http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(5): 332–341 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.898355

RESEARCH ARTICLE

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3-Aminobenzamide – a PARP inhibitor enhances the sensitivity of peripheral blood micronucleus and comet assays in mice Kamran Shekh1*, Sabbir Khan1, Gopabandhu Jena1, Bhavin R. Kansara2, and Sapana Kushwaha3 1

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India, 2Indian Institute of Science Education & Research (IISER) Mohali, Mohali, Punjab, India, and 3Division of biochemistry, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow, Uttar Pradesh, India Abstract

Keywords

Context: DNA repair is an essential outcome of DNA damage, which may compromise the end point of various in vitro and in vivo test systems of the genotoxicity evaluation. poly(ADPribose) polymerase (PARP) enzymes have an essential role in DNA repair. Here, we investigated the effect of 3-AB, a PARP inhibitor on the sensitivity of comet and PBMN assays. Objective: This study aimed to enhance the sensitivity of the comet and peripheral blood micronucleus (PBMN) assays using 3-aminobenzamide (3-AB), a well-characterized PARP inhibitor. Materials and methods: Cyclophosphamide (CP, 50 mg/kg), 5-flourouracil (5-FU, 25 mg/kg), zidovudine (AZT, 400 mg/kg) and furosemide (FUR, 60 mg/kg) were selected as genotoxins. 3-AB was given every 8 h with the first dose given 2 h before the genotoxin treatment. For the PBMN assay, small amount of blood was taken from the tail tip of each animal and smears were prepared. The comet assay was performed in PBL, bone marrow and liver. Results: In the comet as well as PBMN assay, 3-AB pre-treatment enhanced the extent of DNA damage in all the combination groups (3-AB + CP, 3-AB + 5-FU and 3-AB + AZT) compared to CP, 5-FU and AZT per se. 3-AB also enhanced the DNA damage caused by FUR in the bone marrow and liver. Discussion: This study results clearly demonstrate that the pretreatment with 3-AB (30 mg/kg) significantly enhances the sensitivity of the PBMN and comet assays. This model may be useful for the detection of marginally active DNA damaging agents.

DNA damage, genotoxicity, poly(ADP-ribose) polymerase, weak genotoxins

Introduction Various in vitro and in vivo test systems are used for the genotoxicity evaluation of chemicals (Purves et al., 1995). Sensitivity of a genotoxicity test can be defined as its ability to detect known genotoxic chemicals or its ability to avoid false negative results. The DNA repair events following DNA damage may reduce the sensitivity of assays used for genotoxicity determination (Kawaguchi et al., 2010a; Qiu et al., 2011; Zhu et al., 2005). It has been emphasized that the combination of comet and micronucleus (MN) assays in selected tissue can be used as an effective approach for successful detection of genotoxins (Pfuhler et al., 2007; Vasquez, 2010). In the recent years, several developments have been made in the automated scoring of micronuclei; however, *The first two authors contributed equally to this study. Address for correspondence: Dr. Gopabandhu Jena, Assistant Professor, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Mohali – 160062, Punjab, India. Tel: +91-172-2214683 (Extn. 2152). Fax: +91-172-2214692. E-mail: [email protected], [email protected]

History Received 14 November 2013 Revised 22 February 2014 Accepted 23 February 2014 Published online 9 May 2014

very few attempts were made to improve the sensitivity of manual micronuclei detection methods (Dertinger et al., 1996; Torous et al., 2005). In the pursuit of finding more sensitive methods for genotoxicity detection, recently, a novel high throughput yeast-based reporter assay for detection of genotoxins has been developed that utilizes RAD51, an inducible DNA damage sensor, to detect the genotoxicity (Liu et al., 2008). Genotoxicity evaluation is an important part of the preclinical drug development and detection of marginal genotoxins has always been a challenging task. Therefore, increased sensitivity of the genotoxicity screening methods is advantageous for detection of marginal genotoxins in safety evaluation process. Previously, we have reported several approaches for increasing the sensitivity of the PBMN assay. These approaches include the use of prior bleeding alone or in the combination with erythropoietin, and histone deacetylase inhibition by valproic acid. Major limitations with these approaches are physiological stress: loss of blood has a significant effect on erythropoesis. Further, shortcoming of using valproic acid as HDAC inhibitor is its own adverse effects (Ahmad et al., 2013; Vikram et al., 2007, 2008).

