49 results on '"mutation assay"'
Search Results
2. Testing of acetaminophen in support of the international multilaboratory in vivo rat Pig‐a assay validation trial.
- Author
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van der Leede, Bas‐jan, Weiner, Sandy, Van Doninck, Terry, De Vlieger, Kathleen, Schuermans, Ann, Tekle, Fetene, Geys, Helena, van Heerden, Marjolein, De Jonghe, Sandra, and Van Gompel, Jacky
- Subjects
GENETIC toxicology ,SPRAGUE Dawley rats ,ERYTHROCYTES ,LIVER cells ,RETICULOCYTES ,BLOOD cells ,DNA damage - Abstract
Acetaminophen, a nonmutagenic compound as previously concluded from bacteria, in vitro mammalian cell, and in vivo transgenic rat assays, presented a good profile as a nonmutagenic reference compound for use in the international multilaboratory Pig‐a assay validation. Acetaminophen was administered at 250, 500, 1,000, and 2,000 mg·kg−1·day−1 to male Sprague Dawley rats once daily in 3 studies (3 days, 2 weeks, and 1 month with a 1‐month recovery group). The 3‐Day and 1‐Month Studies included assessments of the micronucleus endpoint in peripheral blood erythrocytes and the comet endpoint in liver cells and peripheral blood cells in addition to the Pig‐a assay; appropriate positive controls were included for each assay. Within these studies, potential toxicity of acetaminophen was evaluated and confirmed by inclusion of liver damage biomarkers and histopathology. Blood was sampled pre‐treatment and at multiple time points up to Day 57. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as CD59‐negative RBC and CD59‐negative RET frequencies, respectively. No increases in DNA damage as indicated through Pig‐a, micronucleus, or comet endpoints were seen in treated rats. All positive controls responded as appropriate. Data from this series of studies demonstrate that acetaminophen is not mutagenic in the rat Pig‐a model. These data are consistent with multiple studies in other nonclinical models, which have shown that acetaminophen is not mutagenic. At 1,000 mg·kg−1·day−1, Cmax values of acetaminophen on Day 28 were 153,600 ng/ml and 131,500 ng/ml after single and repeat dosing, respectively, which were multiples over that of clinical therapeutic exposures (2.6–6.1 fold for single doses of 4,000 mg and 1,000 mg, respectively, and 11.5 fold for multiple dose of 4,000 mg) (FDA 2002). Data generated were of high quality and valid for contribution to the international multilaboratory validation of the in vivo Rat Pig‐a Mutation Assay. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
3. EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
- Author
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Marta Rodriguez-Alvarez, Daria Kim, and Andriy Khobta
- Subjects
DNA damage ,DNA damage tolerance ,damage bypass ,translesion synthesis (TLS) ,transcriptional mutagenesis ,mutation assay ,Microbiology ,QR1-502 - Abstract
The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress; however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers; whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed “transcriptional mutagenesis” (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system.
- Published
- 2020
- Full Text
- View/download PDF
4. Mutagenesis of Human p53 Tumor Suppressor Gene Sequences in Embryonic Fibroblasts of Genetically-Enginered Mice
- Author
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Liu, Zhipei, Belharazem, Djeda, Muehlbauer, Karl Rudolf, Nedelko, Tatiana, Knyazev, Yuri, Hollstein, Monica, and Setlow, Jane K., editor
- Published
- 2007
- Full Text
- View/download PDF
5. Genotypic Selection of Age-Related DNA Rearrangements by PCR
- Author
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Wong, Alice, Cortopassi, Gino, Sternberg, Hal, editor, and Timiras, Paola S., editor
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- 1999
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6. Use of Transgenic Mutational Test Systems in Risk Assessment of Carcinogens
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Schmezer, Peter, Eckert, Claudia, Liegibel, Ute M., Klein, Reinhold G., Bartsch, Helmut, Seiler, Jürg P., editor, Autrup, Judith L., editor, and Autrup, Herman, editor
- Published
- 1998
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7. Quantifying In Vivo Somatic Mutations Using Transgenic Mouse Model Systems.
- Author
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Swiger, Roy R.
- Abstract
This chapter describes the use of the bacteriophage
cII positive selection somatic mutational assay with the Muta⠪Mouse transgenic model system. The assay is similar to others involving a transgenic target, including thecII andlacI assays in the Big Blue® Mouse,lacZ in the MutaMouse, and thegpt delta assay. Briefly, high-molecular-weight DNA is purified from the tissue of interest and used as substrate during in vitro packaging reactions, where the λ transgenes are excised from the genome and assembled into viable phage. Phage containing the mutational targets is then adsorbed into an appropriate bacterial host, and mutations sustained in vivo are detected and quantified by either standard recombinant screening or selection assays. Mutant frequencies are reported as the ratio of mutant phage to total phage units analyzed. The λ-based transgenic mouse assays are used to study and characterize in vivo mutagenesis as well as for mutagenicity assessment of chemicals and other agents. These models permit the enumeration of mutations sustained in virtually any tissue of the mouse and are both sensitive and robust. Application of the assays is simple, not requiring resources beyond those commonly found in most academic laboratories. [ABSTRACT FROM AUTHOR]- Published
- 2014
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8. umuC-Independent, recA-Dependent Mutagenesis
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Smith, Kendric C., Sargentini, Neil J., and Riklis, Emanuel, editor
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- 1991
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9. Human Exposure to Airborne Mutagens Indoors and Outdoors Using Mutagenesis and Chemical Analysis Methods
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Matsushita, Hidetsuru, Goto, Sumio, Takagi, Yukihiko, Endo, Osamu, Tanabe, Kiyoshi, Rosenkranz, Herbert S., editor, Waters, Michael D., editor, Daniel, F. Bernard, editor, Lewtas, Joellen, editor, Moore, Martha M., editor, and Nesnow, Stephen, editor
- Published
- 1990
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10. Use of Mutations in Bacteria as Indicators of Carcinogenic Potential
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Callander, R. D., Cooper, Colin S., editor, and Grover, Philip L., editor
- Published
- 1990
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11. Lead(II) Interferes with the Repair and Processing of UV-Induced DNA Damage
- Author
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Hartwig, A., Schlepegrell, R., Beyersmann, D., Seemayer, Norbert H., editor, and Hadnagy, Wolfgang, editor
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- 1990
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12. Induction of Pig-a mutant erythrocytes in male and female rats exposed to 1,3-propane sultone, ethyl carbamate, or thiotepa.
