 0.1b 0.6 6 0.1b 0.7 6 0.0b 0.9 6 0.2b 1.0 6 0.2b 0.8 6 0.1b 1.0 6 0.2b 0.9 6 0.1b 1.0 6 0.3b 0.8 6 0.2b 0.6 6 0.1b

The values are the mean 6 SEM of minimal three independent experiments with four replicates each. Negative control was distilled water. Positive control for assay with (B[a]P) and without (4-NQO) metabolic activation was used at concentrations of 50.0 lM and 0.3 lM, respectively. a Significant differences for I values when compared with the negative control (p < 0.05, Dunnet’s test). b No significant difference was found. c Significant differences for I values when compared with 4-MeQ genotoxic concentration (p < 0.05, Dunnet’s test).

damage producing a small conformational change in the DNA helix would be strong inducers of SOS response. The most common cellular signals activating the SOS response are double-strand breaks or single-stranded DNA regions arising from stalled replication forks (Salles and Defais, 1984; Walker, 1984). Therefore, if there is no DNA replication block, there is no SOS induction. Thus, it is expected that the SOS chromotest would respond more efficiently to compounds that produce DNA adducts which block DNA replication. Previous work (Mersch-Sundermann et al., 1996) using MULTICASE and chemical biophores nature analyses revealed that the accessibility of the electrophile to the nucleophilic site on the DNA and the bulkiness of the DNA adduct were factors determining the probability that chemicals would induce SOS response. To validate the hypothesis in which bulkiness of the DNA adduct is a factor influencing the SOS induction, we compared the SOSIP values of three compounds (AFB1, B[a]P, and 4-MeQ) whose genotoxicity involves epoxide formation (Sims et al., 1974; Raney et al., 1992; Saeki et al., 1996), expecting that DNA adducts produced by these compounds cause different levels of structural distortion in DNA and, therefore, different potency to

induce SOS response. As we described in the “Results” section, SOSIP values depend on compound size as follows: AFB1 > B[a]P > 4-MeQ, suggesting that the SOS chromotest was more sensitive to bulky DNA adducts. Contrasting with AFB1 and B[a]P whose interaction with DNA has been well studied (Johnson and Guengerich, 1997; Perlow and Broyde, 2003), there are little information about how 4-MeQ interacts with DNA. Saeki et al. (1996) have identified the 4-MeQ metabolites (majorly 4-hydroxymethylquinoline) and the plausible intermediates (1,4-hydrated 2,3epoxide) that may react covalently with DNA; however, the resulting 4-MeQ DNA adducts has been not described yet. Suzuki et al. (2000) suggested that quinoline-N2-guanine may be majorly responsible for Q-induced mutagenesis in vivo, although Q binds to poly(A) approximately three times more than to poly(C) or poly(G) in vitro (Tada et al., 1980). Our findings showed lower sensitive of the SOS chromotest to detect 4-MeQ-induced DNA damage than AFB1 and B[a]P, suggesting that 4-MeQ DNA adducts, probably 4-methyl-quinoline-N2-guanine, interfere poorly with DNA replication. The results also supported the hypothesis where bulky DNA adducts that block DNA replication are an




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TABLE III. SOS induction factor (I) in E. coli PQ37 cells treated with 4-NQO derivatives using the SOS chromotest

Compounds

Treatments

Without S9 Fraction

Negative control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141.0 lM 282.0 lM Negative control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141.0 lM 282.0 lM Negative control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141.0 lM 282.0 lM Negative control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM

1.0 6 0.1 24.2 6 2.1a 16.6 6 1.9a 33.9 6 2.8a 32.2 6 2.9a 29.0 6 3.2a 11.8 6 1.8a 3.0 6 0.7a 0.9 6 0.3b 0.5 6 0.0b 0.4 6 0.1b 0.2 6 0.0b 0.2 6 0.0b 0.1 6 0.0b 1.0 6 0.1 16.0 6 3.5a 0.8 6 0.2b,c 1.0 6 0.1b,c 0.9 6 0.1b,c 1.1 6 0.2b,c 1.4 6 0.1b,c 1.6 6 0.2b,c 2.8 6 0.3a 5.2 6 0.5a 11.9 6 0.3a 18.9 6 1.0a 42.6 6 1.2a 35.1 6 2.1a 1.0 6 0.1 34.4 6 13.3a 1.0 6 0.2b,c 1.1 6 0.2b,c 1.2 6 0.2b,c 1.8 6 0.3a,c 2.4 6 0.5a,c 3.4 6 0.6a 4.1 6 1.2a 9.0 6 0.5a 12.2 6 1.2a 10.4 6 1.5a 9.3 6 1.9a 2.5 6 0.6a 1.0 6 0.1 9.8 6 3.5a 0.9 6 0.1b,c 1.0 6 0.1b,c 0.7 6 0.1b,c 0.9 6 0.1b,c 1.1 6 0.1b,c 0.8 6 0.1b,c 0.8 6 0.1b

