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Judice SA, Sussman HE, Walker DM, O’Neill JP, Albertini RJ, Walker VE. Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N-ethyl-N-nitrosourea. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2023; 64:432-457. [PMID: 37957787 PMCID: PMC10842105 DOI: 10.1002/em.22579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/06/2023] [Revised: 11/03/2023] [Accepted: 11/10/2023] [Indexed: 11/15/2023]
Abstract
Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.
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Affiliation(s)
- Stephen A. Judice
- Genetic Toxicology Laboratory, University of Vermont, Burlington, Vermont
- EnviroLogix, Portland, Maine
| | - Hillary E. Sussman
- School of Public Health, University at Albany − SUNY, Albany, NY
- Wadsworth Center, New York State Department of Health, Albany, NY
- Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
| | - Dale M. Walker
- Experimental Pathology Laboratories, Sterling, VA
- The Burlington HC Research group, Inc., Jericho, Vermont
| | - J. Patrick O’Neill
- Genetic Toxicology Laboratory, University of Vermont, Burlington, Vermont
| | - Richard J. Albertini
- Genetic Toxicology Laboratory, University of Vermont, Burlington, Vermont
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Vernon E. Walker
- Wadsworth Center, New York State Department of Health, Albany, NY
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
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Revollo JR, Dad A, Pearce MG, Mittelstaedt RA, Casildo A, Lapidus RG, Robison TW, Dobrovolsky VN. CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2020; 61:797-806. [PMID: 32729949 DOI: 10.1002/em.22402] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Revised: 07/09/2020] [Accepted: 07/24/2020] [Indexed: 06/11/2023]
Abstract
Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation.
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Affiliation(s)
- Javier R Revollo
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA
| | - Azra Dad
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA
| | - Mason G Pearce
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA
| | - Roberta A Mittelstaedt
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA
| | - Andrea Casildo
- Greenbaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Rena G Lapidus
- Greenbaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Timothy W Robison
- Division of Pulmonary, Allergy and Critical Care Products, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA
| | - Vasily N Dobrovolsky
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA
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Walker VE, Fennell TR, Walker DM, Bauer MJ, Upton PB, Douglas GR, Swenberg JA. Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile. Chem Res Toxicol 2020; 33:1609-1622. [PMID: 32529823 DOI: 10.1021/acs.chemrestox.0c00153] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.
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Affiliation(s)
- Vernon E Walker
- Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709, United States.,Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, Vermont 05405, United States.,The Burlington HC Research Group, Inc., Jericho, Vermont 05465, United States
| | - Timothy R Fennell
- Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709, United States.,Center for Bioorganic Chemistry, RTI International, Research Triangle Park, North Carolina 27709, United States
| | - Dale M Walker
- The Burlington HC Research Group, Inc., Jericho, Vermont 05465, United States.,Experimental Pathology Laboratories, Sterling, Virginia 20167, United States
| | | | - Patricia B Upton
- Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709, United States.,Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
| | - George R Douglas
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario K1A 0K9, Canada
| | - James A Swenberg
- Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709, United States.,Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
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Walker VE, Walker DM, Ghanayem BI, Douglas GR. Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile. Chem Res Toxicol 2020; 33:1623-1632. [PMID: 32529832 DOI: 10.1021/acs.chemrestox.0c00154] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.
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Affiliation(s)
- Vernon E Walker
- Wadsworth Center, New York State Department of Health, Albany, New York 12201, United States.,Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, Vermont 05405, United States.,The Burlington HC Research Group, Inc., Jericho, Vermont 05465, United States
| | - Dale M Walker
- The Burlington HC Research Group, Inc., Jericho, Vermont 05465, United States.,Experimental Pathology Laboratories, Sterling, Virginia 20167, United States
| | - Burhan I Ghanayem
- National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, United States
| | - George R Douglas
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario K1A 0K9, Canada
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5
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Knockdown of Rab7a suppresses the proliferation, migration, and xenograft tumor growth of breast cancer cells. Biosci Rep 2019; 39:BSR20180480. [PMID: 29769411 PMCID: PMC6361774 DOI: 10.1042/bsr20180480] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2018] [Revised: 03/28/2018] [Accepted: 05/11/2018] [Indexed: 01/06/2023] Open
Abstract
Breast cancer is a common invasive cancer in women. Ras-related protein Rab-7a (Rab7a) is involved in late endocytic trafficking, while its role in breast cancer is largely unclear. In the present study, we investigated the role of Rab7a in breast cancer. Comparing with adjacent breast tissues, Rab7a expression was increased in breast cancer tissues. Using lentivirus-mediated knockdown strategy, we found that Rab7a silencing inhibited the proliferation and colony formation of MDA-MB-231 cells. Apoptosis and G2 cell cycle arrest were induced in Rab7a knockdown. By contrast, Rab7a suppressed the apoptosis and promoted proliferation and colony formation of MCF-7 cells. The migration of MDA-MB-231 cells was suppressed by Rab7a knockdown. In vivo, depletion of Rab7a inhibited the xenograft tumor development of MDA-MB-231 cells. Altogether, our results highlight the novel function of Rab7a in the proliferation, invasion, and xenograft tumor development of breast cancer cells.