PARP inhibitor and genotoxicity evaluation

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DOI: 10.3109/15376516.2014.898355

The double-strand break (DSB) type DNA damage is extremely hazardous to cells and if left unrepaired may lead to the loss of chromosome fragments and cell death. Two major pathways exist to repair DSB are homologous recombination (HR) and non-homologous end-joining (NHEJ; Hartlerode & Scully, 2009). The HR operates optimally during the S and G2 phases of the cell cycle and NHEJ pathway operates throughout cell cycle (Croset et al., 2013). Poly(ADPribose) polymerase (PARP-1) along with other proteins of HR and single-strand break repair (SSB) operates as an alternative pathway, if the classical mechanism of NHEJ is impeded (Audebert et al., 2004). The SSB is primarily handled by PARP-1 and other PARP family members may also contribute (Croset et al., 2013). Hence, PARP-1 plays an essential role in the SSB and DSB repair of DNA and it is designated as the guardian because it maintains the genomic integrity (D’Amours et al., 1999; Jagtap & Szabo, 2005). The role of PARP-1 in the repair of SSB/BER is well defined. Poly(ADP-ribosylation) of nuclear DNA-binding proteins occurs immediately after DNA damage. PARP catalyses the transfer of the ADP-ribose moiety from the coenzyme NAD+ to a number of protein acceptors at breakage site (Oliver et al., 1999). Several proteins such as X-ray repair cross-complementing protein 1 (XRCC1), DNA polymerase b and PCNA, etc., have been identified to interact with PARP-1 during SSB repair (Swindall et al., 2013). The DNA damage is initially sensed by PARP and poly(ADP-ribosylation) of PARP1 itself (automodification) and of histones H1 and H2B (heteromodification) is triggered, which leads to chromatin relaxation followed by increased access of DNA-repair enzymes at the damage site. This is followed by the recruitment of XRCC1 at the damaged site, which is mediated through its PAR-ylation. This step is further followed by the assembly of the SSB repair complex, which finally converts the DNA ends to 50 -phosphate and 30 -hydroxyl moieties. Then DNA polymerase stimulated by PARP1 fills the gap and ligated by DNA ligase III (Jagtap & Szabo, 2005; Szabo et al., 1996; Zingarelli et al., 1996). A considerable work has been done to understand the role of PARP in the DNA damage and repair (Martin, 2001). PARP inhibitors can increase the sensitivity of chemotherapy as well as radiotherapy (Al-Ejeh et al., 2013; O’Shaughnessy et al., 2011). It has been reported that mutant cells defective in DNA repair pathway provide a sensitive high-throughput assay for genotoxicity (Evans et al., 2010). PARP has attracted attention as a possible target for cancer therapy, because its role in DNA repair and various PARP inhibitors have already been tested as monotherapies against tumors (Michels et al., 2013). 3-AB is a well-characterized competitive PARP inhibitor (Pandya et al., 2010; Zhao et al., 2009). The in vivo as well as in vitro dose of 3-AB for PARP inhibition is also well characterized (Cepeda et al., 2006; McLick et al., 1987; Southan & Szabo, 2003). This study was aimed to increase the sensitivity of the comet and PBMN assays using 3-AB as PARP inhibitor in mice. 3-AB was administrated as a pretreatment and single dose of cyclophosphamide (CP), 5-florouracil (5-FU), zidovudine (AZT) and furosemide (FUR) were administrated in separate treatment groups. The MN assay in the peripheral blood at different time points and the comet assay in PBL, bone marrow and liver were performed. This study results clearly

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demonstrate that 3-AB treatment significantly increases the sensitivity of the PBMN as well as the comet assay.

