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Dose-Response Assessment of Four Genotoxic Chemicals in a Combined Mouse and Rat Micronucleus (MN) and Comet Assay Protocol

Dose-Response Assessment of Four Genotoxic Chemicals in a Combined Mouse and Rat Micronucleus (MN) and Comet Assay Protocol

The Journal of Toxicological Sciences (J. Toxicol. Sci.) 149 Vol.35, No.2, 149-162, 2010

Original Article Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and protocol

Leslie Recio1, Cheryl Hobbs1, William Caspary2 and Kristine L. Witt2

1Genetic Toxicology Division, ILS, Inc., PO Box 13501, RTP, NC 27709, USA 2National Toxicology Program, NIEHS, RTP, NC 27709, USA

(Received October 15, 2009; Accepted November 25, 2009)

ABSTRACT — The in vivo micronucleus (MN) assay has proven to be an effective measure of geno- toxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic dam- DJHXVLQJWKH01DVVD\SURYLGHVDOLPLWHGJHQRWR[LFLW\SUR¿OH7KHin vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues fol- lowing administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophospha- mide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24- hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last admin- LVWUDWLRQ$OOFRPSRXQGVLQGXFHGVLJQL¿FDQWLQFUHDVHVLQPLFURQXFOHDWHGUHWLFXORF\WHV 015(7 LQWKH SHULSKHUDOEORRGRIPLFHDQGDOOEXW$&0LQGXFHG015(7LQUDWV(06DQG$&0LQGXFHGVLJQL¿FDQW increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced '1$GDPDJHZDVGHWHFWHGLQOHXNRF\WHVDQGGXRGHQXPFHOOV96DVSLQGOH¿EHUGLVUXSWLQJDJHQWZDV negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the of substances of public health concern.

Key words: DNA damage, Comet assay, Acrylamide, Ethyl methanesulfonate, Cyclophosphamide, Vincristine sulfate

INTRODUCTION entirely recreated in vitro. The rodent erythrocyte micro- nucleus (MN) assay in peripheral blood or bone marrow Genotoxicity studies in rodents with defined expo- is considered to be the primary assay to assess in vivo sures are useful biological test models for investigative genotoxic potential (Blakey et al., 2008; Eastmond et al., toxicology and mechanistic studies, and they serve as an 2009; ICH, 2008). MN are surrogate measures of structur- important element of the regulatory test battery used in al and numerical chromosomal aberrations that are asso- pre-clinical safety assessment and the evaluation of envi- ciated with increased cancer risk (Bonassi et al., 2007). ronmental agents for genotoxic risk to humans (ICH, MN can also be considered bridging biomarkers of geno- 1996, 1997; U.S. EPA, 2005). Compared with in vit- toxic exposure, since they can be enumerated across mul- ro tests, in vivo tests may provide more relevant data for tiple species including humans (Dertinger et al., 2007). the assessment of DNA damage potential in humans since Although the in vivo rodent MN assay is an effective they take into account dynamic whole-animal physiologi- measure of genotoxicity, the assay is not without limita- cal processes such as uptake and systemic distribution by tions. The assay can only be conducted in rapidly divid- the circulatory system, Phase I and Phase II metabolism, ing cells and typically measures chromosomal damage and intact elimination/excretory systems that cannot be induced in a single tissue (bone marrow), thereby pro-

Correspondence: Kristine L. Witt (E-mail: [email protected])

