Further investigations into the genotoxicity of quinoxaline-di-N-oxides and their primary metabolites

Food and Chemical Toxicology 93 (2016) 145e157

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Further investigations into the genotoxicity of quinoxaline-di-Noxides and their primary metabolites Qianying Liu a, Jianwu Zhang b, Xun Luo b, Awais Ihsan d, Xianglian Liu b, Menghong Dai c, Guyue Cheng c, Haihong Hao c, Xu Wang c, **, Zonghui Yuan a, b, c, * a

National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei 430070, China MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei 430070, China c Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan, Hubei, China d Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 March 2016 Received in revised form 26 April 2016 Accepted 28 April 2016 Available online 8 May 2016

Quinoxaline-di-N-oxides (QdNOs) are potential antibacterial agents with a wide range of biological properties. Quinocetone (QCT), carbadox (CBX), olaquindox (OLA), mequindox (MEQ) and cyadox (CYA) are classical QdNOs. Though the genotoxicity of parent drugs has been evaluated, the genotoxicity of their primary N / O reduced metabolites remains unclear. In the present study, a battery of four different short-term tests, mouse lymphoma assay (MLA), Ames test, chromosomal aberration assay in vitro and bone marrow erythrocyte micronucleus assay in vivo was carried out to investigate the genotoxicity of the six primary N / O reduced metabolites. Additionally, the genotoxicity of five parent drugs was evaluated by the MLA. Strong genotoxicity of N1-MEQ, B-MEQ and B-CBX was found in three of the assays but not in the Ames assay, and the rank order was N1-MEQ>B-MEQ>B-CBX that is consistent with prototype QdNOs. Negative results for the five QdNOs were noted in the MLA. We present for the first time a comparison of the genotoxicity of primary N / O reduced metabolites, and evaluate the ability of five QdNOs to cause mutations in the MLA. The present study demonstrates that metabolites are involved in genetic toxicity mediated by QdNOs, and improve the prudent use of QdNOs for public health. © 2016 Published by Elsevier Ltd.

Keywords: Quinoxaline-di-N-Oxides Metabolites Genotoxicity Mequindox Quinocetone Olaquindox

1. Introduction Quinoxaline-di-N-oxides (QdNOs), acting as inhibitors of deoxyribonucleic acid (DNA) synthesis (Cheng et al., 2015), are a class of synthetic agents with broad biological activity including antibacterial, anticandida, antitubercular, anticancer and antiprotozoal properties (Carta et al., 2005; Cheng et al., 2015; Vicente et al., 2009; Wang et al., 2011a, 2015; Wu et al., 2007). Carbadox (CBX), olaquindox (OLA) are widely used at sub-therapeutic levels to promote growth and improve efficiency of feed conversion in

* Corresponding author. National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei 430070, China. ** Corresponding author. E-mail addresses: [email protected] (X. Wang), [email protected]. edu.cn (Z. Yuan). http://dx.doi.org/10.1016/j.fct.2016.04.029 0278-6915/© 2016 Published by Elsevier Ltd.

animal feeding. Due to carcinogenic, mutagenic and photoallergenic effects of these drugs and their metabolites (FAO/WHO. 1990; JECFA. 1991), the use of CBX and OLA has been prohibited in Europe since 1998 (EC. 1998). Mequindox (MEQ) and quinocetone (QCT), new members of QdNO family developed in China, have been approved for use in animal production for many years (Wang et al., 2015a). Cyadox (CYA) is testing and has been potential replacement for OLA in China (Wang et al., 2015b). A lot of evidence indicated that QdNOs have varying degrees of genetic toxicity. CBX (WHO, 1991a) and OLA (WHO, 1991b) exhibited an obvious genetic effect in bacterial (Ihsan et al., 2013a, b), and mammalian cells (Chen et al., 2008, 2009; Hao et al., 2006). Furthermore, they induced serious allergic and photoallergic dermatitis (He et al., 2006; Woodward, 2008). Compared with CBX, a higher mutagenic effect of MEQ to mammalian cells was found in in vitro and in vivo short-term tests (Ihsan et al., 2013a). QCT invoked DNA fragmentation and reactive oxygen species

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Abbreviations 6-TG ATCC BaP B-CBX B-MEQ B-OLA B-QCT CA CBX CCTCC CP CYX 1,8-DAN DMEM DMSO EMS FCS HAT

6-thioguanine American type culture collection benzo-[a]-pyrene bidesoxy-carbadox bidesoxy-mequindox bidesoxy-olaquindox bidesoxy-quinocetone chromosomal aberration carbadox China Center for Type Culture Collection cyclophosphamide cyadox 1,8-dihydroxy-anthraquinone Dulbecco's modified Eagle's medium dimethyl sulphoxide ethylmethanesulfonate fetal calf serum hypoxanthine aminopterin thymidine

generation in human lymphocytes (Yang et al., 2013). A significant increase in micronuclei formation in human hepatocyte L02 and HepG2 cells was also noted by QCT (Dai et al., 2015; Jin et al., 2009). Genotoxic effects were also found for QCT in various assays such as the Ames, HGPRT gene mutation (HGM) and unscheduled DNA synthesis (UDS) assays (Ihsan et al., 2013b). N / O group reduction is one of the main metabolic pathways of QdNOs (Liu and Sun, 2013). It was reported that N1-QCT and N1MEQ were partially reduced quinoxaline-N-oxide for QCT and MEQ, respectively, while B-QCT, B-MEQ, B-OLA and B-CBX are completely reduced derivatives of QCT, MEQ, OLA and CBX, respectively (Liu et al., 2008, 2010a, b, c, 2011a, b; Liu and Sun, 2013). In previous studies, genotoxicity of QdNOs was found to be closely related to N / O reduction. The N / O reduction metabolite of MEQ, 2isoethanol 4-desoxymequindox (M11), was detected in the testis of Wistar rats accompanying oxidative DNA damage emergence (Ihsan et al., 2011). While 3-methyl-quinoxaline-2-carboxylic acid (MQCA), as a residue of OLA, also causes DNA strand breaks in two human hepatocyte cell lines, L-02 and Chang liver cells (Zhang et al., 2012). It was revealed that the potential genotoxicity of the QdNOs metabolites was decreased by the N / O reduction (Chen et al., 2009; Wang et al., 2011c; Wang et al., 2015a; Yang et al., 2013). DNA damage was significantly decreased after QCT, CBX and OLA incubation with S9 mix in Vero cells (Chen et al., 2009). The same phenomenon was also found in human peripheral lymphocytes after QCT incubation with S9 mix (Yang et al., 2013). Higher cytotoxicity and genotoxicity were caused by QCT than by its metabolites 1,4-bisdesoxyquinocetone (DQCT) and MQCA in human hepatocytes and HepG2 cells (Zhang et al., 2012, 2014). QCT led to apoptosis in mitochondria-dependent and mitochondriaindependent pathways, while DQCT and MQCA were not able to produce such results (Zhang et al., 2015). However, it was also documented that desoxycarbadox had higher tumorigenic potential than its prototype CBX (JECFA, 2003; WHO, 1991a). For the mechanism of genetic toxicity, an unstable oxygen-sensitive radical intermediate that appeared during N / O reduction of QdNOs was regarded as having a crucial role in DNA damage (Cheng et al., 2015; Ganley et al., 2001; Junnotula et al., 2009; Poole et al., 2002). Recent research has demonstrated that structures of QdNO radicals of CYA were similar to those of tirapazamine (TPZ) radicals (Cheng et al.,

HGM M11 MEQ MLA MMC MMS MN N1-MEQ N1-QCT NADP OLA PE QCT QdNOs RS SD TFT Topo II TPZ UDS

HGPRT gene mutation 2-isoethanol 4-desoxymequindox mequindox mouse lymphoma assay mitomycin C methyl methanesulfonate micronucleus assay N1-desoxymequindox N1-desoxyquinocetone nicotinamide adenine dinucleotide phosphate olaquindox plating efficiency quinocetone quinoxaline-di-N-oxides relative survival standard deviation trifluorothymidine topoisomerase II tirapazamine unscheduled DNA synthesis