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Udroiu I, Sgura A. Genotoxic sensitivity of the developing hematopoietic system. MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH 2015; 767:1-7. [PMID: 27036061 DOI: 10.1016/j.mrrev.2015.12.001] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/06/2015] [Revised: 12/02/2015] [Accepted: 12/07/2015] [Indexed: 12/17/2022]
Abstract
Genotoxic sensitivity seems to vary during ontogenetic development. Animal studies have shown that the spontaneous mutation rate is higher during pregnancy and infancy than in adulthood. Human and animal studies have found higher levels of DNA damage and mutations induced by mutagens in fetuses/newborns than in adults. This greater susceptibility could be due to reduced DNA repair capacity. In fact, several studies indicated that some DNA repair pathways seem to be deficient during ontogenesis. This has been demonstrated also in murine hematopoietic stem cells. Genotoxicity in the hematopoietic system has been widely studied for several reasons: it is easy to assess, deals with populations cycling also in the adults and may be relevant for leukemogenesis. Reviewing the literature concerning the application of the micronucleus test (a validated assay to assess genotoxicity) in fetus/newborns and adults, we found that the former show almost always higher values than the latter, both in animals treated with genotoxic substances and in those untreated. Therefore, we draw the conclusion that the genotoxic sensitivity of the hematopoietic system is more pronounced during fetal life and decreases during ontogenic development.
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Affiliation(s)
- Ion Udroiu
- Dipartimento di Scienze, Università Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy.
| | - Antonella Sgura
- Dipartimento di Scienze, Università Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy
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Gollapudi BB, Lynch AM, Heflich RH, Dertinger SD, Dobrovolsky VN, Froetschl R, Horibata K, Kenyon MO, Kimoto T, Lovell DP, Stankowski LF, White PA, Witt KL, Tanir JY. The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2015; 783:23-35. [DOI: 10.1016/j.mrgentox.2014.09.007] [Citation(s) in RCA: 126] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/13/2014] [Accepted: 09/15/2014] [Indexed: 12/01/2022]
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8
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Bhalli JA, Ding W, Shaddock JG, Pearce MG, Dobrovolsky VN, Heflich RH. Evaluating the weak in vivo micronucleus response of a genotoxic carcinogen, Aristolochic acids. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2013; 753:82-92. [DOI: 10.1016/j.mrgentox.2013.03.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/21/2012] [Revised: 03/01/2013] [Accepted: 03/03/2013] [Indexed: 02/08/2023]
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Bajpayee M, Pandey AK, Parmar D, Dhawan A. Current Status of Short-Term Tests for Evaluation of Genotoxicity, Mutagenicity, and Carcinogenicity of Environmental Chemicals and NCEs. Toxicol Mech Methods 2012; 15:155-80. [PMID: 20021080 DOI: 10.1080/15376520590945667] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
The advent of the industrial revolution has seen a significant increase in the number of new chemical entities (NCEs) released in the environment. It becomes imperative to check the toxic potential of NCEs to nontarget species before they are released for commercial purposes because some of these may exert genotoxicity, mutagenicity, or carcinogenicity. Exposure to such compounds produces chemical changes in DNA, which are generally repaired by the DNA repair enzymes. However, DNA damage and its fixation may occur in the form of gene mutations, chromosomal damage, and numerical chromosomal changes and recombination. This may affect the incidence of heritable mutations in man and may be transferred to the progeny or lead to the development of cancer. Hence, adequate tests on NCEs have to be undertaken for the risk assessment and hazard prediction. Compounds that are positive in tests that detect such damages have the potential to be human mutagens/carcinogens. Only long-term animal bioassays, involving lifetime studies on animals, were used earlier to classify substances as mutagens/carcinogens. These tests were cumbersome and time consuming and required a lot of facilities and personnel. Short-term tests, therefore, were brought into practice. A "battery" of three to four of these short-term tests has been proposed now by a number of regulatory authorities for the classification of compounds as mutagenic or carcinogenic. This review deals with the current status of these short-term tests.