Materials and methods Animals All the animal experiments were approved by the Institutional Animal Ethics Committee (IAEC) and experiments were conducted according to Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA) guidelines. Experiments were performed on adult male Swiss albino mice (30 ± 2 g) procured from the Central Animal Facility (CAF) of the institute. Animals were kept under controlled environmental conditions at room temperature (22 ± 2  C), humidity (50 ± 10%) and automatically controlled 12 h light and dark cycles. Standard laboratory animal feed was purchased from a commercial supplier and fed to the animals ad libitum. Animals were acclimatized to the experimental conditions for a period of 2–3 days prior to experiment. Chemicals 3-AB (CAS no. 3544-24-9), CP (CAS no. 6055-19-2), SYBR Green I (CAS no. 163795-75-3), acridine orange (AO, CAS no. 10127-02-3), 5-FU (CAS no. 51-21-8), FUR (CAS no. 5431-9) and corn oil (CAS No. 8001-30-7) were procured from Sigma-Aldrich. Dimethylsulphoxide (DMSO), normal melting agarose (NMA), low melting agarose (LMA), Triton X100, ethylenediamine (EDTA) and Hanks balanced salt solution (HBSS) were obtained from Hi-media Laboratories Ltd. (Mumbai, India). Zidovudine (AZT, CAS no. 30516-871) was gifted by Aurobindo Pharma Ltd. (Hyderabad, India). Experimental design and animal treatment All the animals were randomly divided into different treatment and control groups. Experimental design including different treatment and control groups (saline, alcohol and corn oil) are shown in the Figure 1. All the animals were divided into 12 groups, of which four groups for genotoxin controls (CP, 5-FU, AZT and FUR), four groups for their respective combinations with 3-AB (3-AB + CP, 3-AB + 5FU, 3-AB + AZT and 3-AB + FUR), and remaining four groups were 3-AB, saline, alcohol and corn oil control, respectively. In the combination and 3-AB control groups, 3-AB was administrated every 8 h with the first dose given 2 h prior to genotoxin treatment. For the PBMN assay, small amount of blood was taken from the tail tip and smears were prepared at different time points. 3-AB (30 mg/kg), CP (50 mg/kg) and 5-FU (25 mg/kg) were dissolved in saline while AZT (400 mg/kg) was first dissolved in minimum amount of alcohol followed by required dilution with saline, while FUR (60 mg/kg) was dissolved in corn oil. The doses of CP, 5-FU, AZT, FUR and 3-AB were selected on the basis of previous experiments (Czapski et al., 2006; Dertinger et al., 1996; Mondal et al., 2012; Mughal et al., 2010; Naya et al., 1990; Robbins & Bowen, 1988). All the drugs were injected through intraperitoneal (i.p.) route, immediately after preparation and respective control animals received equal volume of appropriate vehicle.

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Figure 1. Experimental design showing the different treatment groups, time of chemical administration and subsequent time of sampling for the PBMN and comet assays.

PBMN assay The PBMN assay was performed as described by Ahmad et al. (2013) with some modifications. Briefly, blood smears were prepared on pre-cleaned slides. The smears were allowed to dry at room temperature and fixed in absolute methanol for 5 min. After fixation, slides were stained with acridine orange solution and washed thrice with phosphate buffer (pH 6.8). Micronucleus scoring was performed under oil immersion objective (1  100) using an Olympus fluorescent microscope (Model BX 51) connected to digital photomicrograph software (OLYSIA BioReport). For the PBMN assay, randomly 10–15 focuses were observed for each animal under oil immersion, which gave a total of 3000–5000 erythrocytes (ERTs) that includes normochromatic erythrocytes (ERTs) and reticulocytes (RETs) for the determination of total MN (MNERTs and MNRETs) frequency in the peripheral blood and expressed as MNERTs/1000 ERTs or MNRETs/1000 RETs respectively. Further, the RETs-to-ERTs ratio was calculated from the above counted total ERTs and RETs in the peripheral blood for each animal. Comet assay The comet assay was performed in the PBL, bone marrow and liver as described by Khan et al. with some modifications (Khan & Jena, 2014). The entire procedure was conducted in the dark to avoid possible photo-induced DNA damage. Then cell-agarose suspension, 100 ml were spread over a microscope slide (75  25 mm glass slides with 19 mm frosted ends), was pre-coated with 1% NMA. The cells were then lysed in a buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH 10.0) with freshly prepared 1% Triton X-100 and 10% DMSO for 24 h at 4 C. After lysis, the slides were rinsed three times in de-ionized water to remove salt and detergent. The slides were then coded and placed in a specifically designed horizontal electrophoresis tank (Model, CSLCOM20, Cleaver Scientific Ltd, Warwickshire, UK) and the DNA was allowed to unwind for 20 min in an alkaline solution containing 300 mM NaOH and 1 mM EDTA (pH 13) and then electrophoresis was performed at 300 mA and 38 V (0.90 V/cm) for 30 min. The slides were then neutralized with 0.4 M Tris (pH 7.5) for 15 min and stained with SYBR Green I (1:10 000) for 1 h and covered with cover slips. The DNA damage was visualized using an AXIO imager M1 fluorescence microscope (Carl Zeiss, Altlussheim, Germany) and the images were captured with image analysis software (Comet