2015). The stability of the radical intermediate was responsible for the genotoxicity of QdNOs, and was more stable, longer lasting and more damaging to DNA (El-Khatib et al., 2010). Furthermore, the rate of deoxidation and genotoxicity was found to have a close relationship (Wang et al., 2015a). Therefore, it was hypothesized that metabolites might be involved in genotoxicity mediated by QdNOs. However, up to now, the genotoxicity of QdNOs has mainly focused on the parent drugs and subsequent oxidative stress. It still remains undiscovered whether their primary N / O reduction metabolites are involved in genetic toxicity. According to guidelines for genotoxicity testing of pharmaceuticals (VICH, 2000), a standard test battery contains: a. A test for gene mutations in bacteria, b. An in vitro test with cytogenetic evaluation of chromosomal damage using mammalian cells or an in vitro mouse lymphoma thymidine kinaseþ/ gene mutation assay and c. An in vivo test for chromosomal damage using mammalian hematopoietic cells. These assays were considered the best approach for genotoxic hazard identification and constructed a profile of risks/benefits of substances used in food contact materials including QdNOs (EFSA, 2011; European, 2001). The mouse lymphoma assay (MLA) using the thymidine kinase (Tk) gene of L5178Y Tkþ/ -3.7.2C mouse lymphoma cell lines was found to be the closest to the in vivo environment among the different in vitro mammalian and bacterial gene-mutation testings (Maisanaba et al., 2015). The positive results in the Ames test were found irrelevant to the intrinsic genotoxicity of the compounds under test (Zeiger, 2001; Zeiger and Hoffmann, 2012). Many defects in the definition of genotoxicity were found in the Ames test, especially for some compounds that have known interference with mammalian cell replication (e.g. topoisomerase inhibitors, nucleoside analog and DNA metabolic inhibitors) or have outstanding antibacterial activity (e.g. ciprofloxacin and fluoroquinolones). In this case, a validated in vitro test for gene mutation in mammalian cells (e.g. MLA) was required to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity (FDA, 2000; ICH, 2012; Kirkland et al., 2014). QdNOs are widely known as potent antibacterial agents (Cheng et al., 2015; Wang et al., 2015a) against a broad-spectrum of Gram-positive and -negative species. TPZ, a QdNO, is a well-characterized bioreductive anticancer agent that targets topoisomerase II (Hellauer et al.,

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2005; Peters and Brown, 2002; Zhang et al., 2010). Based on this, we therefore evaluated the genotoxicity of five classical QdNOs using the MLA to further investigate the mechanism of genotoxicity. To reveal the relationship between N / O reduction and genotoxicity, and investigate the genotoxicity of primary metabolites, a battery of four tests including the Ames test, chromosome aberration (CA) test, micronucleus (MN) assay in mice bone marrow and the MLA was conducted according to FDA and OECD guidelines (FDA, 2000a, 2000b, 2000c; OECD, 1997, 2014a, 2014b, 2015). N / O group reduction is one of the main metabolic pathways of QdNOs (Liu et al., 2008, 2009a, b, 2010a, b, c; Liu et al., 2011a; Liu et al., 2011b; Wu et al., 2012). Although types and quantities of QdNO metabolites vary in different tissues and species, the six deoxygenate metabolites bidesoxy-carbadox (B-CBX), bidesoxyolaquindox (B-OLA), bidesoxy-mequindox (B-MEQ), N1desoxymequindox (N1-MEQ), bidesoxy-quinocetone (B-QCT) and N1-desoxyquinocetone (N1-QCT) were major and common during N / O metabolic processes, and were chosen for the present study (Fig. 1). 2. Materials and methods 2.1. Chemical reagents CBX (purity 99.5%), OLA (purity 98%), MEQ (purity 98%), QCT (purity 98%), CYA (purity 99.5%), B-CBX (purity 99%), B-OLA (purity 99%), B-MEQ (purity 99%), N1-MEQ (purity 98%), B-QCT (purity 99%) and N1-QCT (purity 99%) were obtained from the Institute of Veterinary Pharmaceuticals, Huazhong Agricultural University (Wuhan, PR China). Mitomycin C (MMC), dimethyl sulfoxide (DMSO), benzo-[a]-pyrene (BaP), ethyl methanesulfonate (EMS), 9aminoacridine, methyl methanesulfonate (MMS), 1,8-dihydroxyanthraquinone (1,8-DAN), 2-aminofluorene, 6-thioguanine (6-TG), nicotinamide adenine dinucleotide phosphate (NADP) and trifluorothymidine (TFT) were procured from Sigma (St. Louis, MO, USA). Carboxymethylcellulose sodium (CMC), cyclophosphamide (CP), histidine and sodium azide were purchased from the Shanghai Chemical Reagent Company (Shanghai, China). RPMI1640 medium was provided by Hyclone (Shanghai, PR China). Fetal calf serum (FCS), fetal bovine serum (FBS) and newborn calf serum (NCS) were produced by Hangzhou Sijiqing Biological Materials Limited (Hangzhou, PR China). Hypoxanthine, aminopterin, thymidine, trypsin and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco (NY, USA). S9 metabolic activation mixture (S9-mix) was prepared from Aroclor 1254-induced rat liver homogenate in a similar manner to that described by Mortelmans and Zeiger (Mortelmans and Zeiger, 2000). S9-mix was used at a final concentration of 1% (1.85 mg protein/mL). All other chemicals were of analytical grade or complied with standards needed for cell culture experiments. 2.2. Cell culture Salmonella typhimurium TA97a, TA98, TA100 and TA102 were procured from the China Center for Type Culture Collection (CCTCC, Wuhan, PR China) while TA1535 was obtained from the American Type Culture Collection (ATCC, Manassas, USA). Mouse lymphoma cells (L5178Y Tkþ/) and Chinese hamster lung fibroblasts (V79) were acquired from the Shanghai Institute of Cell Biology, Chinese Academy of Science (Shanghai, PR China). L5178Y Tkþ/ cells were maintained in RPMI 1640 medium supplemented with 10% horse serum, 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM Lglutamine, 2.5 g/mL amphotericin B and 1 mM sodium pyruvate. To prevent overgrowth, L5178Y Tkþ/ cells were routinely diluted to

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2  105 cells/mL every day. The V79 cells were cultivated under standard conditions in DMEM containing 10% NCS and 1% penicillin-streptomycin solution. Cells were kept 37  C in a humidified atmosphere containing 5% CO2, and density was determined with an automated cell counter. 2.3. MLA test This study was performed according to FDA toxicological principles for the safety assessment of food ingredients and OECD guidelines for the testing of chemicals. IV.C.1.c Mouse Lymphoma Thymidine Kinase Gene Mutation Assay (FDA, 2000d) and Test Guideline 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (OECD, 2015) were used to detect mutagenicity in mammalian cells of five quinoxaline-1,4 dioxides and their primary N / O reduced metabolites. The L5178Y Tkþ/ mouse lymphoma cells were seeded at a density of 2  105 cells/mL for 24 h at 37  C in a 5% CO2 atmosphere before treatment. The highest concentration was determined by solubility in DMSO, of which the final quantity was always less than 0.1%, and a preliminary cytotoxicity test using L5178Y Tkþ/ cells for each compound. The positive controls comprised CP (3 mg/mL) and MMS (10 mg/mL) with and without S9, respectively, and DMSO was chosen as the negative control. Thereby, the same dose range (12.8, 6.4, 3.2 and 1.6 mg/mL) of concentration was selected. The results were analyzed using c2-test with SPSS 11.5 software and shown to be significant for p < 0.05. 2.3.1. Cytotoxicity The cell concentration was adjusted to 5  105 cells/mL and then cultured for 3e4 h. Cytotoxicity was determined by plating efficiency (PE) and relative survival (RS) after a treatment of 0 and 2 days, respectively, with QdNOs and primary metabolites in the presence and absence of S9. Cells were plated at a density of 8 cells/ mL diluted from 2  105 cells/mL in 96-well plates to evaluate the PE on the first day by counting the viable colonies after an incubation of 11e13 days at 37  C and 5% CO2. Cells at a density of 1  104 cells/mL were prepared after seeding for two days and then exposed to TFT (3 mg/mL) in 96-well plates for the mutation analysis. Plates were incubated for 12 days at 37  C and 5% CO2 to obtain the mutation frequency (MF) by calculating the viable colonies. Additionally, cells were diluted from 1  104 cells/mL to a density of 8 cells/mL in 96-well plates after an incubation of 12 days at 37  C and 5% CO2 to evaluate the PE2. The mutant colonies of each plate were counted when the cultured time was reached, and colony size was estimated according to the method described by Honma et al. (Honma et al., 1999a, 1999b). 2.3.2. Calculate MF, RS and PE PE and RS values were used to decide the acceptability of the toxicity at each dose level. MF, RS (RS0, RS2) and PE (PE0, PE2) were calculated as follows:

PEn ¼ lnðEW=TWÞ=N RS% ¼ PEðtestÞ=PEðcontrolÞ  100 where PE0 is the PE of the cells after treatment on the first day, EW is the number of wells without viable colonies in 96-well plates, TW is the total number of wells in 96-well plates and N is the number of cells in every well (usually 1.6).