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Affiliation(s)
- Mahima Bajpayee
- Developmental Toxicology Division, Industrial Toxicology Research Center, M.G. Marg, LucknowIndia
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10
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McDaniel LP, Ding W, Dobrovolsky VN, Shaddock JG, Mittelstaedt RA, Doerge DR, Heflich RH. Genotoxicity of furan in Big Blue rats. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2012; 742:72-8. [DOI: 10.1016/j.mrgentox.2011.12.011] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/27/2011] [Revised: 12/09/2011] [Accepted: 12/10/2011] [Indexed: 01/01/2023]
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Bhalli JA, Shaddock JG, Pearce MG, Dobrovolsky VN, Cao X, Heflich RH, Vohr HW. Report on stage III Pig-a mutation assays using benzo[a]pyrene. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2011; 52:731-737. [PMID: 22052432 DOI: 10.1002/em.20675] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/08/2011] [Revised: 07/15/2011] [Accepted: 07/15/2011] [Indexed: 05/31/2023]
Abstract
Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S. FDA-NCTR and Bayer-Germany. Starting on Day 1, groups of five 6- to 7-week-old male Fischer 344 (F344, used at FDA-NCTR) and Han Wistar rats (Bayer) were given 28 daily doses of 0, 37.5, 75, or 150 mg/kg BaP; blood was sampled on Days -1, 4, 15, 29, and 56. Pig-a mutant frequencies were determined on Days -1, 15, 29, and 56 in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies; percent micronucleated-RETs (%MN-RET) were measured on Days 4 and 29. RBC(CD59-) and RET(CD59-) frequencies increased in a dose- and time-dependent manner, producing significant increases by Day 29 in both rat models. The responses for RETs were stronger than those for RBCs, and the responses in F344 rats were stronger than in Han Wistar rats. BaP also produced significant increases in %MN-RET frequency at Days 4 and 29, with the responses being greater in F344 than Han Wistar rats. The overall findings were consistent with those of the reference laboratory using Han Wistar rats. Finally, mutation assays performed on splenocytes from Day 56 F344 rats indicated that BaP mutant frequencies were three to fivefold higher for the Hprt gene than the Pig-a gene. The results indicate that the Pig-a RET and RBC assays are reproducible, transferable, and show promise for integrating gene mutation into 28-day repeat-dose studies.
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Affiliation(s)
- Javed A Bhalli
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079, USA
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12
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Albertini RJ, Judice SA, Recio L, Walker VE. Hprt mutant frequency and p53 gene status in mice chronically exposed by inhalation to benzene. Chem Biol Interact 2010; 184:77-85. [DOI: 10.1016/j.cbi.2009.12.019] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2009] [Revised: 12/11/2009] [Accepted: 12/16/2009] [Indexed: 11/30/2022]
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13
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Accumulation and persistence of Pig-A mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl-N-nitrosourea. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2009; 677:86-92. [DOI: 10.1016/j.mrgentox.2009.05.014] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/18/2009] [Revised: 05/21/2009] [Accepted: 05/22/2009] [Indexed: 11/20/2022]
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14
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Von Tungeln LS, Churchwell MI, Doerge DR, Shaddock JG, McGarrity LJ, Heflich RH, Gamboa da Costa G, Marques MM, Beland FA. DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tkmice treated neonatally with acrylamide or glycidamide. Int J Cancer 2009; 124:2006-15. [DOI: 10.1002/ijc.24165] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Jones IM, Burkhart-Schultz K, Strout CL, Nelson DO. Studies of thioguanine-resistant lymphocytes induced by in vivo irradiation of mice. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2008; 49:343-350. [PMID: 18418873 DOI: 10.1002/em.20388] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/26/2023]
Abstract
The frequency of Hprt-deficient lymphocytes in mice after in vivo gamma irradiation, has been found to vary as a function of time elapsed after exposure and irradiation dose. The frequency of mutant lymphocytes in spleen was determined using an in vitro, clonogenic assay for thioguanine-resistant T-lymphocytes. Mice were exposed to single doses of 0-400 cGy from cesium-137 or to eight daily doses of 50 cGy. The time to maximum-induced mutant frequency was 3 weeks. The dose response was strikingly curvilinear at 3-5 weeks after irradiation, but less precisely defined for 10-53 weeks after exposure, being fit by either linear or quadratic dependence. Three weeks after eight daily 50 cGy exposures, mutant frequency was elevated above controls and mice exposed to 50 cGy (which were not distinct from the nonirradiated controls), but only 17% in that of mice given a single 400 cGy fraction. This fractionation effect and the curvilinearity of the early dose-response curve suggested that saturation of repair increased the yield of mutations at higher acute doses. The decline of spleen mutant frequency in mice observed between 5 and 10 weeks after irradiation may reflect selection against some mutants. The marked variation of mutant frequency, as a function of time after irradiation and of dose rate, emphasize the need to evaluate these variables carefully and consistently in future studies.