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L. Recio et al. viding a limited assessment of genotoxic potential for 1) examine DNA damage in the same accessible cell type a chemical. Since direct measurements of chromosom- used in human biomonitoring (leukocytes), 2) assess gen- al aberrations or endogenous gene mutations in most tis- otoxicity in a major site of xenobiotic metabolism (liv- sues other than blood or bone marrow are not currently er), and 3) evaluate genotoxicity in the gut region where technologically feasible, a number of surrogate endpoints most drug absorption takes place (duodenum). Addition- are used to assess mutagenicity and genotoxicity in other al tissues were assessed in ACM-treated animals based rodent tissues. These surrogate endpoints permit the eval- RQSULRUNQRZOHGJHRIVSHFL¿FWDUJHWV LHWHVWLFXODUWLV- uation of DNA damage, chromosomal damage, genom- sue). Here, we report the data from the alkaline (pH > ic responses to DNA damage, and mutation in mark- 13) Comet assay from the same B6C3F1 mice and Fish- er genes (Guyton et al., 2009; ICH, 2008; Kirkland and er 344/N rats used in MN assay studies reported earlier Speit, 2008; Lambert et al., 2005). The alkaline (pH > 13) (Witt et al., 2008). Comet assay is being proposed by testing and regulatory agencies as a second (additional) in vivo genotoxicity bio- MATERIALS AND METHODS assay, to complement the in vivo MN assay, since it can detect DNA repair and a broad spectrum of DNA damage, Chemicals including DNA breaks, apurinic sites, alkali-labile DNA Details of chemicals, animal husbandry, and dos- adducts, and a spectrum of reactive oxygen/lipid peroxi- ing were reported previously (Witt et al. %ULHÀ\ dation species-induced DNA lesions in virtually any tis- EMS (CASRN: 62-50-0), ACM (79-06-1), CP (50-18-0), sue (Fortini et al., 1996; Gedik and Collins, 2005). Fur- and VS (57-22-7) were purchased from Sigma-Aldrich thermore, the Comet assay requires small numbers of (St. Louis, MO, USA) and assigned code numbers prior cells and importantly, it does not require cell division for to use in the experiments described below. ACM, EMS the evaluation of DNA damage. However, uncertainties (both direct-acting ), and CP (a that with respect to the origin and fate of the detectable tran- requires metabolic activation) were dissolved in phos- sient DNA lesions produced by xenobiotics may limit the phate buffered saline (pH 7.4) and administered by oral XVHRIWKH&RPHWDVVD\WRKD]DUGLGHQWL¿FDWLRQ )RUWLQL et gavage. VS (an aneugen) was dissolved in phosphate al., 1996). The Comet assay is recommended as a follow- buffered saline (pH 7.4) and administered by intraperi- up to a negative or equivocal in vivo MN assay, as a con- toneal injection due to the limited bioavailability of VS ¿UPDWLRQWRDSRVLWLYH01DVVD\DQGDVDPHDQVWRPHDV- when administered by oral gavage. For these well-char- ure genotoxicity in a target tissue other than bone marrow acterized genotoxic compounds, dose-setting information (e.g., liver) (Eastmond et al., 2009; ICH, 2008). An exten- was available from previous studies conducted at ILS, sive international effort, led by the Japanese Center for Inc. or from published studies. the Validation of Alternative Methods (JaCVAM), is cur- rently underway to conduct an in vivo Comet assay vali- Animal husbandry dation study in rats (http://jacvam.jp). Male B6C3F1 mice and male Fisher 344/N rats were The National Toxicology Program (NTP) is evaluating used for this study. All studies were approved by the ILS, an “acute” genotoxicity testing protocol in rodents that Inc. Institutional Animal Use and Care Committee. Proce- combines the MN and alkaline (pH > 13) Comet assays dures were completed in compliance with the Animal Wel- for a more comprehensive assessment of genotoxicity in fare Act Regulations, 9 CFR 1-4, and animals were handled tissues of mice or rats (MN/Comet assay) than could be and treated according to the Guide for the Care and Use achieved with either assay alone. This manuscript reports of Laboratory Animals (ILAR, 1996). Animals were accli- results from initial studies evaluating a combined MN/ mated for 7 days after receipt from the supplier (Charles Comet assay protocol using dose-response experiments River Laboratories, Portage, MI, USA). Animals were 8- to test four model genotoxic chemicals in male B6C3F1 16 weeks of age at the beginning of treatment; treatment mice and male Fisher 344/N rats. The four chemicals JURXSVFRQVLVWHGRI¿YHDQLPDOV$QLPDOVZHUHPDLQWDLQHG used in the studies were ethyl methanesulfonate (EMS), in constant temperature rooms (71 ± 3°F) with a relative acrylamide (ACM), cyclophosphamide (CP), and vin- humidity of 30-70% on a 12:12 (5:00 AM - 5:00 PM) light: cristine sulfate (VS). These four chemicals induce MN dark cycle. Animals were housed individually in polycar- by different mechanisms. ACM and EMS are both direct- bonate cages with Sanichip Laboratory hardwood bedding acting clastogens, CP is clastogenic after metabolic acti- (P.J. Murphy Forest Products Corp., Montvale, NJ, USA) vation, and VS induces (whole chromosome DQGSURYLGHGIRRG 3XULQD&HUWL¿HG5RGHQW&KRZ loss). The tissues or cell types examined were selected to: Ralston Purina, St. Louis, MO, USA) and tap water ad libi-