TABLE III. Continued Compounds

Treatments

Without S9 Fraction

17.6 lM 35.2 lM 70.5 lM 141.0 lM 282.0 lM Negative control Positive control 0.13 lM 0.27 lM 0.55 lM 1.1 lM 2.2 lM 4.4 lM 8.8 lM 17.6 lM 35.2 lM 70.5 lM 141.0 lM 282.0 lM

1.0 6 0.0b 0.9 6 0.1b 1.2 6 0.2b 1.0 6 0.2b 1.1 6 0.2b 1.0 6 0.1 26.2 6 5.2a 0.8 6 0.1b,c 0.8 6 0.1b,c 0.8 6 0.1b,c 0.8 6 0.1b,c 0.7 6 0.1b,c 0.8 6 0.1b,c 1.1 6 0.1b 1.0 6 0.2b 1.0 6 0.2b 1.1 6 0.2b 1.1 6 0.2b 1.1 6 0.1b

The values are the mean 6 SEM of minimal three independent experiments with four replicates each. Negative control was distilled water. Positive control for assay without (4-NQO) metabolic activation was used at concentrations of 0.3 lM. a Significant differences for I values when compared with the negative control (p < 0.05, Dunnet’s test). b No significant difference was found. c Significant reduction for I values when compared with 4-NQO genotoxic concentration (p < 0.05, Dunnet’s test).

essential factor for triggering the SOS response in E. coli cells. In fact, He et al. (2005), based on multiple classifier system analysis, concluded that the size and shape of the Q derivatives and their hydrophobic/hydrophilic characters are the main determinants of Qs genotoxicity. However, this hypothesis could be experimentally demonstrated for a higher number of Qs carefully selected based on their size. As expected, Q or 4-MeQ derivatives with group adding at position C-2 of pyridine ring (i.e., 2-phenylquinolines, 2(3,4-methylenedioxyphenyl)quinolines, and 2-aryl-4-methylquinolines) showed negative results in the SOS chromotest. In addition, the incorporation of halogen atoms or alkyl groups into the benzene ring of these Q or 4-MeQ derivatives either modify their genotoxicity. Contrasting with 4- AQ5 MeQ, the 2-aryl-4-methylquinolines showed a reduced genotoxicity, as it was observed in the case of the 2-fluoro-4methylquinolines (Kato et al., 2000). Previous works have indicated that positions C-2 and C-3 in Q and 4-MeQ skeletons are critical sites associated with its genotoxicity (Nagao et al., 1977; Takahashi et al., 1988; LaVoie et al., 1991; Saeki et al., 1993, 1996; Hirano et al., 2002). The results suggest that aryl ring incorporation at position C-2 of 4MeQ inhibited the formation of the enamine epoxide in the pyridine moiety and reduced the genotoxic potential of this




