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

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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 Comet assay 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 genotoxicity 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]) Vol. 35 No. 2 150 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 clastogens), and CP (a clastogen 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 aneuploidy (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- Vol. 35 No. 2 151 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
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