Imager V.2.0.0). Duplicate slides were prepared for each animal/treatment and 50 comets per slide at random were scored to quantify the DNA damage. The parameters for the DNA damage analysis include: tail length (TL, in mM), tail moment (TM), olive tail moment (OTM) and % tail DNA (% DNA). The edges of the slides, any damaged part of the gel, any debris, superimposed comets and comets without distinct head (‘‘hedgehogs’’ or ‘‘ghost’’ or ‘‘clouds’’) were not considered for the analysis. All the slides were blindly coded and scored without knowledge of the code. Statistical analysis Results were shown as mean ± standard error of the mean (SEM) for each group. Statistical analysis was performed using Jandel Sigma Stat Version 3.5 software (Jandel Scientific Software, San Rafael, CA). For multiple comparisons, one-way analysis of variance (ANOVA) was used. Where ANOVA showed significant differences, post-hoc analysis was performed with Tukey’s test. Simple linear correlation analysis was performed in order to establish correlation between PBMN and comet assays as described previously Khan et al. (2011). The significance of correlation was determined using Pearson’s correlation coefficient. p50.05 was considered to be statistically significant.

Results Effect of 3-AB treatment on CP, 5-FU and AZT-induced MN frequency Combination treatment (3-AB + CP, 3-AB + 5-FU and 3AB + AZT) significantly increased the frequency of MNRETs and MNERTs as compared to CP, 5-FU and AZT control, respectively (Figures 2–4a and b). Further, CP, 5-FU and AZT alone as well as in the combination with 3-AB, significantly decreased the RETs-to-ERTs ratio as compared to saline and 3-AB control (Figures 2–4c). However, no significant change in the RETs-to-ERTs ratio was observed in combination as compared to CP, 5-FU and AZT control (Figures 2–4c). Effect of 3-AB treatment on FUR-induced MN frequency No significant increase in the MNRETs and MNERTs frequency was observed in the combination treatment (3AB + FUR) as compared to FUR control (Figure 5a and b). FUR alone as well as in the combination with 3-AB

PARP inhibitor and genotoxicity evaluation

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Figure 2. Effect of 3-AB treatment on CP-induced MN formation in the peripheral blood (PB) of the mice. (a) frequency of MN in ERTs (MNERTs/1000 ERTs), (b) frequency of MN in RETs (MNRETs/1000 RETs) and (c) RETs-to-ERTs ratio. All the values were shown as mean ± standard error of mean (n ¼ 5), *p50.05, @p50.01 and #p50.001, ‘‘a’’ versus control, ‘‘b’’ versus 3-AB and ‘‘c’’ versus CP.

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Figure 3. Effect of 3-AB treatment on 5-FU-induced MN formation in the PB of the mice. (a) frequency of MN in ERTs (MNERTs/1000 ERTs), (b) frequency of MN in RETs (MNRETs/1000 RETs) and (c) RETs-to-ERTs ratio. All the values were shown as mean ± standard error of mean (n ¼ 5), *p50.05 and #p50.001, ‘‘a’’ versus control, ‘‘b’’ versus 3-AB and ‘‘c’’ versus 5-FU.