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molecule when compared with the substitutions with chlorine (Nagao et al., 1977) and fluorine atoms (LaVoie et al., 1991; Kato et al., 2000) and methyl group (Takahashi et al., 1988; LaVoie et al., 1991; Saeki et al., 1996) substitutions. Altogether, these data suggest that 2-phenyl-, 2-(3,4-methylenedioxyphenyl)-, and 2-aryl-quinolines may have reduced genotoxic potential in comparison with the parent compounds and should be further evaluated for therapeutic potential in the treatment of parasitic and fungal disease. On the other hand, 4-NQO, 2-methyl-4-NQO, and 2-(4nitrophenyl)24-NQO resulted potent inducers of the SOS response in E. coli PQ37. The incorporation of methyl and 4-nitrophenyl groups at position C-2 of 4-NQO significantly reduced its SOSIP value, indicating the importance of position C-2 to genotoxic effect of these compounds. However, these substitutions did not completely eliminate the genotoxic effect of 4-NQO derivatives, which demonstrated SOS induction at higher concentrations when compared with 4NQO. In addition, we showed that 4-NQO derivatives such as 2-methyl-quinoline-1-oxide and 2-(4-nitrophenyl)-quinoline-1-oxide, with the absence of NO2 group at position C-4 of Q skeleton, did not induce the SOS response. These results support previous findings (Araki et al., 1970; Paul et al., 1971) and confirm the importance of NO2 group at position C-4 to genotoxicity of 4-NQO derivatives. When compared with Qs, whose genotoxicity involves formation of an enamine epoxide such as Q and 4-MeQ, little is known on the importance of group substitutions on mutagenicity of 4-NQO. Mifuchi et al. (1963) studied different 4NQO analogs and found that 2-methyl derivative of 4-NQO was as potent as the same 4-NQO to induce respirationdeficient mutant in Sacharomyces cerevisiae. Kawazoe et al. (1967, 1969) showed that chlorine, methyl, and methoxy incorporations at position C-3 of 4-NQO remove totally the carcinogenicity of this molecule in mice, whereas C-3 bromine atom and C-2 methyl incorporations do not. Paul et al. (1971) proposed a model of the interaction of 4-NQO metabolite with DNA that highlights the importance of the bulky substituents (i.e., butyl, ethyl, and methyl) in positions C-2 or C-3 of this molecule to its carcinogenicity. On the basis of the molecular orbital data and steric factors, they postulated that 4-HAQO, a metabolic reduction product of 4-NQO, is a proximal carcinogen that forms complex with deoxyguanosine in DNA molecule. In light of our findings, we supposed that the incorporation of methyl and 4-nitrophenyl groups at position C-2 of 4-NQO might interfere with the formation of this reactive metabolite, thus reducing the genotoxicity of 2methyl-4-NQO and 2-(4-nitrophenyl)24-NQO in E. coli PQ37 cells. This explains the reduction of SOSIP values observed with these compounds. The fact that the incorporation of the methyl and 4-nitrophenyl groups at the C-2 position of the 4-NQO reduced their genotoxicity independently on compound size confirms that other factors different to bulkiness are also important determinants for Qs genotoxicity, as previously indicated by He et al. (2005).

13

This work has shown that Q and 4-MeQ derivatives whose genotoxicity involve enamine epoxide formation are not genotoxic in the SOS chromotest. In contrast, 4-NQO derivatives induced SOS response in E. coli PQ37 cells, indicating DNA damage in these bacterial cells. C-2-substituted quinolines (i.e., phenyl, 3,4-methylenedioxyphenyl, pyridinyl, methyl, and 4-nitrophenyl quinolines) demonstrated reduced induction of the SOS response, suggesting that these derivatives have reduced genotoxic potential. The incorporation of halogen atoms or alkyl groups into the benzene ring of these compounds did not significantly modify its genotoxicity. Harmonized studies on genotoxicity of these compounds using a battery of in vivo assays that evaluate different levels of DNA damage expression will be required for practical use of these compounds in therapeutics. The importance of chemical nature substitutions at position C-2, as well as the reduction of NO2 group at position C-4 of the 4-NQO, to genotoxicity of this compound was demonstrated. The results suggest that introduction of a sub- AQ6 stituent atom or group at this position in question may be an useful tool to modify their genotoxic potency and to better understand the mechanism of genotoxicity. Thus, usefulness of SOS chromotest to study structure–genotoxicity relationships as well as to bioassay-guided syntheses of 4-NQO derivatives seems to be very high. The authors thank the anonymous reviewers who helped to improve the quality of the manuscript.

AUTHOR CONTRIBUTIONS V.V.K. and J.L.F.L. designed and prepared the framework project and applied for obtaining research funds. The published data are part of results obtained in the abovementioned project and these constitute the B.Sc. (Biology) thesis of L.T.D.D. and N.O.R. They developed the experimental work, collected and analyzed the data, and prepared draft figures and tables. L.T.D.D. also prepared the manuscript draft with important intellectual input from V.V.K. and J.L.F.L. The synthesis of 4-NQO derivatives as well as the preparation de novo of other functionalized quinolines AQ7 was conducted by C.E.P.G. under the supervision of V.V.K. All authors approved the final manuscript and had complete access to the study data.

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AQ1: Please confirm whether the short title is OK as given. AQ2: Please confirm that all author names are OK and are set with first name first, surname last. AQ3: Please check whether the author name “Jorge Luis Fuentes Lorenzo” is OK as given. AQ4: Please note that the sentence beginning as “In addition, change of nitrogen position…” is unclear as given; please modify as appropriate to make it understandable to the reader. AQ5: Please note that the sentence beginning as “In addition, the incorporation of halogen atoms…” is unclear as given; please modify as appropriate to make it understandable to the reader. AQ6: Please note that the sentence beginning as “The importance of chemical nature substitutions…” is unclear as given; please modify as appropriate to make it understandable to the reader. AQ7: Please clarify the use of “de novo” in the author contribution information. AQ8: Please check whether the grant information is OK as given.