MF% ¼ PEðmutantÞ=PE2  100

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Fig. 1. Structures of quinoxaline-di-N-oxides and primary N / O reduction metabolites.

PEðmutantÞ ¼ lnðEW=TWÞ=NðmutantÞ where PE2 is the PE of cells incubated for two days, EW is the number of wells without viable colonies in 96-well plates after exposure to TFT, TW is the total number of wells and N is the average number of cells (1.6). 2.3.3. Criterion of date acceptance The definition of a positive result should meet the following conditions: a. The MF and PE of the solvent and positive controls are within a specified range. With respect to the solvent control, the range from 60  106 to 180  106 was required for MF, and 60% to 140% for PE0 and 70% to 130% for PE2, respectively. The MF of the solvent control should not exceed three times than in the basic

culture medium. The MF gap between the solvent and positive controls is at least more than half of that between the solvent and basic culture medium; b. At least one dose of MF increases significantly and, once this appears, it should reproduce simultaneously; c. The MFs in every group need doseeresponse relationships. Caution should be taken to identify the result once a positive response is only found in the highest dose, especially a dose higher than that of more than 90% cytotoxicity (Clements, 2000; OECD, 2015; Omori et al., 2002).

2.4. Ames test The Ames test was performed by a plate incorporation procedure as outlined by OECD No.471, 46 Redbook 2000 IV.C.1.a. (OECD,

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1997), Redbook 2000: IV.C.1.a. (FDA, 2000b), and Chinese standard guidelines (Chinese, 2003), to evaluate the mutagenicity of B-QCT, N1-QCT, B-OLA, B-CBX, N1-MEQ and B-MEQ. The following fi-M Salmonella strains were used for the bacterial reverse mutation assay: TA97a, TA98, TA100, TA102 and TA1535. All strains were checked for maintenance of genetic markers prior to study. The proper concentration levels of these six metabolites were determined by a preliminary test and solubility on Salmonella strains. Therefore, three different amounts, (5.0e0.312, 1.25e0.078 and 2.5e0.156 mg/plate) were selected as the tested concentrations in the presence or absence of S9-mix for three metabolites (B-OLA, BMEQ and B-CBX), two metabolites (N1-MEQ and N1-QCT) and one metabolite (B-QCT), respectively. These compounds were dissolved in DMSO, and stock solution was prepared. With S9-mix, 2aminofluorene (10 mg/plate) was used as the positive control for all strains except TA102, where 1,8-DAN (50 mg/plate) was selected as the positive control. Without S9-mix, the positive controls were 9-aminoacridine (50 mg/plate) for TA97a, 2-nitrofluorene (50 mg/ plate) for TA98, MMC (50 mg/plate) for TA102 and NaN3 (50 mg/ plate) for TA100 and TA1535. Before plating onto minimal agar, the assay tubes were incubated at 37  C for 20 min. Three test plates per concentration were incubated at 37  C for 48 h. A positive response was defined only when compared to the solvent control in at least one strain. A greater than two fold increase in average plate count was found for at least one concentration level, and a dose response was found over the range of tested concentrations with or without S9. The difference between the control and treated cultures were tested by the one-way ANOVA and q-test with SPSS 11.0 software. Results were considered statistically significant when the P-value was <0.05.

2.5. Chromosomal aberration (CA) assay in vitro The potential of B-CBX, B-OLA, B-MEQ, N1-MEQ, B-QCT and N1QCT to induce structural and numerical chromosome aberrations was evaluated in Chinese hamster lung fibroblast cells (V79) according to OECD No.473 (OECD, 2014b), Redbook 2000 IV.C.1.b In Vitro Mammalian Chromosomal Aberration Test (FDA, 2000c) and methods described by Swierenga et al. (Swierenga et al., 1991). The cells were cultured at a concentration of 1  106 cells per 100 mm plate in a 5% CO2 atmosphere at 37  C for approximately 24 h before treatment. Based on the preliminary cytotoxicity test and solubility in DMSO, the same dose range (67, 33.5 and 16.75 mg/ mL) was determined for each metabolite. A vehicle control was set with 0.5% DMSO. The positive controls were CP (50 mg/mL) and MMC (0.2 mg/mL) used with and without S9, respectively. The negative control was DMSO and the final concentration was less than 0.1%. For testing without S9, cells were incubated with test metabolites, solvent and positive controls for 6 and 18 h, respectively. In the presence of S9, cells were incubated with test metabolites, solvent, and positive controls for 6 h. After incubation, cells were washed and placed in fresh culture medium containing 0.3 mg/mL colchicine and then exposed for an additional 4 h before harvesting. After treating with trypsin, cells were incubated in 0.075 M KCl for 20e25 min and fixed twice in 1:3 glacial acetic acid/methanol. To prepare chromosomes, cells were resuspended in 0.1e0.2 mL of cold fixative after centrifugation (270  g 5 min). Two slides per plate were prepared and stained with 10% Giemsa for 20 min. Metaphases having 21 ± 2 centromeres were scored per concentration and control. Fisher's exact test was used to analyze the statistical differences between the different controls and data was expressed as the mean ± SD.

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2.6. Bone marrow erythrocyte micronucleus (MN) assay in vivo The in vivo MN assay was conducted in Kunming mice in accordance with OECD Guideline No.474 (OECD, 2014a) and Redbook 2000 IV.C.1.d. Mammalian Erythrocyte Micronucleus Test (FDA, 2000b). Two hundred and forty SPF Kunming mice (7e9 weeks old) weighing 25e32 g were purchased from the Center of Laboratory Animals of Hubei Province (Wuhan, PR China) and randomly divided into 24 groups containing 10 mice each (five males and five females). The animals were maintained in a room conditioned at 22 ± 3  C, a relative humidity of 50% ± 20%, and a 12 h light/dark cycle. Use of animals in this study was in accordance with the NIH publication “The Development of Science Based Guidelines for Laboratory Animal Care” (NRC, 2004). According to related guideline (OECD, 2014a), the highest dose was determined by LD50 values and solubility in CMC. The dose levels were 1.25, 2.5 and 5 mg/kg b.w. of B-MEQ, B-OLA and B-CBX, and 0.08, 0.16 and 0.31 mg/kg b.w. of N1-MEQ, N1-QCT and B-QCT, respectively. The positive and negative controls were CP (i.p. 40 mg/kg b.w.) and 0.5% CMC solution, respectively. All these metabolites were administered through gavage twice (0.1 mL/10 g) in 30 h with a 24 h interval. Six hours after the second time treatment, the mice were sacrificed by cervical dislocation to obtain cell suspensions from femur bone marrow. Bone marrows were flushed with 1 mL NCS and then dropped onto microscope slides. After drying, cells were fixed with absolute methanol and stained with 5% Giemsa stain for 3e5 min before microscopic analysis. For each mouse, at least 1000 polychromatic erythrocytes (PCE) were counted to determine the micronucleus frequencies and record the micronucleus occurrence rate per one thousand PCE, and the proportion of PCE to normochromatic erythrocytes (NCE) was evaluated by counting a total of 1000 erythrocytes. The data of micronucleus occurrence rate and PCE/NCE ratio was compared using t-test with SPSS 17.0 software and expressed as the mean ± SD of the number of individuals examined. 3. Results 3.1. MLA results The MLA results obtained after treatment of L5178Y Tkþ/ cells with 11 compounds (including five QdNOs and their primary metabolites) are shown in Table 1. As indicated, the PE0, PE2 and MF of negative control were 73.2%, 86.4%, 79.6  106 with S9 and 66.3%, 72.5%, 58.2  106 without S9, respectively. Compared with the solvent controls, the MFs of the positive groups increased significantly: 286.2  106 for MMS and 395.4  106 for CP. According to the OECD guideline for results acceptance, MF, PE0 and PE2 should be in a range of 60  106e180  106, 60%e140% and 70%e130%, respectively (OECD, 2015). Thereby, the MLA test in the present study was established successfully and could be used to analyze the tested compounds. Five QdNOs, including CBX, OLA, MEQ, QCT and CYA, and three N / O reduced metabolites (B-OLA, B-QCT and N1-QCT) did not present a mutagenic response at all doses (12.8, 6.4, 3.2 and 1.6 mg/ mL). However, at the highest concentration (12.8 mg/mL), the other three metabolites, B-CBX, B-MEQ and N1-MEQ, showed statistically pronounced increases in MF, which significantly increased after incubation with S9-mix. The reproducibility of the results should be considered when the results of the experiment showed no dose response relationship, and only one dose of the mutation results. When the relative survival rate of cells is lower than 10%, the positive mutation results in this dose may be due to the secondary reaction of cytotoxic effect, which needs to be treated with caution (Clements, 2000). In the present study, the highest concentration