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parameters (olive tail moment and % DNA in tail) in the PBL, liver and bone marrow as compared to CP, 5-FU and AZT control, respectively, while peak damage were observed at 36 h (Figures 6–8) in all organs with all the tested agents.

Effect of 3-AB treatment on the comet assay sensitivity in PBL, bone marrow and liver using CP, 5-FU and AZT

Effect of 3-AB treatment on the comet assay sensitivity in PBL, bone marrow and liver using FUR

3-AB treatment significantly increased the comet assay sensitivity in the combination (3-AB + CP, 3-AB + 5-FU and 3-AB + AZT) as revealed by significant increase in the comet

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Figure 4. Effect of 3-AB treatment on AZT-induced MN formation in the PB of the mice. (a) frequency of MN in ERTs (MNERTs/1000 ERTs), (b) frequency of MN in RETs (MNRETs/1000 RETs) and (c) RETs-to-ERTs ratio. All the values were shown as mean ± standard error of mean (n ¼ 5), *p50.05, @p50.01 and #p50.001, ‘‘a’’ versus control, ‘‘b’’ versus 3-AB and ‘‘c’’ versus AZT.

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combination treatment (3-AB + FUR) significantly increased the extent of DNA damage induced by FUR alone in liver and bone marrow. Further, in liver and bone marrow, the effect was most significant at 24 h followed by gradual decrease in the DNA damage (Figure 9B and C).

PBL, bone marrow and liver for all tested agents. Correlation analysis results indicate that 3-AB treatment increases the sensitivity of both assays equally as revealed by R2 value for the organs tested (Figures 10 and 11a–c).

Discussion Correlation analysis between MNERTs and OTM as well as % DNA in PBL, bone marrow and liver Linear correlation analysis was performed between MNERTs and olive tail moment (OTM) as well as % DNA in tail, in the

This study focuses on the use of 3-AB, a PARP inhibitor, to increase the sensitivity of the comet and micronucleus assays for detection of weak genotoxins. It has been reported that PARP inhibitors increase the DNA damage by impeding the

PARP inhibitor and genotoxicity evaluation

DOI: 10.3109/15376516.2014.898355

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Figure 6. (A–C) Effect of 3-AB treatment on CP-induced DNA damage in the PBL, liver and bone marrow of the mice, respectively, as analyzed by the comet assay at 24, 36 and 48 h. [A–C (a)] olive tail moment, [A–C (b)] % DNA in the PBL, liver and bone marrow respectively. All the values were shown as mean ± standard error of mean (n ¼ 5), #p50.001, ‘‘a’’ versus vehicle control, ‘‘b’’ versus 3-AB and ‘‘c’’ versus CP.

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DNA repair mechanisms and this approach is already being utilized for increasing the efficacy of anticancer agents (AlEjeh et al., 2013; Booth et al., 2013; Do & Chen, 2013; Lord & Ashworth, 2008). In various studies, DNA resynthesis inhibitors have been reported to enhance the genotoxicity by inhibition of DNA repair systems in mammalian cells (Kawaguchi et al., 2010b; Lim et al., 2012). In mammalian cells, PARP is a nuclear protein and abundantly expressed at physiological conditions (Herceg & Wang, 2001). When cells are exposed to alkylating agents, ionizing radiation or free radicals, PARP binds rapidly to DNA strand breaks and undergoes automodification, which leads to the formation of poly(ADP-ribose) polymers on the target sites, using NAD+ as a substrate (Herceg & Wang, 2001). Hence, PARP acts as a DNA damage detector in the cells during various genotoxin/ pathological insults. Poly(ADP-ribosylation) of the acceptor proteins helps in the DNA repair process by modifying target proteins at DNA strand breaks, which relaxes the condensed chromatin, thereby facilitates the recruitment of DNA repair complex (Surjana et al., 2010). It has been reported that cells treated with PARP inhibitors or reduced PARP expression exhibit augmented sensitivity of DNA damaging agents (Herceg & Wang, 2001). PARP inhibitors have been shown to prevent the depletion of NAD+ and render the cells susceptible to genotoxins (D’Amours et al., 1999; Shall, 1995). In this study, different genotoxins were selected on the