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Combined micronucleus/Comet assay protocol tum. With the exception of VS in rats, the animals were with 1% Triton X-100 (Sigma) and 10% DMSO). Fol- administered each test article once daily over four con- lowing at least 1-2 hr of incubation in lysis solution, two secutive days (at 24-hr intervals), euthanized by carbon slides per sample were rinsed with 0.4 M Trizma base and dioxide anesthesia/sedation, and then exsanguinated 4 hr incubated in alkaline conditions (300 mM NaOH (Fisher), after the last dosing. Due to cage-side observations indic- 1 mM EDTA, pH > 13) for 20 min, followed by electro- ative of toxicity in the rats, VS was administered for only phoresis in the same buffer for 20 min at 0.7 V/cm (elec- 3 days and rats were euthanized 4 hr after the last expo- trode to electrode) and 300 mA. After electrophoresis, sure. slides were immersed in an excess amount of 0.4 M Triz- Peripheral blood samples were processed for flow PDEDVHWRQHXWUDOL]HWKHDONDOLDQGWKHQ¿[HGLQ cytometry evaluation of micronucleated reticulocytes HWKDQRO 0F&RUPLFN:HVWRQ0286$ )ROORZLQJ¿[- (MN-RET) (Witt et al., 2008), and blood and tissue sam- ation, the slides were air dried and stored at room tem- ples (blood, liver, duodenum, and additional tissues as perature in a desiccator until they were scored. Prior to noted below) were processed for assessment of DNA scoring slides, the DNA was stained with SYBR Gold™, damage using the Comet assay (Burlinson et al., 2007; following the supplier’s directions (Molecular Probes, Hartmann et al., 2003; Tice et al., 2000). Inc., Eugene, OR, USA). The slides were scored without knowledge of the dose group. The extent of DNA migra- Alkaline (pH > 13) Comet assay tion was determined for each sample by simultaneous The alkaline (pH > 13) Comet assay was conduct- image capture and scoring of 100 cells (50 cells on each ed according to published recommendations (Burlinson RIWZRVOLGHV DWîPDJQL¿FDWLRQXVLQJWKH.LQHWLF et al., 2007; Hartmann et al., 2003; Tice et al., 2000). At Imaging, Ltd., Komet© 5.5 image analysis system. necropsy, samples (1 cm2) of liver, duodenum, and blood During the scoring of testes cell preparations from (20 μl) were collected from each animal and maintained rats and mice, two distinct sizes of nuclei were noted and in cold mincing buffer (Mg++ and Ca++ free Hanks’ Bal- these were scored independently for each slide. The larger anced Salt Solution (Gibco, Carlsbad, CA, USA) with 20 nuclei, similar in size to somatic cell nuclei in leukocytes, mM Na2EDTA (EDTA) (Sigma) and 10% v/v dimethyl- liver, and duodenum, were presumed to be from somat- sulfoxide (DMSO) (Fisher, Waltham, MA, USA)). Liv- ic cells. The smaller nuclei were presumed to contain er and duodenum samples were minced in cold mincing one-half the DNA content of the somatic cells, reflect- buffer. For ACM-treated animals, additional tissues were LQJVSHUPFHOO'1$1RIXUWKHUFHOOSXUL¿FDWLRQRUFKDU- collected as follows. A testis was removed from each ani- acterization of cell origin was done on these mixed cell mal and placed into mincing solution. Tubules were col- samples. These assumptions were used to classify each lected from the testis by dissection, and the contents of data set as DNA damage in presumptive somatic cells or the tubules were flushed out into mincing solution. In sperm cells. ACM-treated rats only, thyroid was collected and placed The extent of DNA migration for all samples was eval- into cold mincing solution and minced. uated according to the following endpoint measurements: Aliquots of blood or minced tissue samples were imme- % Tail DNA: intensity of all tail pixels divided by the GLDWHO\SODFHGLQWRPLFURFHQWULIXJHWXEHVDQGÀDVKIUR]HQ total intensity of all pixels in the Comet, expressed as a LQOLTXLGQLWURJHQWXEHVZHUHVWRUHGIUR]HQEHORZíƒ& percentage. until the cells could be processed further. This proc- Tail Length: the horizontal distance from the center of ess of cryopreservation allowed for control of a uniform the head (start of tail) to the end of the tail. time interval between tissue collection and electrophore- Olive Tail Moment (OTM): the distance between the sis (Witt et al., 2009). Slides for the Comet assay were center of gravity of the DNA distribution in the tail and prepared from blood and minced tissue as follows. Blood the center of gravity of the DNA distribution in the head, and tissue samples were thawed at room temperature and multiplied by the fraction of DNA in the tail. allowed to sit momentarily to permit large tissue pieces to settle. A 10 μl aliquot of suspension containing approx- Statistical analysis imately 10,000 cells was mixed with 0.5% low melting Data from 100 cells per animal were collected and point agarose (Sigma) and spread on standard micro- assessed for significant (P < 0.05) increases in DNA scope slides (Fisher) pre-dipped in agarose. Slides were migration endpoints by statistical analysis using Analyse- then allowed to harden on a cold surface prior to further it® Standard Edition software (http://www.analyse-it.com/). processing. All slides were placed in cold lysis solution Using individual animal data, the Shapiro-Wilk test was (2.5 M NaCl, 100 mM EDTA, and 10 mM Trizma base, used to assess normality of the negative control group.

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Data that were not normally distributed were analyzed B6C3F1 male mice (Table 1) and F344/N male rats (Table by the Mann-Whitney test (Mann and Whitney, 1947) to 2) treated with 50, 100, 200, or 300 mg/kg EMS. One compare each dose level to the concurrent control, and by hundred cells were scored for each tissue examined per the Kendall rank correlation test (Kendall, 1938) to deter- animal, and three measures of DNA damage were made mine the presence of a dose response. Data that were nor- for each sample: % Tail DNA, Tail Length, and OTM. mally distributed were analyzed using the F test to deter- The mean values from all animals per treatment group mine homogeneity of variances, an independent one-tailed per tissue are presented in the data tables along with the t-test to compare each dose level to the concurrent control, results of the statistical analyses. and linear regression to determine the presence of a dose %DVHGRQWKH270HQGSRLQW(06LQGXFHGVLJQL¿FDQW response. increases in DNA damage in mice at all dose levels in all of the tissues examined. In rats, EMS also induced a sig- RESULTS QL¿FDQWGRVHUHODWHGLQFUHDVHLQ'1$GDPDJHLQDOOWLV- sues examined. In leukocytes and in liver cells of rats, EMS VLJQL¿FDQWLQFUHDVHVLQ'1$GDPDJHZHUHGHWHFWHGDWDOO The alkaline (pH > 13) Comet assay was conducted dose levels. However, in contrast to the response seen in in blood leukocytes and in liver and duodenum cells of PLFHVLJQL¿FDQWLQFUHDVHVLQ270LQWKHUDWGXRGHQXP

Table 1. Comet assay data for mice exposed to EMS Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 9.1 1.18 25.8 4.09 1.9 0.40 50 5 29.0 1.47 48.8 3.46 7.4 a 0.79 100 5 26.9 5.59 57.0 5.82 7.8 a 1.32 200 5 33.6 7.17 87.8 13.24 12.6 a 2.70 300 5 21.2 3.91 90.4 8.07 9.7 a 1.15 Trend Test b: p = 0.0020 Liver 0 5 20.3 2.40 7.2 0.49 0.7 0.10 50 5 27.3 1.97 7.8 0.36 1.1 a 0.08 100 5 36.3 1.44 9.1 0.50 1.5 a 0.09 200 5 57.2 3.94 10.7 1.10 2.5 a 0.19 300 5 64.3 3.63 12.5 0.32 3.0 a 0.23 Trend Test b: p < 0.0001 Duodenum 0 5 42.9 2.65 9.9 0.40 2.2 0.20 50 5 58.8 2.61 9.1 0.08 2.9 a 0.16 100 5 72.0 1.05 10.3 0.36 4.0 a 0.11 200 5 71.6 2.10 10.4 0.34 4.0 a 0.19 300 5 71.3 1.16 11.4 0.48 4.4 a 0.16 Trend Test b: p < 0.0001 Data based on 100 cells scored per animal. a p < 0.05; one-tailed t-test. b /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05.