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Table 1 Results of the toxicity and mutagenicity of quinoxalines and key metabolites in mouse lymphoma cells (L5178Y Tkþ/) with/without S9-mix. Substance (mg/mL)

DMSO MMSa/CPb CYA

QCT OLA

CBX

MEQ

N1-QCT

B-QCT

B-OLA

B-CBX

N1-MEQ

B-MEQ

Concentration

0.2 10.0a/3.0b 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8 1.6 3.2 6.4 12.8

þS9

-S9 PE0 (%)

PE2 (%)

RS0 (%)

RS2 (%)

MF (106)

PE0 (%)

PE2 (%)

RS0 (%)

RS2 (%)

MF (106)

66.3 41.7 47.4 50.6 51.7 38.3 64.9 43.3 33.7 24.4 38.2 63.1 44.6 66.7 57.9 68.7 48.8 57.9 51.7 57.9 48.8 22.5 26.3 22.2 23.7 22.3 43.3 16.3 12.5 4.8 27.3 19.7 27.3 35.3 12.2 15.4 19.7 24.4 27.3 54.7 51.7 29.4 19.7 44.0 25.3 16.3

72.5 43.6 14.6 11.6 11.2 32.1 14.6 39.7 36.8 37.6 11.6 16.3 18.2 19.8 33.7 36.8 21.9 42.0 14.9 19.7 18.2 13.0 36.0 32.6 23.4 26.0 18.2 18.4 17.7 27.3 18.7 17.1 15.3 13.6 17.3 23.7 24.7 16.5 36.0 66.1 43.3 36.0 40.8 28.3 43.3 25.3

100.0 62.8 71.5 76.3 78.0 57.8 97.9 65.3 50.8 36.8 57.6 95.2 67.3 66.0 87.3 63.0 73.6 87.3 78.0 87.3 73.6 33.9 39.7 33.5 35.7 33.6 65.3 24.6 18.9 7.2 41.2 29.7 41.2 53.2 18.4 23.2 29.7 36.8 41.2 82.5 78.0 44.3 29.7 66.4 38.2 24.6

100.0 60.1 20.1 16.0 15.4 44.3 20.1 54.8 50.8 51.9 16.0 22.5 25.1 27.3 46.5 50.8 30.2 57.9 20.6 27.2 25.1 17.9 49.7 45.0 32.3 35.9 25.1 25.4 24.4 37.7 25.8 23.6 21.1 18.8 23.9 32.7 34.1 22.8 49.7 91.2 59.7 49.7 56.3 39.0 59.7 34.9

58.2 286.2* 81.2 61.0 90.0 88.9 99.2 82.0 63.5 71.9 75.9 65.0 64.5 89.8 65.6 66.8 88.9 89.0 65.4 85.0 68.0 87.4 83.3 82.5 64.0 73.0 83.5 63.4 70.5 97.4 67.7 61.3 72.0 96.7 86.2 93.4 62.5 132.6* 63.4 59.8 80.0 155.0* 90.0 72.3 96.2 164.5*

73.2 32.6 45.5 41.2 47.0 23.1 58.4 43.0 29.8 20.1 35.0 60.3 42.2 59.6 59.6 66.3 50.0 48.9 48.5 33.6 37.2 18.7 16.4 17.4 19.6 19.9 52.3 19.3 17.5 12.6 23.6 18.2 26.1 31.2 14.2 13.1 16.4 19.7 22.6 52.1 58.9 30.2 18.8 36.5 21.4 18.6

86.4 36.6 12.4 14.7 13.2 29.4 13.9 37.4 36.9 36.5 12.1 13.8 15.2 17.4 33.2 35.1 23.3 42.3 16.3 22.4 17.6 14.5 31.4 28.6 33.5 24.6 14.6 17.5 15.6 32.8 17.4 16.4 16.6 14.8 18.9 18.7 19.6 18.9 29.6 54.6 39.4 29.0 36.7 25.4 45.0 24.2

100.0 86.3 84.7 56.7 84.5 66.7 89.7 75.2 61.2 42.8 65.7 58.9 34.7 45.8 74.1 36.7 76.8 78.5 65.8 74.2 68.9 31.5 65.4 42.3 57.8 42.3 69.8 35.4 24.7 32.5 37.5 48.5 35.4 46.8 41.1 31.5 39.4 46.5 39.7 75.9 83.1 52.6 38.4 75.2 43.2 32.6

100.0 58.4 27.9 26.3 56.8 35.9 32.8 68.2 49.6 48.1 21.0 31.9 37.8 42.6 38.9 49.6 29.8 61.3 31.7 31.2 26.9 22.3 50.2 33.6 43.1 25.9 40.1 32.6 31.1 29.9 24.1 21.8 20.1 19.6 21.1 39.6 40.1 23.9 43.2 94.4 57.4 39.9 51.0 42.2 50.9 39.2

79.6 395.4* 75.6 58.2 82.3 84.2 85.6 83.4 53.3 61.3 68.4 67.4 66.7 81.3 58.4 56.9 76.5 75.0 54.9 83.6 63.0 76.9 86.4 96.3 67.3 72.8 89.6 68.3 76.5 101.2 64.3 66.0 79.8 110.3 78.6 105.6 63.9 196.3* 46.3 55.0 82.3 214.6* 94.4 84.6 85.3 203.4*

Note: apositive control is MMS (10.0 mg/mL) without S9; bpositive control is CP (3.0 mg/mL) with S9. B-CBX, bidesoxy-CBX; B-MEQ, bidesoxy-MEQ; B-OLA, bidesoxy-OLA; B-QCT, bidesoxy-QCT; CBX, carbadox; CP, cyclophosphamide; CYA, cyadox; DMSO, dimethylsulfoxide; MEQ, mequindox; MF, mutation frequency; MMS, methyl methanesulfonate; N1-MEQ, N1-desoxy mequindox; N1-QCT, N1-desoxy quinocetone; OLA, olaquindox; PE, plating efficiency; QCT, quinocetone; RS, relative survival. The means of differences were analyzed in c2-test. *Significantly different from negative control value (p < 0.05).

was used according to the cytotoxicity of the subjects in the pre experiments and the solubility in DMSO. At the highest dose of BCBX, N1-MEQ, and B-MEQ, the MFS increased significantly, which has a good repeatability. Furthermore, the cell relative survival (RS) rate at the highest dose of drugs was more than 20% (Table 1). Then, it was suggested that the results of the experiment were reliable. The PE of the negative controls on the first day (PE0) and second day after (PE2) cells were exposed to reagents were 66.3% and 72.5% (without S9) and 73.2% and 86.4% (with S9), respectively. The MF of the negative controls was 58.2  106 in the absence of S9, and 79.6  106 in the presence of S9. Significant increases in MF were observed in the positive controls of MMS and CP, which was more than half of the base value of the negative controls. Data in the control group were consistent with the acceptance criteria. These results demonstrated that B-CBX, B-MEQ and N1-MEQ were mutagenic for L5178Y Tkþ/ cells in the test conditions of this study.