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Affiliation(s)
- Irene M Jones
- Chemistry, Materials, Earth and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California, USA.
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16
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Dobrovolsky VN, Shaddock JG, Mittelstaedt RA, Bishop ME, Lewis SM, Lee FW, Aidoo A, Leakey JEA, Dunnick JK, Heflich RH. Frequency of Hprt mutant lymphocytes and micronucleated erythrocytes in p53-haplodeficient mice treated perinatally with AZT and AZT in combination with 3TC. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2007; 48:270-82. [PMID: 17358030 DOI: 10.1002/em.20280] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
Azidothymidine (AZT) is a nucleoside reverse transcriptase inhibitor (NRTI) that is used for reducing mother-to-child transmission of human immunodeficiency virus I. Combinations of AZT and 3'-thiacytidine (3TC) are even more effective than AZT alone. AZT, however, is a mutagen and carcinogen in rodent models and 3TC can increase the genotoxicity of AZT. Since p53 plays a key role in human and mouse tumorigenesis, p53-haplodeficient mice are currently being evaluated as a model for assessing the carcinogenicity of perinatal exposure to NRTIs. In the present study, male C57BL/6 p53(+/+) and p53(-/-) mice were mated with C3H p53(+/+) females; the pregnant females were treated on gestation day 12 through parturition with 40, 80, and 160 mg/kg of AZT or a combination of 160 mg/kg AZT and 100 mg/kg 3TC (AZT-3TC); the p53(+/+) and p53(+/-) offspring were treated daily after birth through postnatal day (PND) 28. The frequencies of micronucleated reticulocytes (MN-RETs) and micronucleated normochromatic erythrocytes (MN-NCEs) were determined on PND1, PND10, and PND28; the frequency of Hprt mutant lymphocytes was measured on PND28. The frequencies of MN-RETs and MN-NCEs were increased in treated animals at all time points; there were no differences in the responses of p53(+/+) and p53(+/-) animals treated with identical doses of NRTIs. After correction for clonal expansion, both AZT and AZT-3TC treatments induced small but significant increases in the frequency of Hprt mutant lymphocytes in p53(+/-) mice, but not in p53(+/+) mice. The data indicate that p53 haplodeficiency affects the genotoxicity of NRTIs; thus, p53(+/-) mice may be a sensitive model for evaluating the carcinogenicity of perinatal exposure to NRTIs.
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Affiliation(s)
- Vasily N Dobrovolsky
- US Food and Drug Administration, Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, Arkansas, USA
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Torres SM, Walker DM, Carter MM, Cook DL, McCash CL, Cordova EM, Olivero OA, Poirier MC, Walker VE. Mutagenicity of zidovudine, lamivudine, and abacavir following in vitro exposure of human lymphoblastoid cells or in utero exposure of CD-1 mice to single agents or drug combinations. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2007; 48:224-38. [PMID: 17358033 DOI: 10.1002/em.20264] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
Experiments were performed to investigate the impact of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using cell cloning assays for assessing the effects of individual drugs/drug combinations in (1) TK6 human lymphoblastoid cells exposed in vitro and (2) splenic lymphocytes from male CD-1 mice exposed transplacentally on days 12-18 of gestation. In TK6 cells, dose-related increases in HPRT and TK mutant frequencies were found following 3 days of exposure to AZT or 3TC alone (33, 100, or 300 microM), or to equimolar amounts of AZT-3TC. Compared with single drug exposures, AZT-3TC coexposures generally yielded enhanced elevations in HPRT and TK mutant frequencies. Mutagenicity experiments with ABC alone, or in combination with AZT-3TC, were complicated by the extreme cytotoxicity of ABC. Exposure of cells either to relatively high levels of AZT-3TC short-term (100 microM, 3 days), or to peak plasma-equivalent levels of AZT-3TC for an extended period (10 microM, 30 days), resulted in similar drug-induced mutagenic responses. Among sets of mice necropsied on days 13, 15, or 21 postpartum, Hprt mutant frequencies in T-cells were significantly elevated in the AZT-only (200 mg/kg bw/day) and AZT-3TC (200 mg AZT + 100 mg 3TC/kg bw/day) groups at 13 days of age. These results suggest that the mutagenicity by these nucleoside analogs is driven by cumulative dose, and raises the question of whether AZT-3TC has greater mutagenic effects than AZT alone in perinatally exposed children.