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Table 2. Comet assay data for rats exposed to EMS Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 8.2 1.41 26.2 4.09 1.6 0.38 50 5 44.1 8.45 91.3 8.85 12.9 a 2.13 100 5 55.6 7.55 107.7 7.64 16.3 a 1.92 200 5 47.7 13.28 111.2 15.18 16.1 a 4.22 300 4 57.5 10.88 140.3 12.15 25.1 a 3.21 Trend Test b: p < 0.0001 Liver 0 5 30.2 2.86 9.0 0.95 1.3 0.18 50 5 58.1 1.33 13.8 0.89 2.7 a 0.13 100 5 57.8 0.94 14.1 0.60 2.6 a 0.07 200 5 61.2 1.32 14.7 0.47 2.9 a 0.08 300 5 71.8 2.52 19.0 3.23 4.3 a 0.80 Trend Test b: p < 0.0001 Duodenum 0 5 62.6 10.46 51.1 5.89 15.8 3.54 50 5 82.7 2.75 65.4 5.12 23.8 2.73 100 5 81.8 1.74 60.0 3.37 21.7 1.19 200 5 79.5 1.24 61.0 3.08 20.8 1.14 300 5 85.5 2.02 88.9 21.46 32.7 a 7.73 Trend Test b: p = 0.0229 Data based on 100 cells scored per animal. a p < 0.05; one-tailed t-test. b /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05. were observed only at the highest administered dose of $OWKRXJKVWDWLVWLFDOO\VLJQL¿FDQWLQFUHDVHVLQ'1$GDP- EMS (300 mg/kg). age were observed in multiple tissues of rats, the overall magnitude of the increases was lower than that observed ACM for the corresponding tissues in mice. In blood leukocytes The Comet assay was conducted in blood leukocytes from ACM-treated rats, low variability within the vehicle and in cells of the liver, duodenum, and testes of B6C3F1 control group allowed the detection of small but statisti- mice (Table 3) and F344/N rats (Table 4) administered FDOO\VLJQL¿FDQWLQFUHDVHVLQ'1$GDPDJH,QGXRGHQXP 12.5, 25, 37.5 or 50 mg/kg ACM. For rats, cells from testicular somatic cells, and thyroid cells, clear statistical- the thyroid were also examined (Table 4). In mice, ACM O\VLJQL¿FDQWLQFUHDVHVLQ'1$GDPDJHZHUHREVHUYHG LQGXFHGVLJQL¿FDQWLQFUHDVHVLQ'1$GDPDJHEDVHGRQ OTM in all of the tissues examined at all dose levels test- CP ed, with the single exception of testicular somatic cells of The Comet assay was conducted in blood leukocytes animals administered the lowest dose of ACM (12.5 mg/ and in cells of the liver and duodenum of B6C3F1 mice kg). In rats, the DNA damage response was more vari- and F344/N rats administered CP at doses of 25, 50, 75, DEOHWKDQLQPLFH$&0LQGXFHGVLJQL¿FDQWLQFUHDVHVLQ or 100 mg/kg (Table 5) and 2.5, 5.0, 10 or 20 mg/kg DNA damage based on the OTM measurement in blood 7DEOH UHVSHFWLYHO\,QPLFH&3LQGXFHGVLJQL¿FDQW P leukocytes, testicular somatic cells, and cells of the thy- < 0.05) increases in DNA damage in blood leukocytes, as roid and duodenum; no increases in DNA damage were indicated by OTM measurements, at all dose levels. No detected in rat liver cells or in presumptive sperm cells. increase in DNA damage was seen in liver of CP-treated

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Table 3. Comet assay data for mice exposed to ACM Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 8.7 0.77 2.8 0.53 0.2 0.03 12.5 4 20.0 2.73 7.5 1.70 0.6 a 0.07 25.0 4 19.8 2.38 8.6 1.77 0.7 a 0.17 37.5 4 20.7 1.26 9.8 1.16 0.9 a 0.07 50.0 5 25.4 2.80 9.8 0.84 1.1 a 0.16 Trend Test b: p < 0.0001 Liver 0 5 14.2 1.40 24.5 2.68 2.4 0.33 12.5 5 20.3 0.89 33.9 1.77 3.8 a 0.37 25.0 5 27.2 2.26 40.4 1.53 6.0 a 0.70 37.5 5 24.9 1.30 40.9 1.99 5.2 a 0.50 50.0 5 27.9 0.92 38.3 1.83 5.8 a 0.33 Trend Test b: p < 0.0001 Duodenum 0 5 17.0 1.48 24.6 3.01 2.6 0.31 12.5 5 32.8 2.59 45.6 2.78 6.9 a 0.74 25.0 5 42.5 4.84 53.8 6.41 10.8 a 2.15 37.5 5 41.3 4.13 53.0 6.09 10.8 a 2.12 50.0 5 44.1 3.85 57.7 3.53 11.4 a 1.35 Trend Test b: p = 0.0003 Testes - Germ Cells 0 5 7.0 0.72 14.3 1.12 0.8 0.13 12.5 5 18.9 3.63 27.3 5.50 4.0 a 1.36 25.0 5 23.5 1.78 35.6 5.32 5.8 a 0.54 37.5 5 16.5 1.78 27.7 2.89 3.6 a 0.51 50.0 5 21.5 2.05 32.3 2.89 5.3 a 0.71 Trend Test b: p < 0.0070 Testes - Somatic Cells 0 5 14.9 2.13 27.5 1.93 2.4 0.40 12.5 5 15.5 0.75 25.0 2.62 2.4 0.19 25.0 5 22.0 1.38 32.8 1.82 3.8 a 0.36 37.5 5 20.3 0.97 30.9 1.34 3.6 a 0.29 50.0 5 24.7 2.67 36.4 2.98 4.9 a 0.83 Trend Test b: p = 0.0004 Data based on 100 cells scored per animal. a p < 0.05; one-tailed t-test. b /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05.