3.2. Ames test Compared with the negative control with and without S9-mix, the number of revertants in the positive controls was more than two fold, while no increase greater than two fold was observed with S. typhimurium TA97a, TA98, TA100, TA102 and TA1535 strains following treatment with B-CBX, B-OLA, B-MEQ, N1-MEQ, B-QCT and N1-QCT at the tested concentrations. These results suggested that the six main N / O reduced metabolites of QdNOs did not produce a mutagenic effect in S. typhimurium at the tested dose levels (Tables 2e7). 3.3. Chromosomal aberration (CA) test in vitro Compared to the negative control, positive controls MMC (S9) and CP (þS9) induced significant increases in the frequency of

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Table 2 Results of the bacterial reverse mutation test of B-CBX. Drug

Concentration (mg/plate) Revertant colonies per plate (mean ± standard deviation) TA97a

TA98 þS9

-S9 B-CBX

Blank DMSO 9-Aminoacridine 2-Nitrofluorene NaN3 Mitomycin C 2-Aminofluorene 1,8-DAN

0.312 0.625 1.25 2.5 5

0.05 0.01 0.0015 0.0005 0.01 0.05

17 6 7 19 38 54 115 1487 e e e e e

± ± ± ± ± ± ± ±

TA100 þS9

-S9

2 91 3 75 3 90 3 84 11 80 0 86 6 81 205a e e e e 1509 e

± ± ± ± ± ± ±

21 18 8 3 4 23 2

33 30 31 30 32 31 27 e 562 e e ± 177a e e

± ± ± ± ± ± ±

6 4 8 3 18 6 6

35 40 37 36 38 36 35 e a ± 99 e e e 973 e

TA102 þS9

-S9 ± ± ± ± ± ± ±

3 0 1 5 1 1 1

187 207 186 183 171 189 222 e e 1785 e ± 64a e e

± ± ± ± ± ± ±

11 5 5 18 0 25 3

TA1535 þS9

-S9

178 152 159 162 74 173 157 e e ± 59a e e 1427 e

± ± ± ± ± ± ±

± ± ± ± ± ± ±

35 18 2 3 30 6 10

261 235 256 243 245 227 254 e e e 2156 ± 90a e e

8 5 40 1 16 16 49

295 277 272 302 306 285 278 e e e ± 194a e e 1831

þS9

-S9 ± ± ± ± ± ± ±

0 27 19 65 74 20 16

18 22 26 16 17 26 30 e e 329 e e ± 129a e

± ± ± ± ± ± ±

1 1 5 1 5 1 8

19 29 23 24 23 17 22 e e ± 49a e e 495 e

± ± ± ± ± ± ±

4 3 1 12 7 9 8

± 138a

Note: B-CBX, bidesoxy-carbadox; DMSO, dimethylsulfoxide; 1,8-DAN, 1,8-dihydroxy-anthraquinone. The means of differences were analyzed in one-way ANOVA and q-test. a Number of revertant colonies induced was double or more in the sample/number of spontaneous in negative control.

Table 3 Results of the bacterial reverse mutation test of B-OLA. Drug

Concentration mg/plate Revertant colonies per plate (mean ± standard deviation) TA97a

TA98 þS9

-S9 B-OLA

Blank DMSO 9-Aminoacridine 2-Nitrofluorene NaN3 Mitomycin C 2-Aminofluorene 1,8-DAN

0.312 0.625 1.25 2.5 5

0.05 0.01 0.0015 0.0005 0.01 0.05

66 83 80 77 46 74 102 1487 e e e e e

± ± ± ± ± ± ± ±

6 72 7 80 2 83 17 72 30 73 30 78 2 88 205a e e e e 1509 e

TA100 þS9

-S9 ± ± ± ± ± ± ±

5 12 2 11 1 17 8

± 177a

30 30 31 26 24 30 32 e 562 e e e e

± ± ± ± ± ± ±

6 1 5 4 2 1 1

± 99a

35 32 36 32 23 27 33 e e e e 973 e

TA102 þS9

-S9 ± ± ± ± ± ± ±

0 6 13 4 2 1 1

± 64a

133 130 131 136 125 103 135 e e 1785 e e e

± ± ± ± ± ± ±

3 5 33 7 17 1 10

± 59a

189 163 150 164 157 164 194 e e e e 1427 e

TA1535 þS9

-S9 ± ± ± ± ± ± ±

4 29 33 12 12 6 14

± 90a

239 241 228 244 224 237 267 e e e 2156 e e

± ± ± ± ± ± ±

28 28 18 19 15 1 16

± 194a

229 201 228 215 244 242 238 e e e e e 1831

þS9

-S9 ± ± ± ± ± ± ±

41 38 18 47 23 16 18

± 129a

14 10 14 15 12 16 13 e e 329 e e e

± ± ± ± ± ± ±

0 7 6 1 1 6 7

± 49a

21 21 22 22 22 21 20 e e e e 495 e

± ± ± ± ± ± ±

8 0 2 2 2 1 2

± 138a

Note: B-OLA, bidesoxy-olaquindox; DMSO, dimethylsulfoxide; 1,8-DAN, 1,8-dihydroxy-anthraquinone. The means of differences were analyzed in one-way ANOVA and q-test. a Number of revertant colonies induced was double or more in the sample/number of spontaneous in negative control.

Table 4 Results of the bacterial reverse mutation test of B-MEQ. Drug

Concentration mg/plate Revertant colonies per plate (mean ± standard deviation) TA97a

TA98 þS9

-S9 B-MEQ

Blank DMSO 9-aminoacridine 2-Nitrofluorene NaN3 Mitomycin C 2-Aminofluorene 1,8-DAN

0.312 0.625 1.25 2.5 5

0.05 0.01 0.0015 0.0005 0.01 0.05

125 109 89 104 127 106 121 1487

± ± ± ± ± ± ± ±

10 6 11 5 0 21 7 205a

83 81 87 85 75 84 92

TA100 þS9

-S9 ± ± ± ± ± ± ±

4 4 7 1 13 13 16

36 38 41 32 30 41 34

± ± ± ± ± ± ±

6 6 7 3 8 2 1

31 37 33 43 34 44 38

± ± ± ± ± ± ±

2 6 2 1 2 10 8

562 ± 99a

1509 ± 177a

TA102 þS9

-S9 109 121 109 120 31 123 111

± ± ± ± ± ± ±

7 15 3 8 10 1 21

161 180 175 161 169 180 169

± ± ± ± ± ± ±

14 12 7 7 5 15 3

1785 ± 59a 973 ± 64a

TA1535 þS9

-S9

1427 ± 90a

245 244 240 332 229 233 253

± ± ± ± ± ± ±

21 7 21 11 20 5 11

228 241 230 232 197 221 239.0

4 8 30 16 12 11 25

7 8 11 5 2 4 3

± ± ± ± ± ± ±

1 0 1 3 1 3 1

18 26 21 24 26 23 21

± ± ± ± ± ± ±

3 1 3 2 4 6 4

329 ± 49a

2156 ± 194a

Note: B-MEQ, bidesoxy-mequindox; DMSO, dimethylsulfoxide; 1,8-DAN, 1,8-dihydroxy-anthraquinone. The means of differences were analyzed in one-way ANOVA and q-test. a Number of revertant colonies induced was double or more in the sample/number of spontaneous in negative control.

þS9

-S9 ± ± ± ± ± ± ±

1831 ± 129a

495 ± 138a

152

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Table 5 Results of the bacterial reverse mutation test of N1-MEQ. Drug

Concentration mg/plate Revertant colonies per plate (mean ± standard deviation) TA97a

TA98 þS9

-S9 N1-MEQ

Blank DMSO 9-aminoacridine 2-Nitrofluorene NaN3 Mitomycin C 2-Aminofluorene 1,8-DAN

0.078 0.156 0.312 0.625 1.25

0.05 0.01 0.0015 0.0005 0.01 0.05

116 122 99 103 95 106 144 1487 e e e e e

± ± ± ± ± ± ± ±

28 87 13 80 13 92 25 87 26 90 45 83 13 90 205a e e e e 1509 e

TA100 þS9

-S9 ± ± ± ± ± ± ±

6 4 4 0 9 5 1

39 35 38 29 40 30 48 e 562 e e ± 177a e e

± ± ± ± ± ± ±

1 4 4 16 21 1 16

25 40 35 39 36 32 41 e a ± 99 e e e 973 e

TA102 þS9

-S9 ± ± ± ± ± ± ±

8 0 2 1 2 5 18

90 97 101 86 76 92 103 e e 1785 e ± 64a e e

± ± ± ± ± ± ±

9 14 6 6 14 6 16

156 163 133 151 148 157 167 e e ± 59a e e 1427 e

TA1535 þS9

-S9 ± ± ± ± ± ± ±

19 24 11 29 6 4 1

244 256 252 244 249 239 255 e e e 2156 ± 90a e e

± ± ± ± ± ± ±

18 3 0 12 1 0 6

259 266 282 264 260 272 264 e e e ± 194a e e 1831

þS9

-S9 ± ± ± ± ± ± ±

0 8 11 1 13 2 13

15 13 7 12 8 17 18 e e 329 e e ± 129a e

± ± ± ± ± ± ±

1 5 1 4 1 3 2

15 18 17 21 19 20 17.0 e e a ± 49 e e 495 e

± ± ± ± ± ± ±

6 6 6 4 9 0 1.41

± 138a

Note: N1-MEQ, N1-desoxy mequindox; DMSO, dimethylsulfoxide; 1,8-DAN, 1,8-dihydroxy-anthraquinone. The means of differences were analyzed in one-way ANOVA and q-test. a Number of revertant colonies induced was double or more in the sample/number of spontaneous in negative control.