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Affiliation(s)
- Salina M Torres
- Lovelace Respiratory Research Institute, Albuquerque, New Mexico, USA
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Meng Q, Walker DM, McDonald JD, Henderson RF, Carter MM, Cook DL, McCash CL, Torres SM, Bauer MJ, Seilkop SK, Upton PB, Georgieva NI, Boysen G, Swenberg JA, Walker VE. Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene. Chem Biol Interact 2007; 166:121-31. [PMID: 16945358 DOI: 10.1016/j.cbi.2006.07.005] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2005] [Revised: 07/18/2006] [Accepted: 07/20/2006] [Indexed: 10/24/2022]
Abstract
Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.
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Affiliation(s)
- Quanxin Meng
- Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr. SE, Albuquerque, NM 87108, USA
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Lambert IB, Singer TM, Boucher SE, Douglas GR. Detailed review of transgenic rodent mutation assays. Mutat Res 2005; 590:1-280. [PMID: 16081315 DOI: 10.1016/j.mrrev.2005.04.002] [Citation(s) in RCA: 259] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2005] [Revised: 04/04/2005] [Accepted: 04/12/2005] [Indexed: 11/17/2022]
Abstract
Induced chromosomal and gene mutations play a role in carcinogenesis and may be involved in the production of birth defects and other disease conditions. While it is widely accepted that in vivo mutation assays are more relevant to the human condition than are in vitro assays, our ability to evaluate mutagenesis in vivo in a broad range of tissues has historically been quite limited. The development of transgenic rodent (TGR) mutation models has given us the ability to detect, quantify, and sequence mutations in a range of somatic and germ cells. This document provides a comprehensive review of the TGR mutation assay literature and assesses the potential use of these assays in a regulatory context. The information is arranged as follows. (1) TGR mutagenicity models and their use for the analysis of gene and chromosomal mutation are fully described. (2) The principles underlying current OECD tests for the assessment of genotoxicity in vitro and in vivo, and also nontransgenic assays available for assessment of gene mutation, are described. (3) All available information pertaining to the conduct of TGR assays and important parameters of assay performance have been tabulated and analyzed. (4) The performance of TGR assays, both in isolation and as part of a battery of in vitro and in vivo short-term genotoxicity tests, in predicting carcinogenicity is described. (5) Recommendations are made regarding the experimental parameters for TGR assays, and the use of TGR assays in a regulatory context.
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Affiliation(s)
- Iain B Lambert
- Mutagenesis Section, Environmental Health Sciences Bureau, Healthy Environments and Consumer Safety Branch, 0803A, Health Canada, Ottawa, Ont., Canada K1A 0L2.
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20
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Walker DM, Seilkop SK, Scott BR, Walker VE. Hprt mutant frequencies in splenic T-cells of male F344 rats exposed by inhalation to propylene. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2004; 43:265-272. [PMID: 15141366 DOI: 10.1002/em.20020] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Propylene is a major industrial intermediate and atmospheric pollutant to which humans are exposed by inhalation. In this study, 6-week-old male F344 rates were exposed to 0, 200, 2000, or 10,000 ppm propylene by inhalation for 4 weeks (6 h/day, 5 days/week), and mutant frequencies were determined in the Hprt gene of splenic T-lymphocytes. Twenty milligrams of cyclophosphamide monohydrate (CPP)/kg bw, given on the penultimate day of propylene exposure, was used as a positive control mutagen. Rats (n = 8/group) were necropsied for isolation of T-cells 8 weeks after the last dose, a sampling time that produced peak spleen Hprt mutant frequencies (Mfs) in a preliminary mutant manifestation study using CCP treatment. Hprt Mfs were measured via the T-cell cloning assay, which was performed without knowledge of the animal treatment groups. Mean Hprt Mfs were significantly increased over control values (mean Mf = 5.24 +/- 1.55 (SD) x 10(-6)) in CPP-treated rats (10.37 +/- 4.30 x 10(-6), P = 0.007). However, Hprt Mfs in propylene-exposed rats were not significantly increased over background, with mean Mfs of 4.90 +/- 1.84 x 10(-6) (P = 0.152), 5.05 +/- 3.70 x 10(-6) (P = 0.895), and 5.95 +/- 2.49 x10(-6) (P = 0.500) for animals exposed to 200, 2000, or 10,000 ppm propylene, respectively. No significant increase in F344 rat or B6C3F1 mouse cancer incidence was reported in the National Toxicology Program carcinogenicity studies of propylene across this same exposure range. Taken together, these findings support the conclusion that inhalation exposure of rats to propylene does not cause mutations or cancer.