PLFH,QGXRGHQXPDVWDWLVWLFDOO\VLJQL¿FDQWLQFUHDVHZDV cant increases in DNA damage in blood leukocytes at the measured only in the highest dose group (100 mg/kg); the highest dose tested (20 mg/kg). No increase in DNA dam- WUHQGWHVWZDVQRWVLJQL¿FDQW,QUDWV&3LQGXFHGVLJQL¿- age was detected in liver of CP-treated rats; in duodenum,

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Table 4. Comet assay data for rats exposed to ACM Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 7.3 1.04 3.0 0.58 0.2 0.04 12.5 5 8.3 0.95 3.8 0.29 0.2 0.02 25.0 5 10.2 0.72 4.2 0.39 0.3 a 0.03 37.5 5 12.6 1.64 4.7 0.58 0.4 a 0.08 50.0 5 13.6 0.80 4.9 0.35 0.4 a 0.03 Trend Test b: p = 0.0004 Liver 0 5 15.8 1.10 27.8 1.63 2.9 0.31 12.5 5 16.8 2.45 29.4 1.75 3.3 0.67 25.0 5 18.0 1.09 30.5 2.12 3.2 0.37 37.5 5 22.6 2.52 38.9 4.72 4.6 1.14 50.0 5 18.8 1.97 31.2 3.22 3.5 0.57 Trend Test b: p = 0.2613 Duodenum 0 5 27.8 2.63 31.7 2.28 4.9 0.55 12.5 5 36.5 2.45 47.7 4.63 8.5 a 1.12 25.0 5 31.9 2.95 41.3 2.30 7.6 a 1.12 37.5 5 31.5 2.92 42.7 2.89 6.5 1.05 50.0 5 42.3 2.86 57.4 4.34 11.3 a 1.00 Trend Test b: p = 0.0067 Testes - Germ Cells 0 5 18.8 1.36 15.9 1.07 2.5 0.21 12.5 5 17.3 3.12 14.7 2.47 2.2 0.64 25.0 5 19.6 2.92 19.7 2.95 2.9 0.63 37.5 5 17.4 3.04 17.3 3.71 2.5 0.89 50.0 5 15.3 1.98 15.1 1.61 1.8 0.36 Trend Test b: p = 0.3920 Testes - Somatic Cells 0 5 8.8 1.13 16.4 1.79 1.0 0.14 12.5 5 11.8 1.31 22.9 2.28 1.6 a 0.30 25.0 5 12.8 1.34 25.9 1.72 1.8 a 0.28 37.5 5 14.2 1.31 27.1 2.65 2.1 a 0.40 50.0 5 19.3 1.80 29.6 1.57 3.1 a 0.41 Trend Test b: p < 0.0001 Thyroid 0 5 18.5 1.20 36.3 5.56 4.2 0.79 12.5 5 24.4 1.68 47.5 6.44 6.2 c 0.87 25.0 5 26.9 1.74 50.2 5.11 7.2 c 0.69 37.5 5 27.3 2.96 48.9 4.93 6.9 c 1.11 50.0 5 33.1 4.32 52.6 4.52 9.3 c 2.02 Trend Test: p = 0.0074 d Data based on 100 cells scored per animal. a p < 0.05; one-tailed t-test. b /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05. c p < 0.05; one-tailed Mann-Whitney test. d .HQGDOO&RUUHODWLRQVLJQL¿FDQWDWp < 0.05.

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Table 5. Comet assay data for mice exposed to CP Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 4.6 0.30 2.0 0.12 0.1 0.01 25 5 7.7 0.62 3.0 0.16 0.2 a 0.01 50 5 10.3 0.44 3.9 0.16 0.3 a 0.02 75 5 14.2 1.18 4.8 0.26 0.5 a 0.06 100 5 15.5 1.51 5.2 0.35 0.6 a 0.08 Trend Test b: p < 0.0001 Liver 0 5 18.6 2.08 27.9 3.10 3.6 0.69 25 5 21.6 1.71 32.3 3.98 4.4 0.49 50 5 19.8 1.14 29.7 2.39 3.9 0.40 75 5 22.8 1.89 32.0 2.56 4.5 0.59 100 5 19.5 1.00 30.2 1.30 3.9 0.33 Trend Test b: p = 0.7330 Duodenum 0 5 23.5 3.82 6.8 0.92 0.9 0.19 25 5 30.6 7.23 7.8 1.57 1.3 0.41 50 5 34.2 6.68 8.1 1.32 1.4 0.38 75 5 23.5 2.68 6.8 0.42 0.9 0.13 100 5 38.9 6.35 8.7 1.02 1.5 a 0.28 Trend Test b: p = 0.3519 Data based on 100 cells scored per animal. a p < 0.05; one-tailed t-test. b /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05.

VLJQL¿FDQWLQFUHDVHVZHUHGHWHFWHGEXWWKHPDJQLWXGHRI Witt et al. (2008). The data for the MN assays, along with the increases was similar across doses. the corresponding Comet assay results, are summarized in Table 9. VS The Comet assay was conducted in blood leukocytes DISCUSSION and in cells of the liver and duodenum of B6C3F1 mice and F344/N rats administered VS by intraperitoneal injec- There is a clear need to identify compounds having tion at doses of 0.0125, 0.0250, 0.0500 or 0.0750 mg/kg the potential to permanently alter the human genome. (Table 7) and 0.00625, 0.01250, 0.02500 or 0.03125 mg/ The genetic toxicology test battery addresses this need kg (Table 8), respectively. Under the conditions used in by evaluating genotoxicity resulting from chemical expo- WKLVVWXG\96GLGQRWLQGXFHVLJQL¿FDQWO\LQFUHDVHGOHY- sures (ICH, 1996, 1997; U.S. EPA, 2005). However, this els of DNA damage in any of the tissues examined in test battery is used in the hazard identification step in mice or rats. risk assessment and is not designed to be a quantitative SUHGLFWRURIRUJDQRUWLVVXHVSHFL¿FWXPRULQGXFWLRQLQ Summary of combined MN/Comet assay in mice rodents (Elespuru et al., 2009). As the test battery contin- and rats XHVWRXQGHUJRUHYLVLRQDQGUH¿QHPHQWWKH&RPHWDVVD\ As indicated previously, the MN assay was conduct- is being considered for use as a second in vivo genotoxic- ed in the same animals used in the studies conducted by ity assay (Eastmond et al., 2009; ICH, 2008). The Com-