Table 6 Results of the bacterial reverse mutation test of B-QCT. Drug

Concentration mg/plate Revertant colonies per plate (mean ± standard deviation) TA97a

TA98 þS9

-S9 B-QCT

Blank DMSO 9-aminoacridine 2-Nitrofluorene NaN3 Mitomycin C 2-Aminofluorene 1,8-DAN

0.156 0.312 0.625 1.25 2.5

0.05 0.01 0.0015 0.0005 0.01 0.05

96 113 121 113 99 110 125 1487

± ± ± ± ± ± ± ±

13 3 12 23 23 6 0 205a

109 98 95 89 91 88 102

TA100 þS9

-S9 ± ± ± ± ± ± ±

4 20 24 1 16 16 11

29 30 23 28 30 28 23

± ± ± ± ± ± ±

1 4 1 2 3 1 1

32 31 31 29 24 35 35

þS9

-S9 ± ± ± ± ± ± ±

3 1 4 1 8 4 3

562 ± 99a

1509 ± 177a

TA102

229 230 244 176 185 221 248

± ± ± ± ± ± ±

15 82 48 29 24 25 23

131 143 158 144 143 127 163

þS9

-S9 ± ± ± ± ± ± ±

38 8 17 3 5 12 3

1785 ± 59a 973 ± 64a

TA1535

286 308 271 278 291 305 319

± ± ± ± ± ± ±

15 10 22 15 22 10 22

261 239 213 214 147 241 311

37 9 4 36 35 23 18

24 21 22 22 20 21 25

± ± ± ± ± ± ±

6 4 8 1 1 1 1

28 23 20 23 24 25 23

± ± ± ± ± ± ±

1 1 4 4 6 6 5

329 ± 49a

2156 ± 194a 1427 ± 90a

þS9

-S9 ± ± ± ± ± ± ±

495 ± 138a

1831 ± 129a

Note: B-QCT, bidesoxy-quinocetone; DMSO, dimethylsulfoxide; 1,8-DAN, 1,8-dihydroxy-anthraquinone. The means of differences were analyzed in one-way ANOVA and q-test. a Number of revertant colonies induced was double or more in the sample/number of spontaneous in negative control.

Table 7 Results of the bacterial reverse mutation test of N1-QCT. Drug

Concentration mg/plate Revertant colonies per plate (mean ± standard deviation) TA97a

TA98 þS9

-S9 N1-QCT

Blank DMSO 9-aminoacridine 2-Nitrofluorene NaN3 Mitomycin C 2-Aminofluorene 1,8-DAN

0.078 0.156 0.312 0.625 1.25

0.05 0.01 0.0015 0.0005 0.01 0.05

64 70 75 77 86 54 99 1487

± ± ± ± ± ± ± ±

4 5 6 7 18 0 1 205a

64 78 70 56 70 85 144

TA100 þS9

-S9 ± ± ± ± ± ± ±

19 11 1 2 2 5 13

36 32 34 28 35 30 34

± ± ± ± ± ± ±

2 4 2 2 4 4 1

32 42 36 35 30 35 31

þS9

-S9 ± ± ± ± ± ± ±

6 4 13 6 0 5 2

562 ± 99a

1509 ± 177a

TA102

143 148 138 134 136 118 155

± ± ± ± ± ± ±

3 1 6 8 6 12 7

199 183 165 170 149 182 195

þS9

-S9 ± ± ± ± ± ± ±

8 4 9 6 7 11 12

1785 ± 59a 973 ± 64a

TA1535

222 176 185 203 183 170 200

± ± ± ± ± ± ±

11 5 13 8 1 29 13

303 329 303 305 303 352 328

33 62 26 4 18 30 21

Note: N1-QCT, N1-desoxy quinocetone; DMSO, dimethylsulfoxide; 1,8-DAN, 1,8-dihydroxy-anthraquinone. The means of differences were analyzed in one-way ANOVA and q-test. a Number of revertant colonies induced was double or more in the sample/number of spontaneous in negative control.

10 14 13 13 11 10 12

± ± ± ± ± ± ±

2 0 5 7 0 3 1

29 31 33 29 30 26 21

± ± ± ± ± ± ±

8 1 1 5 3 4 3

329 ± 49a

2156 ± 194a 1427 ± 90a

þS9

-S9 ± ± ± ± ± ± ±

1831 ± 129a

495 ± 138a

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153

4. Discussion

chromosome aberrations (Table 8). B-QCT, N1-QCT and B-OLA did not produce any significant increase in the ratio of chromosome aberrations in cultured V79 cells after 6 and 18 h treatments at any of the concentrations tested, with or without S9-mix. However, a significant increase in the incidence of chromosome aberrations was noted at a dose of 67.00 mg/mL in the absence of S9-mix for BCBX after 18 h of treatment, and for B-MEQ after 6 and 18 h treatments. N1-MEQ induced statistically significant increases in the ratio of chromosome aberrations after 6 and 18 h treatments at dose levels of 67.00 and 33.50 mg/mL, and the aberration frequency was higher in the presence of S9-mix. These results indicated that B-QCT, N1-QCT and B-OLA were not mutagenic in V79 cells, while B-CBX, N1-MEQ and B-MEQ could produce chromosome aberration in V79 cells. The different types of chromosome aberrations induced by these metabolites included breaks, exchanges, fragments and other multiple damages.

Regarding the potential genotoxicity of QdNOs, many studies have been conducted with different results, based on a range of concentrations and testing systems. As we mentioned above, CBX, OLA and MEQ had strong mutagenicity in in vivo and in vitro systems (Ihsan et al., 2013a, 2013b) while genotoxicity of QCT and CYA was found only in in vitro genotoxicity assays (Ihsan et al., 2013a, 2013b). Previously, CBX, OLA, MEQ, QCT and CYA produced Hisþ mutants to varying degrees in S. typhimurium strains (Ihsan et al., 2013a, 2013b). In the MLA, we used L5178Y Tkþ/ -3.7.2 cells to investigate the genotoxicity of QdNOs. Irrespective of S9-mix, none of the tested prototype QdNOs gave a positive response at the concentration levels considered, which was inconsistent with previous results for the Ames test. There is a growing body of evidence that the hormetic effects of compound can result in the positive response in Ames test that are not related to the intrinsic mutagenicity of the compound (Zeiger and Hoffmann, 2012). For example, compounds identified as non-genotoxic such as rifampicin (IARC, 1980), AMP397 (Suter et al., 2002), Kojic acid (Nohynek et al., 2004), HI-6-dimethanesulfonate (Nakab et al., 2014) and Triallate (Healy et al., 2003) were tested as positive in the Ames test. Genotoxic compounds such as 4-aminosalicylic acid and pyrazinamide were recorded as negative in the Ames test (Ishidate et al., 1988; Zeiger et al., 1987). The MLA had higher sensitivity than any other in vitro genotoxicity assay (Maisanaba et al., 2015). It can not only detect mutations in the Tk locus, but also identify multiple types of genetic poisons including a wide range of chromosome aberration and recombination agents, spindle poisons, and non-ploidy induction agents (Kirkland and Clements, 1998). A high degree of consistency between the MLA and carcinogenic activity was reported in recent reviews (Brambilla and Martelli, 2009; Brambilla et al., 2010; Kirkland et al., 2006), demonstrating that the genotoxicity in mammals is a multi-step phenomenon, and of which, occurrence of a pre-mutagenic lesion may be only a single first step. The