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basis of their DNA damaging mechanisms such as alkylating agent, anti-metabolites and nucleoside reverse transcriptase inhibitor (CP, 5-FU and AZT, respectively). Further, FUR was also included in this study because of its weak genotoxicity potential as well as organ-specific genotoxicity in the rodents (Mondal et al., 2012). The organ-specific DNA damaging potential of FUR might be the result of its bioactivation into reactive metabolites (Williams, 2006). The bioactivation of FUR generates three metabolites furosemide-g-ketocarboxylic acid, furosemide-glutathione conjugate and mixed N-acetyl lysine and N-acetyl cysteine conjugate in vitro and in vivo (Williams et al., 2007). Exact molecular mechanism of DNA damage induced by FUR is not characterized, but reactive metabolite production seems to play a crucial role. Overall, in the present test system, CP and 5-FU are considered as strong genotoxins, while AZT and FUR are considered as weak or marginal genotoxin at the tested doses. The weak genotoxic potential of AZT and FUR has already been reported using the same in vivo model without PARP inhibition (Ayers, 1988; Mondal et al., 2012; Motimaya et al., 1994; Mughal et al., 2010). In this study, CP, 5-FU and AZT showed significant DNA damage in comet assay at 24 h as compared to respective controls. The DNA damage induced by these genotoxins attain peak level at 36 h followed by gradual decrease, but still significant at 48 h in PBL, liver and bone marrow. 3-AB pre-

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Figure 8. (A–C) Effect of 3-AB treatment on AZT-induced DNA damage in the PBL, liver and bone marrow of the mice, respectively, as analyzed by the comet assay at 24, 36 and 48 h. [A–C (a)] olive tail moment, [A–C (b)] % DNA in tail in PBL, liver and bone marrow respectively. All the values were shown as mean ± standard error of mean (n ¼ 5), #p50.001, ‘‘a’’ versus control, ‘‘b’’ versus 3-AB and ‘‘c’’ versus AZT.

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Figure 7. (A–C) Effect of 3-AB treatment on 5-FU-induced DNA damage in the PBL, liver and bone marrow of the mice, respectively, as analyzed by the comet assay at 24, 36 and 48 h. [A–C (a)] olive tail moment, [A–C (b)] % DNA in tail in PBL, liver and bone marrow, respectively. All the values were shown as mean ± standard error of mean (n ¼ 5), #p50.001, ‘‘a’’ versus control, ‘‘b’’ versus 3-AB and ‘‘c’’ versus 5-FU.

Toxicol Mech Methods, 2014; 24(5): 332–341

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PARP inhibitor and genotoxicity evaluation

DOI: 10.3109/15376516.2014.898355

Oil

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10 R2 = 0.9402 R2 = 0.9458

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48

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Figure 9. (A–C) Effect of 3-AB treatment on FUR-induced DNA damage in the PBL, liver and bone marrow of the mice, respectively, as analyzed by the comet assay at 24, 36 and 48 h. [A–C (a)] olive tail moment, [A–C (b)] % DNA in tail in PBL, liver and bone marrow respectively. All the values were shown as mean ± standard error of mean (n ¼ 5), #p50.001, ‘‘a’’ versus control, ‘‘b’’ versus 3-AB and ‘‘c’’ versus FUR.

339

15 R2 = 0.9308

10

R2 = 0.993 4 R2 = 0.971

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4

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Figure 10. Correlation analysis showing the positive correlation between MNERTs and olive tail moment (OTM) in the PBL, bone marrow and liver of the mice induced by 3-AB treatment and single dose of CP/5-FU/AZT. (a–c) Represents correlation between MNERTs and OTM in the PBL, bone marrow and liver of mice respectively (n ¼ 5).