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Table 6. Comet assay data for rats exposed to CP Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 6.0 0.90 11.7 0.70 0.7 0.17 2.5 5 5.8 0.11 11.6 0.57 0.6 0.03 5.0 5 5.7 0.07 13.1 0.54 0.7 0.06 10 5 7.6 0.86 13.9 1.12 0.9 0.13 20 5 8.8 0.86 15.7 0.92 1.1 a 0.13 Trend Test: p = 0.0011 b Liver 0 5 20.4 4.41 22.8 3.14 3.8 1.17 2.5 5 28.6 2.53 29.3 1.14 6.4 0.82 5.0 5 25.5 3.31 28.8 1.44 5.2 1.00 10 5 21.6 1.93 27.1 0.67 4.2 0.55 20 5 36.1 9.25 30.5 3.69 8.5 2.40 Trend Test: p = 0.0565 c Duodenum 0 5 21.7 3.99 23.7 2.00 3.9 0.78 2.5 5 32.0 5.30 32.4 3.99 6.5 d 1.11 5.0 5 33.9 9.43 32.0 3.08 6.7 1.90 10 5 28.5 2.66 29.5 1.98 5.7 d 0.47 20 5 37.9 5.75 32.2 2.15 7.4 d 1.13 Trend Test: p = 0.1331 c Data based on 100 cells scored per animal. a p < 0.05; one-tailed Mann-Whitney test. b .HQGDOO&RUUHODWLRQVLJQL¿FDQWDWp < 0.05. c /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05. d p < 0.05; one-tailed t-test. et assay was recently shown to detect nearly 90% of car- mice and rats, a dose response for EMS genotoxicity was cinogens that were negative or equivocal in the MN assay detected in all of the mouse tissues analyzed and in rat and therefore, a combined MN/Comet assay has been rec- blood leukocytes and liver cells. In rat duodenum, EMS ommended to broadly assess in vivo genotoxic potential was positive only at the top dose of 300 mg/kg. Although (Kirkland and Speit, 2008; Pfuhler et al., 2007). EMS-induced DNA damage in the rat duodenum was not The purpose of the studies reported here was to eval- detectable with the Comet assay at doses up to 200 mg/ uate a combined in vivo genetic toxicity testing proto- kg, the increases in DNA damage seen in leukocytes and col using the MN/Comet assay. The four model geno- liver cells at these doses suggest that EMS absorption and toxic compounds chosen-EMS, ACM, CP, and VS-are entry into systemic circulation may occur proximal to the well described in the literature and dose-response stud- duodenum and reflect EMS-induced “first-pass” DNA ies were conducted to provide causal links to exposure. damage in blood leukocytes and liver. If so, then the EMS, ACM, CP, and VS have all been previously tested increased DNA damage observed in the rat duodenum at in the Comet assay in vivo or in vitro, and they represent the top dose of EMS (300 mg/kg) may be due to incom- a variety of genotoxic modes of action (Anderson et al., plete absorption of EMS in the stomach, thus allowing 'HDU¿HOG et al., 1988; Eastmond and Tucker, 1989; some EMS to reach the duodenum at biologically effec- Gocke and Müller, 2009; Tice et al., 2000). tive levels detectable by the Comet assay. Using this combined MN/Comet assay protocol in Recent studies have examined the dose response for

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Table 7. Comet assay data for mice treated with VS Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 9.2 0.75 12.6 1.29 1.2 0.12 0.0125 5 8.8 0.88 11.3 1.38 1.3 0.20 0.0250 5 11.9 2.13 14.5 2.12 2.0 0.50 0.0500 5 11.2 1.62 14.3 2.01 1.9 0.42 0.0750 5 11.4 1.30 15.0 0.94 1.9 0.33 Trend Test a: p = 0.1297 Liver 0 5 20.2 1.83 24.6 1.28 3.7 0.37 0.0125 5 20.4 1.60 24.2 1.73 3.8 0.42 0.0250 5 20.3 1.80 26.4 0.84 3.9 0.41 0.0500 5 20.8 2.19 27.2 1.89 4.1 0.51 0.0750 5 19.1 1.84 24.8 1.50 3.6 0.41 Trend Test a: p = 0.0980 Duodenum 0 5 19.2 6.40 6.6 2.26 0.9 0.43 0.0125 5 17.3 5.72 6.1 2.11 0.7 0.36 0.0250 5 20.6 6.61 6.9 2.42 1.0 0.39 0.0500 5 18.1 3.62 7.0 1.63 0.8 0.22 0.0750 5 29.8 5.72 10.5 1.16 1.6 0.40 Trend Test a: p = 0.2227 Data based on 100 cells scored per animal. a /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05.