3.4. MN assay in vivo The results for micronucleated polychromatic erythrocytes (MN-PCE) in male and female mice after treatment with primary metabolites of QdNOs are presented in Tables 9 and 10, respectively. There was no significant increase in the proportion of MN-PCE observed in B-QCT, N1-QCT and B-OLA treatment groups compared to the control group. In male mice, a significant increase in the ratio of MN-PCE was found when B-CBX and B-MEQ were administered by gavage at 5 mg/kg b.w., and N1-MEQ at 0.31 and 0.16 mg/kg b.w. In female mice, B-CBX (5 and 2.5 mg/kg b.w) and N1-MEQ (0.31, 0.16 and 0.08 mg/kg b.w) induced a statistically significant increase in the proportion of MN-PCE. A statistical difference in the ratio of MN-PCE to PCE was observed in the positive control group. Additionally, the ratios of PCE/NCE in each group ranged from 0.6 to 1.2 and no decrease in PCE/NCE was noted at any doses of these metabolites. The results indicated that B-CBX, BMEQ and N1-MEQ were mutagenic for mice in vivo.

Table 8 Chromosomal aberrations induced by metabolites of quinoxalins metabolites in Chinese hamster V79 cells with/without S9. Substance

Concentration (mg/mL)

Cells scored -S9

B-CBX

B-OLA

B-MEQ

N1-MEQ

B-QCT

N1-QCT

DMSO MMC CP

16.75 33.50 67.00 16.75 33.50 67.00 16.75 33.50 67.00 16.75 33.50 67.00 16.75 33.50 67.00 16.75 33.50 67.00 e 0.2 50.0

200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200

Aberrant metaphases þS9

200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200

-S9

Aberrations (%) þS9

-S9

þS9

6h

18 h

6h

6h

18 h

6h

9 12 16 4 7 11 11 13 24 13 26 33 4 4 5 4 6 7 6 86 e

12 16 26 3 5 8 7 12 33 16 30 36 3 5 7 3 8 9 8 80 e

7 9 13 5 8 14 11 14 17 17 28 39 3 5 7 6 7 9 6 e 95

4.5 6.0 8.0 2.0 3.5 5.5 5.5 6.5 12.0* 6.5 13.0* 16.5* 2.0 2.0 2.5 2.0 3.0 3.5 3 43*

6.0 8.0 13.0* 1.5 2.5 4.0 3.5 6.0 16.5* 8.0 15.0* 18.0* 1.5 2.5 3.5 1.5 4.0 4.5 4 40* e

3.5 4.5 6.5 2.5 4.0 7.0 5.5 7.0 8.5 8.5 14.0* 19.5* 1.5 2.5 3.5 3.0 3.5 4.5 3 e 47.5*

Note: B-CBX, bidesoxy-carbadox; B-MEQ, bidesoxy-mequindox; B-OLA, bidesoxy-olaquindox; B-QCT, bidesoxy-quinocetone; CP, cyclophosphamide; DMSO, dimethylsulfoxide; MMC, mitomycin C; N1-MEQ, N1-desoxymequindox; N1-QCT, N1-desoxyquinocetone. The means of differences were tested with Fisher's exact test. *Significantly different from negative control value (p < 0.01).

154

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Table 9 Micronucleated polychromatic erythrocytes (MN-PCEs) in male mice bone marrow after treatment of quinoxalins metabolites. Substance

Concentration (mg/kg b.w.)

PCE

MN-PCE

MN-PCE/PCE (‰)

CMC B-OLA

e 1.25 2.5 5 1.25 2.5 5 1.25 2.5 5 0.08 0.16 0.31 0.08 0.16 0.31 0.08 0.16 0.31 40

5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000

9 13 15 16 13 21 36 12 17 37 20 28 41 12 10 11 13 16 19 177

1.8 2.5 3.2 3.3 2.6 4.2 8.2 2.4 3.4 7.4 4.0 5.6 8.2 2.4 2.0 2.2 2.6 3.2 3.8 35.4

B-CBX

B-MEQ

N1-MEQ

B-QCT

N1-QCT

CP

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.3 0.7 2.3 1.5 2.3 1.9** 1.1 0.5 2.2** 0.7 2.7* 2.1** 1.1 0.7 0.8 1.1 1.3 1.9 2.7**

PCE/NCE 0.94 1.01 1.00 1.04 1.01 1.05 0.91 1.05 0.97 1.06 1.01 1.03 0.92 1.06 1.02 0.96 0.84 1.04 0.92 0.99

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.13 0.03 0.09 0.12 0.09 0.14 0.18 0.11 0.21 0.12 0.18 0.14 0.14 0.12 0.11 0.11 0.17 0.11 0.16 0.18

Note: B-CBX, bidesoxy-carbadox; B-MEQ, bidesoxy-mequindox; B-OLA, bidesoxy-olaquindox; B-QCT, bidesoxy-quinocetone; CMC, carboxymethyl-cellulose; CP, cyclophosphamide; N1-MEQ, N1-desoxymequindox; N1-QCT, N1-desoxyquinocetone; NCE, normochromatic erythrocytes; PCE, polychromatic erythrocytes. The means of differences were analyzed in t-test. *Significantly different from negative control value (p < 0.05). **Significantly different from negative control value (p < 0.01).

Table 10 Micronucleated polychromatic erythrocytes (MN-PCEs) in female mice bone marrow after treatment of quinoxalins metabolites. Substance

Concentration (mg/kg b.w.)

PCE

MN-PCE

MN-PCE/PCE (‰)

CMC B-OLA

e 1.25 2.5 5 1.25 2.5 5 1.25 2.5 5 0.08 0.16 0.31 0.08 0.16 0.31 0.08 0.16 0.31 40

5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000

10 12 14 16 18 21 33 13 11 15 27 25 39 11 10 12 11 15 24 157

2.0 2.5 2.8 3.3 3.6 4.2 6.6 2.6 2.2 3.0 5.4 7.0 7.8 2.2 2.0 2.4 2.2 3.0 4.8 31.4

B-CBX

B-MEQ

N1-MEQ

B-QCT

N1-QCT

CP

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.4 0.7 0.9 1.1 1.1 1.8* 1.1** 0.9 0.8 0.7 1.1* 3.1** 1.9** 0.8 0.7 1.1 1.1 1.2 3.6 5.6**

PCE/NCE 0.98 1.01 1.03 1.01 0.94 1.04 1.05 1.03 1.03 1.06 1.03 0.98 1.03 1.02 1.08 1.01 1.08 1.01 1.03 1.01

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.03 0.02 0.01 0.10 0.08 0.05 0.03 0.08 0.04 0.07 0.08 0.16 0.13 0.10 0.12 0.11 0.16 0.14 0.19

Note: B-CBX, bidesoxy-carbadox; B-OLA, bidesoxy-olaquindox; B-MEQ, bidesoxy-mequindox; N1-MEQ, N1-desoxymequindox; B-QCT, bidesoxy-quinocetone; N1-QCT, N1desoxyquinocetone; CMC, carboxymethyl-cellulose; CP, cyclophosphamide; NCE, normochromatic erythrocytes; PCE, polychromatic erythrocytes. The means of differences were analyzed in t-test. *Significantly different from negative control value (p < 0.05). **Significantly different from negative control value (p < 0.01).

appearance of different results in bacterial and mammalian cells could result from distinctions between prokaryotic and eukaryotic cells, such as xenobiotic metabolism, effective doses, metabolic activation pathways and metabolizing enzymes. The MLA was one of the required genotoxicity tests prior to commercialization (FDA, 2000; ICH, 2008) and, usually, negative or weak positive results in the Ames test should be confirmed again in the MLA. False positive responses occurred when the tested compound had powerful toxicity to S. typhimurium strains in the Ames test. Fluoroquinolones, developed as strong antibacterial agents, gave negative results in various in vitro and in vivo genotoxicity assays, while they had “mutagenicity” to S. typhimurium TA102 (Hu et al.,