treatment further enhanced the extent of DNA damage in all the combination groups as evident from the results of comet and PBMN assays (3-AB + CP, 3-AB + 5-FU and 3AB + AZT) when compared to CP, 5-FU and AZT, respectively. Interestingly, 3-AB per se did not induce any DNA damage (genotoxicity) neither in the comet nor in the PBMN assays as compared to control, indicating lack of inherent genotoxic effect. The comet assay parameters show peak level damage at 36 h and hence were not measured beyond 48 h,

whereas MNERTs frequency was highest at 48 h. The most likely explanation for the difference in peak time in the comet and PBMN assays is the possible time lag between erythrocytes maturation and their appearance in the peripheral blood (Savill et al., 2009). The most plausible explanation for increase in the sensitivity of the comet and PBMN assays by 3-AB treatment is the inhibition of DNA repair process through PARP inhibition and/or involvement of PARP in the micronucleus formation. FUR results show the genotoxicity in

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Toxicol Mech Methods, 2014; 24(5): 332–341

(a)

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60

R2

= 0.9537

%DNA

40

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15 R2 = 0.9966 10 5

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R2 = 0.9348

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R2 = 0.994

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Figure 11. Correlation analysis showing the positive relation between MNERTs and % DNA in tail in the PBL, bone marrow and liver of the mice induced by 3-AB treatment and single dose of CP/5-FU/AZT. (a–c) Represents correlation between MNERTs and % DNA in tail in the PBL, bone marrow and liver of the mice (n ¼ 5).

liver and bone marrow, but not in PBL as evaluated by comet assay. Further, FUR did not increase the MN frequency as revealed by PBMN assay (Figure 5). It is interesting to note that 3-AB failed to increase the DNA damage in the combination with FUR (3-AB + FUR) as compared to FUR alone in peripheral blood for both PBMN and comet assays. Results with FUR support the reliability of this model due to the lack of the false positive. Since, DNA damage in bone marrow and liver caused by FUR was further enhanced by 3AB, as evident by comet assay results, which confirmed that this approach can enhance the genotoxic effect of marginal genotoxins. Correlation analysis between comet assay (OTM and % DNA) and the MNERTs in peripheral blood was performed. All the correlation coefficient (except FUR) shows significant positive correlation between comet and PBMN assay not only in the PBL but also in the liver and bone marrow. Correlation analyses demonstrate the reliability of the present approach and indicate that 3-AB increases the weak signal of genotoxic insult, which can be detected by either of the assays. Currently, comet assay is not a prerequisite for regulatory submissions; however, it is now rapidly gaining attention as a supportive assay for standard genetic toxicology test battery. Recently, ICH steering committee approved S2(R1) ‘‘Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use’’. In this draft guidance, the comet assay is recommended as one of the options for suitable in vivo tests (ICH Harmonised Tripartite Guideline, 2011) and the final guideline is expected to be available soon. Further, no OECD guideline yet exists for comet assay; however, a draft OECD guideline is already available (Draft OECD Guideline for the Testing of Chemicals, 2012) and the final guideline is expected to be available soon. Moreover, European Food Safety authority (EFSA) also accepts the comet assay data to assess the DNAdamaging potential of chemicals in various organs and tissues of exposed animals. Recently, EFSA has laid down the minimum requirements for conducting and reporting comet assay to ensure consistency in the acceptability and interpretation of results (European Food Safety Authority, 2012). Taken together, it is apparent that the comet assay is rapidly gaining attention primarily due to its simple test methodology and its capability to detect DNA damage in various types of mammalian organs.

Finally, any approach that could further enhance the sensitivity of the comet or MN assays might be helpful in the detection of marginal/weak genotoxins in the early preclinical studies and hence prevent the development of unpromising molecules, which might help reduce the futile investment of time and money. Our results with FUR clearly demonstrate the ability of this approach to avoid false positive results as well as to augment the genotoxic response, if any. In addition, PARP inhibition by 3-AB is unlikely to produce any adverse affect on the normal physiology, as PARP-1 null mice are viable and fertile (Stro¨m & Helleday, 2012). Further, mechanistic studies can provide new understandings on the influence of the DNA repair process and its inhibitors on the stability of nucleic acid and can advance the knowledge in the field of genotoxicity testing.

Acknowledgements The authors are grateful to Aurobindo Pharma Ltd. (Andhra Pradesh, India) for providing the generous gift sample of zidovudine.

Declaration of interest The authors report no declarations of interest. This study was supported by funds of the National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Mohali, Punjab 160062, India.

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