genotoxicity and mutagenicity of EMS administered for kg) may produce greater numbers of DNA lesions that are 28 days by oral gavage in the MutaTM mouse using the QRWDVHI¿FLHQWO\UHSDLUHGWUDQVODWLQJLQWRFKURPRVRPDO in vivo MN assay and the lacZ mutation assay (Gocke damage detectable as MN. and Müller, 2009). No increases in lacZ mutations were DNA damage, detected by the Comet assay, in mul- observed with EMS doses of 25-50 mg/kg/day, and dos- tiple tissues of mice and rats administered ACM has es up to 80 mg EMS/kg/day did not induce a signifi- EHHQUHSRUWHGSUHYLRXVO\ 'REU]\ĔVND0DQLqUH cant increase in MN-RET in bone marrow. In the present et al., 2005). In the present study, ACM induced a sig- study, the Comet assay detected EMS-induced DNA dam- QL¿FDQWGRVHGHSHQGHQWLQFUHDVHLQ'1$GDPDJHLQDOO age in multiple tissues of mice and rats after four daily of the tissues examined in mice (blood leukocytes, liv- exposures of 50 mg/kg/day in the absence of an increased er, duodenum, and gonadal cells). In ACM-treated rats, a frequency of MN-RET. These results indicate that the dose-dependent increase in DNA damage detected by the Comet assay detects EMS-induced DNA lesions at lower Comet assay was observed in thyroid and presumptive levels in these tissues than are required for the MN assay testicular tubule somatic cells, and in blood leukocytes; to detect increases in MN-RET due to bone marrow dam- no DNA damage was seen in liver or presumptive germ age (Table 9; Witt et al., 2008). This difference in sen- cells. The DNA damage seen in testes of mice and rats VLWLYLW\PD\EHGXHWRWKHHI¿FLHQWUHSDLURI³ORZGRVH´ exposed to ACM is consistent with the well-document- EMS lesions prior to the cell division required for the for- ed germ cell genotoxicity and testicular toxicity of ACM mation of a chromosomal aberration detectable as a MN LQURGHQWV 'HDU¿HOG et al., 1988; Yang et al., 2005). A in reticulocytes. Higher EMS exposure levels (> 50 mg/ variable but dose-responsive increase in DNA damage

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Table 8. Comet assay data for rats exposed to VS Number of % Tail DNA Tail Length (Microns) Olive Tail Moment Dose (mg/kg) Animals Mean S.E. Mean S.E. Mean S.E. Blood 0 5 8.2 0.63 15.4 0.64 1.1 0.08 0.00625 5 9.3 0.43 14.7 1.23 1.4 a 0.11 0.01250 5 8.1 1.05 13.6 0.99 1.1 0.17 0.02500 5 9.3 1.56 14.8 1.39 1.4 0.33 0.03125 5 8.7 1.03 14.4 1.29 1.3 0.23 Trend Test b: p = 0.5610 Liver 0 5 31.5 6.79 9.1 0.94 1.6 0.49 0.00625 5 25.6 4.48 8.2 0.62 1.2 0.25 0.01250 5 25.1 4.90 6.9 0.90 1.1 0.28 0.02500 5 34.0 2.57 9.4 0.33 1.8 0.17 0.03125 5 28.2 2.52 8.6 0.47 1.3 0.15 Trend Test b: p = 0.8742 Duodenum 0 5 35.5 2.14 5.9 0.42 1.3 0.07 0.00625 5 36.5 3.38 6.1 0.52 1.4 0.14 0.01250 5 34.2 0.31 6.0 0.20 1.3 0.12 0.02500 5 36.0 5.03 6.0 0.50 1.4 0.17 0.03125 5 37.9 3.31 6.7 0.31 1.4 0.13 Trend Test b: p = 0.6632 Data based on 100 cells scored per animal. a p < 0.05; one-tailed t-test. b /LQHDUUHJUHVVLRQVLJQL¿FDQWDWp < 0.05. was seen in rat duodenum, with a clear increase above sitive to these effects than mice, necessitating the use of the background detected at the top dose of 50 mg/kg/day. lower top doses in rats. These two antitumor drugs operate ACM-induced genetic damage in somatic and germ cells through two distinct modes of action: CP is bioactivated is dependent upon the extent of metabolism of the par- to reactive metabolites that produce a spectrum of DNA ent compound to the genotoxic metabolite glycidamide, lesions resulting in chromosomal breakage and forma- PHGLDWHGE\&<3( 'HDU¿HOG et al.'REU]\ĔVND tion of MN. In contrast, VS-induced chromosomal dam- 2007; Ghanayem et al.0DQLqUH et al., 2005; Witt et age is primarily numerical in nature (chromosome loss) al., 2008). Compared to rats, mice produce higher levels and results from impaired microtubule assembly and sub- of hemoglobin adducts derived from the bioactivation of sequent chromosome malsegregation and loss (Anderson ACM to glycidamide, likely accounting for the more exten- et al., 1995; Cushnir et al., 1990; Eastmond and Tucker, sive distribution of DNA damage observed in ACM-treated 1989). Thus, VS does not induce DNA damage (negative mice compared to rats (Doerge et al., 2005; Ghanayem et al., results in the Comet assay) but does, through non-DNA 2005; Sumner et al., 2003). Consistent with what is known reactive mechanisms, induce aberrant mitoses, result- about metabolism of ACM, MN-RET frequencies were ele- ing in chromosome loss (aneuploidy) and production of vated in mice, but not rats, treated with ACM (Table 9). MN. As predicted by their known modes of action, eval- The antitumor agents CP and VS induced genotoxicity uation of the nature of the CP-induced MN in mice and and bone marrow toxicity in mice and rats based on eval- rats revealed that the MN were primarily due to breakage uations of MN-RET frequencies and percent RET data events and contained chromosomal fragments, while VS- reported previously (Witt et al., 2008); rats were more sen- induced MN were likely due to aneuploidy events, since