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Affiliation(s)
- Dale M Walker
- Lovelace Respiratory Research Institute, Albuquerque, New Mexico, USA
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Liu SX, Cao J, An H, Shun HM, Yang LJ, Liu Y. Analysis of spontaneous, gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR. World J Gastroenterol 2003; 9:578-83. [PMID: 12632522 PMCID: PMC4621586 DOI: 10.3748/wjg.v9.i3.578] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and 60Co γ-rays.
METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and 60Co γ-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique.
RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200 μg/mL, P < 0.01; in the group with dosage of 60Co γ-ray with 2-4 Gy, P < 0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 μg/mL, P < 0.05; in the group of 60Co γ-ray with 1-4 Gy, P < 0.05) significantly. In the 13 spontaneous mutants analyzed, 92.3% of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7%, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7% of ENU-induced mutations (62.5%-89.4%, P < 0.01) and in 61.7% of gamma-ray-induced mutations (28.6%-76.5%, P < 0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9% in gamma-ray-induced mutants, 88.1% in ENU-induced mutants).
CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or 60Co γ-ray. Although both ENU and γ-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.
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Affiliation(s)
- Sheng-Xue Liu
- Department of Healath Toxicology, Preventive Medical College, Third Military Medical University, Chongqing 400038, China
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Abstract
Historically, fish have played significant roles in assessing potential risks associated with exposure to chemical contamination in aquatic environments. Considering the contributions of transgenic rodent models to biomedicine, it is reasoned that the development of transgenic fish could enhance the role of fish in environmental toxicology. Application of transgenic fish in environmental studies remains at an early stage, but recent introduction of new models and methods demonstrates progress. Rapid advances are most evident in the area of in vivo mutagenesis using fish carrying transgenes that serve as recoverable mutational targets. These models highlight many advantages afforded by fish as models and illustrate important issues that apply broadly to transgenic fish in environmental toxicology. Development of fish models carrying identical transgenes to those found in rodents is beneficial and has revealed that numerous aspects of in vivo mutagenesis are similar between the two classes of vertebrates. Researchers have revealed that fish exhibit frequencies of spontaneous mutations similar to rodents and respond to mutagen exposure consistent with known mutagenic mechanisms. Results have demonstrated the feasibility of in vivo mutation analyses using transgenic fish and have illustrated their potential value as a comparative animal model. Challenges to development and application of transgenic fish relate to the needs for improved efficiencies in transgenic technology and in aspects of fish husbandry and use. By taking advantage of the valuable and unique attributes of fish as test organisms, it is anticipated that transgenic fish will make significant contributions to studies of environmentally induced diseases.