2001; Wu et al., 1991; Zhou and Lin, 1996). It was speculated that positive results in the Ames test for QdNOs derived from strong antibacterial activity that may be toxic to bacterial test strains, and subsequently led to misleading results that were not related to the intrinsic genotoxicity of the compound. The negative results for CYA in the MLA confirmed earlier findings that the previous weak mutagenicity to bacterial strains was not reflected in other in vivo and in vitro genotoxicity (Burke et al., 1996; Cihak and Srb, 1983; Cihak and Vontorkova, 1985; Ihsan et al., 2013b). Additionally, the in vitro genotoxicity of QCT was not detected in vivo (Ihsan et al., 2013a, 2013b), which was consistent with results in the MLA. The metabolism of QdNOs was characterized by the rapid

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reduction of the N-oxide groups, the cleavage and hydroformylation of QdNOs side-chain (Liu et al., 2009a, 2009b, 2010b, 2010c, 2011b; Liu and Sun, 2013). After incubation with S9 enzymes, N1-MEQ was deoxided to generate B-MEQ. The mutagenicity of BMEQ was higher than N1-MEQ. After hydroxyl reaction of the side chain, 2-isoethanol 4-desoxymequindox (M11) and 2-isoethanol 1, 4-desoxymequindox (M14) were produced from N1-MEQ and BMEQ, respectively. 2-Isoethanol 4-desoxymequindox (M11) was detected in the testis accompanying oxidative DNA damage emergence (Ihsan et al., 2011), suggesting the potential genotoxicity of M11 and M14. Hydrazine, a metabolite derived from the cleavage of B-CBX side chain, had been found strong genetic toxicity on bacterial and mammalian cell (Ames, 1971; Von wright and Tikkanen, 1980; Rogers and Back, 1981). Therefore, it was suspected that the potential mutagenic products during the reaction with S9 enzymes were hydrazine from B-CBX, M14 from B-MEQ, B-MEQ and M11 from N1-MEQ, respectively. However, further investigation should be carried out. There is a growing body of evidence suggesting that genotoxicity may be derived from metabolites, especially for drugs that are theoretically nitrosatable in the presence of amines, the interaction formed of genotoxic-carcinogenic N-nitroso compounds (Brambilla and Martelli, 2007). The N / O groups of QdNOs played an important role in genotoxicity (Chen et al., 2009; Wang et al., 2011b, 2016, 2015; Yang et al., 2013). In the present study, N1MEQ gave positive results in the MLA and CA tests irrespective of S9-mix. The MF and incidence of chromosomal aberrations were significantly increased after incubation with S9-mix. This finding demonstrated that intermediate metabolites of N1-MEQ exerted higher genotoxicity than N1-MEQ itself. B-MEQ, as one of the deoxidized metabolites of N1-MEQ, was also found to be positive in the MLA and CA tests, and its mutagenicity was higher than N1MEQ in the MLA in the absence of S9-mix. We supposed that BMEQ was the main metabolite of N1-MEQ in the presence of S9-mix and involved in the genotoxicity caused by N1-MEQ. In a preliminary experiment of the MN test, when N1-MEQ was administered to mice by gavage at 5 and 2.5 mg/kg b.w., mice died within 24 h (data not shown), indicating strong toxicity of N1-MEQ. A significant increase in the ratio of PCE to NCE was observed for N1MEQ in a dose-dependent relationship. B-MEQ produced a significantly increased proportion of MN-PCE at the highest dose and only in male mice. In a sub-chronic study of MEQ in Wistar rats, degeneration of seminiferous tubules in the testis was apparent in two rats fed a 275 mg MEQ/kg diet (Ihsan et al., 2010). The metabolite 2-isoethanol 4-desoxymequindox (M11) was found in the testis by LC/MS-IT-TOF analysis along with a significant increase in 8-OhdG, suggesting that M11 might be genotoxic to rats (Ihsan et al., 2011). In the present study, a positive result for B-MEQ in the male MN test and MLA was observed, indicating that B-MEQ might also have genotoxicity. Previous research demonstrated that B-CBX, the metabolite of CBX, was mutagenic and had apparently greater tumorigenic potential than CBX (JECFA, 2003). In the present genotoxicity assays, B-CBX was found to be positive in the MLA, CA and MN tests and thus confirmed earlier reports and extended knowledge about the genotoxicity of QdNOs. Interestingly, the MFs of B-MEQ and B-CBX significantly increased after incubation with S9-mix in the MLA. This result indicated that other desoxy metabolites of CBX and MEQ might have potential genotoxicity, and the toxicity of desoxy metabolites might be higher than the parent compounds of B-MEQ and B-CBX. However, the positive findings given by B-CBX and BMEQ in the MN test suggested that there might be another factor mediating their toxicities related to their metabolism in vivo, which still remains unclear. The genotoxicity of QdNOs is related to N / O group reduction (Liu et al., 2012; Wang et al., 2015a; Zhang et al.,

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2014). Recent research implied that the deoxidation rates of QdNOs, especially bidesoxy rates, had a higher correlation with their genotoxicity (Wang et al., 2015a) and these findings indicated the possibility of bidesoxy metabolites as genotoxic items. The hypoxia-selective DNA cleavage and unstable oxygen-sensitive radical intermediates of QdNOs play important roles in the genotoxicity of QdNOs (El-Khatib et al., 2010; Ganley et al., 2001; Junnotula et al., 2009; Khaled Ghattass and Gali-Muhtasib, 2014). The reason for the higher genotoxicity of dichloroquinoxaline (DCQ) than TPZ was the radical stability of DCQ. It was found that the DCQ radical was more stable than TPZ, which induced longer lasting and more serious damage to DNA (El-Khatib et al., 2010; Junnotula et al., 2009). In the present study, our results suggest that not only the presence of N / O groups but also the side chains of QdNOs play a critical role in their mutagenicity. Further study should be focused on the different side chains in bidesoxy QdNOs (B-MEQ and B-CBX) to interpret and evaluate the genotoxic characteristics of QdNOs. Based on the current studies, three QdNO metabolites have potential genotoxicity in the MLA, CA and MN tests. Thus, genotoxicity mediated by QdNOs was closely related to N / O reduced metabolites. The side chains of QdNOs may affect and be involved in the genotoxicity of QdNOs because of the bidesoxy metabolites BMEQ and B-CBX having genotoxicity in the present study. In addition, the genotoxicity of these three metabolites was confirmed in the MLA, MN and CA tests, while it was negative in the Ames test. Our work for first time provides a systematic genotoxicity of QdNO metabolites according to current guidelines, and provides a scientific justification for antimicrobial agents to select a standard battery of genotoxicity tests. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by Natural Science Foundation of China (grant no. 31272614 and 31502115), Grants from 2015 National Risk Assessment of Quality and Safety of Livestock and Poultry Products (GJFP2015008) and Research on the detection standard of veterinary drug residue (2662015PY021). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2016.04.029. References Ames, B., 1971. In: Hollaender, A. (Ed.), Chemical Mutagens, vol. 1. Plenum Press, New York, p. 267. Brambilla, G., Martelli, A., 2007. Genotoxic and carcinogenic risk to humans of drugnitrite interaction products. Mutat. Res. 635, 17e52. Brambilla, G., Martelli, A., 2009. Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals. Mutat. Res. 681, 209e229. Brambilla, G., Mattioli, F., Robbiano, L., Martelli, A., 2010. Genotoxicity and carcinogenicity testing of pharmaceuticals: correlations between induction of DNA lesions and carcinogenic activity. Mutat. Res. 705, 20e39. Burke, D.A., Wedd, D.J., Burlinson, B., 1996. Use of the miniscreen assay to screen novel compounds for bacterial mutagenicity in the pharmaceutical industry. Mutagenesis 11, 201e205. Carta, A., Corona, P., Loriga, M., 2005. Quinoxaline 1,4-dioxide: a versatile scaffold endowed with manifold activities. Curr. Med. Chem. 12, 2259e2272. Chen, Q., Chen, Y., Qi, Y., Hao, L., Tang, S., Xiao, X., 2008. Characterization of carbadox-induced mutagenesis using a shuttle vector pSP189 in mammalian cells. Mutat. Res. 638, 11e16. Chen, Q., Tang, S., Jin, X., Zou, J., Chen, K., Zhang, T., Xiao, X., 2009. Investigation of the genotoxicity of quinocetone, carbadox and olaquindox in vitro using Vero

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