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Table 9. Summary of combined MN/Comet assay data in B6C3F1 mice and F344/N rats Administered Dose MN Assay a Comet (pH > 13) Assay b (Mice/Rats) Compound (mg/kg/day in Mice/Rats) (Mice/Rats) Blood Liver Duodenum Other c EMS 50 -/- +/+ +/+ +/- NA 100 +/+ +/+ +/+ +/- NA 200 +/+ +/+ +/+ +/- NA 300 +/+ +/+ +/+ +/+ NA

ACM 12.5 +/- +/- +/- +/+ GSp: +/-; GS: -/+; Tr: + 25.0 +/- +/+ +/- +/+ GSp: +/-; GS: +/+; Tr: + 37.5 +/- +/+ +/- +/- GSp: +/-; GS: +/+; Tr: + 50.0 +/- +/+ +/- +/+ GSp: +/-; GS: +/+; Tr: +

CP 25/2.5 +/+ +/- -/- -/+ NA 50/5.0 +/+ +/- -/- -/+ NA 75/10.0 +/+ +/- -/- -/- NA 100/20.0 +/+ +/+ -/- +/+ NA

VS 0.0125/0.00625 +/+ -/+ -/- -/- NA 0.025/0.0125 +/+ -/- -/- -/- NA 0.050/0.025 +/+ -/- -/- -/- NA 0.075/0.03125 +/+ -/- -/- -/- NA a Data from Witt et al., 2008. b Comet assay: + or - based on Olive Tail Moment. c GSp = gonad sperm cells ; GS = gonad somatic cells ; Tr = Thyroid (rat only) ; NA = not analyzed. they contained larger amounts of chromosomal material, of the genotoxicity of environmental agents of concern. pointing to the presence of whole chromosomes, rather The three-day exposure regimen adopted by the NTP for than fragments (Witt et al., 2008). the in vivo MN assay was extended to include a fourth A dose response for CP-induced DNA damage was GD\RIGRVLQJZLWK¿QDOGRVLQJRFFXUULQJKUSULRUWR detectable in mice by the Comet assay in blood leuko- euthanasia, to meet the sample time requirements of the cytes, and at the top dose in duodenum cells; DNA dam- Comet assay (Tice et al., 1998). With this design, sam- age was not observed in liver cells of CP-treated mice. In ple times for the assessment of these two genotoxicity UDWVVLJQL¿FDQWLQFUHDVHVLQ'1$GDPDJHZHUHREVHUYHG endpoints comply with regulatory requirements stipulat- in duodenum cells and leukocytes at the top dose of CP. ed for the MN assay (OECD 474) and recent recommen- The absence of CP-induced DNA damage in liver of mice dations for the conduct of the Comet assay (Burlinson et DQGUDWVVXJJHVWVHI¿FLHQWGHWR[LFDWLRQRIUHDFWLYHPHWDE- al., 2007). This combined MN/Comet assay protocol per- olites. In the case of VS, there was no indication of DNA mits the evaluation of two distinct genotoxicity endpoints damage in blood leukocytes, liver, or duodenum cells in the same animal, thereby reducing animal usage and under the conditions used in this study. The lack of detect- cost, and providing a basis for integrating genotoxici- able DNA damage using the Comet assay in mice or rats ty endpoints into traditional subacute toxicological stud- administered VS is consistent with microtubulin, rather ies in animals. The MN/Comet assay appears to be a use- than DNA, as a primary cellular target of VS. ful combination of in vivo genotoxicity endpoints for The overall objective of this study was to initiate KD]DUGLGHQWL¿FDWLRQLQSUHFOLQLFDOVDIHW\DVVHVVPHQWDQG development of a database from which to evaluate the the evaluation of environmental agents. Based on these usefulness of integrating the Comet assay into the suba- results, the NTP is presently using this combined proto- cute dosing regimens used by the NTP in the evaluation col as part of its efforts to evaluate the genetic toxicity of

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Combined micronucleus/Comet assay protocol substances of public health concern. rats. Mutat. Res., 580, 131-141. Eastmond, D.A., Hartwig, A., Anderson, D., Anwar, W.A., Cimino, M.C., Dobrev, I., Douglas, G.R., Nohmi, T., Phillips, D.H. and ACKNOWLEDGMENTS Vickers, C. (2009): Mutagenicity testing for chemical risk assess- ment: update of the WHO/IPCS harmonized scheme. Mutagenesis, This work was supported by the National Institute of 24, 341-349. Environmental Health Sciences/National Toxicology Pro- Eastmond, D.A. and Tucker, J.D. (1989): Identification of ane- gram [contract number NO1-ES-35514]. uploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody. Environ. Mol. The authors appreciate the skilled assistance of Cathy ., 13, 34-43. Baldetti and John Winters at ILS, Inc. in conducting (OHVSXUX5.$JDUZDO5$WUDNFKL$+%LJJHU&$+HÀLFK the Comet assay. The authors are grateful to Drs. David R.H., Jagannath, D.R., Levy, D.D., Moore, M.M., Ouyang, Y., DeMarini (US EPA), Daniel Shaughnessy (NIEHS), and 5RELVRQ7:6RWRPD\RU5(&LPLQR0&DQG'HDU¿HOG Raymond Tice (NTP) for helpful comments and useful K.L. (2009): Current and future application of genetic toxicity assays: the role and value of in vitro mammalian assays. Toxicol. discussions during the preparation of this manuscript, and Sci., 109, 172-179. Claudine Gregorio at ILS, Inc. for critical review and edit- Fortini, P., Raspaglio, G., Falchi, M. and Dogliotti, E. (1996): Anal- ing of this manuscript. ysis of DNA alkylation damage and repair in mammalian cells by the Comet assay. Mutagenesis, 11, 169-175. REFERENCES Gedik, C.M. and Collins, A. (2005): Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J., 19, 82-84. 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