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Affiliation(s)
- R N Winn
- Aquatic Biotechnology and Environmental Laboratory, Warnell School of Forest Resources, University of Georgia, Athens, Georgia, USA
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Meng Q, Henderson RF, Long L, Blair L, Walker DM, Upton PB, Swenberg JA, Walker VE. Mutagenicity at the Hprt locus in T cells of female mice following inhalation exposures to low levels of 1,3-butadiene. Chem Biol Interact 2001; 135-136:343-61. [PMID: 11397400 DOI: 10.1016/s0009-2797(01)00222-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
A study was conducted to test the hypothesis that repeated low level exposures to 1,3-butadiene (BD), approaching the OSHA occupational threshold for this chemical, produce a significant mutagenic response in mice. Female B6C3F1 mice (4-5 weeks of age) were exposed by inhalation for 2 weeks (6 h/day, 5 days/week) to 0 or 3 ppm BD, and then necropsied at 4 weeks after the cessation of exposures to measure the frequency of mutations (MF) at the Hprt locus using the T-lymphocyte clonal assay. At necropsy, T cells were isolated from spleen and cultured in the presence of mitogen, growth factors, and a selection agent. Cells were scored for growth on days 8-9 after plating to determine cloning efficiencies (CEs) and Hprt MFs. There was a marginal but significant reduction in the growth of splenic T cells from mice exposed to 3 ppm (n=27) compared with control mice (n=24) (P=0.004), suggesting the occurrence of BD-induced cytotoxicity at this low exposure concentration. In addition, the average Hprt MF in mice exposed to 3 ppm BD [1.54+/-0.82 (S.D.)x10(-6)] was significantly increased by 1.6-fold over the average control value of 0.96+/-0.51 (S.D.)x10(-6) (P=0.004). Comparisons of these data to earlier Hprt mutagenicity studies of mice exposed to high concentrations of BD (where significant mutagenic but not cytotoxic effects were observed) indicate that the ability to detect the cytotoxic and mutagenic responses of T cells to low levels of BD was enhanced by using a much larger sample size than usual for both the control and treatment groups. Additional analyses of the quantitative relationships between CE and MF demonstrated that CE had no significant effect upon MF values in sham-exposed control mice or mice exposed to low-level BD. Furthermore, the approaches for assessing the impact of CE and clonality on Hprt MFs in these control and BD-exposed mice were applied with the same rigor as in in vivo Hprt mutagenicity studies in human children. The overall study results support the conclusion that short-term low-level BD exposure is mutagenic in the mouse.
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Affiliation(s)
- Q Meng
- Wadsworth Center for Laboratories and Research, New York State Department of Health, P.O. Box 509, Empire State Plaza, Albany, NY 12201-0509, USA
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Sussman HE, Bauer MJ, Shi X, Judice SA, Albertini RJ, Walker VE. Transplacental mutagenicity of N-ethyl-N-nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 mice. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2001; 38:30-37. [PMID: 11473385 DOI: 10.1002/em.1047] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13-16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 +/- 0.3 (SE) x 10(-6). In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 +/- 15.8 (SE) x 10(-6)] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 +/- 16.3 (SE) x 10(-6)] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 x 10(3), respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life.
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Affiliation(s)
- H E Sussman
- Wadsworth Center, New York State Department of Health, Albany, New York,, USA
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Chen T, Mittelstaedt RA, Aidoo A, Hamilton LP, Beland FA, Casciano DA, Heflich RH. Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2001; 37:195-202. [PMID: 11317337 DOI: 10.1002/em.1028] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.
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Affiliation(s)
- T Chen
- Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
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Winn RN, Norris MB, Brayer KJ, Torres C, Muller SL. Detection of mutations in transgenic fish carrying a bacteriophage lambda cII transgene target. Proc Natl Acad Sci U S A 2000; 97:12655-60. [PMID: 11035814 PMCID: PMC18819 DOI: 10.1073/pnas.220428097] [Citation(s) in RCA: 68] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage lambda vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for lambda transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by lambda in vitro packaging. After infecting and plating phage on a hfl- bacterial host, cII mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on lambda transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 x 10(-5) in liver, 2.9 x 10(-5) in whole fish, to 1.8 x 10(-5) in testes, were comparable to ranges in lambda transgenic rodents. Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action. Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7- and 6. 7-fold above background at 15, 20, and 30 days, respectively. Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment. Spontaneous and induced mutational spectra in the fish were also consistent with those of lambda transgenic rodent models. Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models.
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Affiliation(s)
- R N Winn
- Warnell School of Forest Resources, University of Georgia, Athens, GA 30602, USA.
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27
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Walker VE, Wu KY, Upton PB, Ranasinghe A, Scheller N, Cho MH, Vergnes JS, Skopek TR, Swenberg JA. Biomarkers of exposure and effect as indicators of potential carcinogenic risk arising from in vivo metabolism of ethylene to ethylene oxide. Carcinogenesis 2000; 21:1661-9. [PMID: 10964097 DOI: 10.1093/carcin/21.9.1661] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
The purposes of the present study were: (i) to investigate the potential use of several biomarkers as quantitative indicators of the in vivo conversion of ethylene (ET) to ethylene oxide (EO); (ii) to produce molecular dosimetry data that might improve assessment of human risk from exogenous ET exposures. Groups (n = 7/group) of male F344 rats and B6C3F1 mice were exposed by inhalation to 0 and 3000 p. p.m. ET for 1, 2 or 4 weeks (6 h/day, 5 days/week) or to 0, 40, 1000 and 3000 p.p.m. ET for 4 weeks. N:-(2-hydroxyethyl)valine (HEV), N:7-(2-hydroxyethyl) guanine (N7-HEG) and HPRT: mutant frequencies were assessed as potential biomarkers for determining the molecular dose of EO resulting from exogenous ET exposures of rats and mice, compared with background biomarker values. N7-HEG was quantified by gas chromatography coupled with high resolution mass spectrometry (GC-HRMS), HEV was determined by Edman degradation and GC-HRMS and HPRT: mutant frequencies were measured by the T cell cloning assay. N7-HEG accumulated in DNA with repeated exposure of rodents to 3000 p.p.m. ET, reaching steady-state concentrations around 1 week of exposure in most tissues evaluated (brain, liver, lung and spleen). The dose-response curves for N7-HEG and HEV were supralinear in exposed rats and mice, indicating that metabolic activation of ET was saturated at exposures >/=1000 p.p.m. ET. Exposures of mice and rats to 200 p.p.m. EO for 4 weeks (as positive treatment controls) led to significant increases in HPRT: mutant frequencies over background in splenic T cells from exposed rats and mice, however, no significant mutagenic response was observed in the HPRT: gene of ET-exposed animals. Comparisons between the biomarker data for both unexposed and ET-exposed animals, the dose-response curves for the same biomarkers in EO-exposed rats and mice and the results of the rodent carcinogenicity studies of ET and EO suggest that too little EO arises from exogenous ET exposure to produce a significant mutagenic response or a carcinogenic response under standard bioassay conditions.
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Affiliation(s)
- V E Walker
- Department of Pathology and Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA.
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Meng Q, Singh N, Heflich RH, Bauer MJ, Walker VE. Comparison of the mutations at Hprt exon 3 of T-lymphocytes from B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene or the racemic mixture of 1,2:3,4-diepoxybutane. Mutat Res 2000; 464:169-84. [PMID: 10648904 DOI: 10.1016/s1383-5718(99)00157-6] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Experiments were conducted to define the spectra of mutations occurring in Hprt exon 3 of T-cells isolated from spleens of female B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene (BD) or its reactive metabolite, (+/-)-diepoxybutane (DEB). Hprt mutant frequencies (Mfs) in BD-exposed (1250 ppm for 2 weeks or 625 ppm for 4 weeks; 6 h/day, 5 days/week) and DEB-exposed (2 or 4 ppm for 4 weeks or 5 ppm for 6 weeks; 6 h/day, 5 days/week) mice and rats were significantly increased over concurrent control values. Mutant T-cell colonies from control and treated animals were screened for mutations in Hprt exon 3 using PCR amplification of genomic DNA and denaturing gradient gel electrophoresis, followed by sequence analysis. Exon 3 mutations were found at the following frequencies: 20/394 (5%) in control mice, 56/712 (8%) in BD-exposed mice, 59/1178 (5%) in BD-exposed rats, 66/642 (10%) in DEB-exposed mice, and 51/732 (7%) in DEB-exposed rats. Mutations in exposed animals included base substitutions, small deletions (1 to 74 bp), and small insertions (1 to 8 bp), with base substitutions predominating. Among the types of base substitutions observed in mice, the proportions of G.C-->A.T transitions (p=0.035, Fisher's Exact Test) and G.C-->C.G transversions (p=0.05) were significantly different in control vs. BD-exposed animals. Given the small number of exon 3 mutants analyzed, there was a high degree of overlap in the mutational spectra between BD-exposed mice and rats, between BD- and DEB-exposed mice, and between BD- and DEB-exposed rats in terms of the sites with base substitutions, the mutations found at those mutated sites, the relative occurrence of the most frequently observed base substitutions, and the occurrence of a consistent strand bias for the most frequently observed base substitutions. The spectra data suggest that adduction of both G.C and A.T bps is important in the induction of in vivo mutations by BD metabolites in exposed mice and rats.
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Affiliation(s)
- Q Meng
- Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY, USA
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Tates AD, van Dam FJ, Natarajan AT, van Teylingen CM, de Zwart FA, Zwinderman AH, van Sittert NJ, Nilsen A, Nilsen OG, Zahlsen K, Magnusson AL, Törnqvist M. Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide. Mutat Res 1999; 431:397-415. [PMID: 10636004 DOI: 10.1016/s0027-5107(99)00182-7] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.
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Affiliation(s)
- A D Tates
- Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, Netherlands.
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