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Labash, Carson, Carlson, Kristine, Avlasevich, Svetlana L., Berg, Ariel, Bemis, Jeffrey C., MacGregor, James T., and Dertinger, Stephen D.
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ERYTHROCYTES , *URETHANE , *THIOTEPA , *GENETIC mutation , *BIOLOGICAL assay , *PHENOTYPES , *LABORATORY rats - Abstract
Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, groups of five male and female Sprague Dawley rats were exposed to the mutagens 1,3-propane sultone (80 mg/kg/day), ethyl carbamate (600 mg/kg/day), or thiotepa (7.5 mg/kg/day) for three consecutive days (study days 1–3). Pig-a mutant phenotype reticulocyte (RET CD59− ) and mutant phenotype erythrocyte (RBC CD59− ) frequencies were determined on study days −4, 15, 30 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis ( In Vivo MutaFlow ® ). While the percentage of reticulocytes (%RET) was markedly higher for pre-treatment blood samples from males compared to females (6.6% vs. 3.5%), this sex effect was slight or nonexistent at later time points. Treatment-related effects to %RET were generally modest owing to the 12-day interval between last exposure and blood sampling. Mean RET CD59− and RBC CD59− frequencies were consistently low in vehicle control animals of both sexes, with 77% of samples exhibiting mutant cell frequencies ≤1 × 10 −6 over study days 15–46. Treatment with each mutagen caused significant increases to mean RET CD59− and RBC CD59− frequencies. Whereas genotoxicant-induced RET CD59− values were maximal on day 15, induced RBC CD59− frequencies were highest at the last sampling time. Sex did not affect 1,3-propane sultone- or thiotepa-induced mutant cell frequencies. While ethyl carbamate-exposed females exhibited higher mean mutant cell frequencies compared to like-treated males, statistical significance was achieved only for RBC CD59− at one time point (7.6 ± 1.0 × 10 −6 compared to 4.7 ± 0.6 × 10 −6 on day 30). Thus, while some quantitative differences were evident, there were no qualitative differences in how males and females responded to three diverse mutagens. These data support the use of both sexes for Pig-a gene mutation studies. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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13. Detection of ultra-low levels of DNA changes by drinking water: epidemiologically important finding.
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Kumari, Parmila, Meiko Kamiseki, Biyani, Manish, Miho Suzuki, Naoto Nemoto, Takuyo Aita, and Koichi Nishigaki
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DRINKING water , *MUTAGENS , *GENETIC mutation , *ECOSYSTEM management , *MUTAGENICITY testing - Abstract
The safety of drinking water is essential to our health. In this context, the mutagenicity of water needs to be checked strictly. However, from the methodological limit, the lower concentration (less than parts per million) of mutagenicity could not be detected, though there have been of interest in the effect of less concentration mutagens. Here, we describe a highly sensitive mutation assay that detects mutagens at the ppb level, termed genome profiling-based mutation assay (GPMA). This consists of two steps; (i) Escherichia coli culture in the medium with/without mutagens and (ii) Genome profiling (GP) method (an integrated method of random PCR, temperature gradient gel electrophoresis and computer-aided normalization). Owing to high sensitivity of this method, very low concentration of mutagens in tap water could be directly detected without introducing burdensome concentration processes, enabling rapid measurement of low concentration samples. Less expectedly, all of the tap waters tested (22 samples) were shown to be significantly mutagenic while mineral waters were not. Resultantly, this article informs two facts that the GPMA method is competent to measure the mutagenicity of waters directly and the experimental results supported the former reports that the city tap waters contain very low level of mutagenicity reagent trihalomethanes. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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14. Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup
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Robert H. Heflich, Takafumi Kimoto, David P. Lovell, Daniel J Roberts, Stephen D. Dertinger, Leon F. Stankowski, Daishiro Miura, B. Bhaskar Gollapudi, Javed A. Bhalli, and Leslie Recio
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Male ,Pig‐a gene ,Reticulocytes ,Epidemiology ,Health, Toxicology and Mutagenesis ,Rat model ,Pig a gene ,Rodentia ,Review ,010501 environmental sciences ,Bioinformatics ,01 natural sciences ,03 medical and health sciences ,mutation assay ,Dose group ,Medicine ,Animals ,Statistical analysis ,Workgroup ,Genetics (clinical) ,030304 developmental biology ,0105 earth and related environmental sciences ,0303 health sciences ,mutagen ,business.industry ,Mutagenicity Tests ,flow cytometry ,genotoxicity ,Guideline ,Rats ,Mutation ,Biological Assay ,business ,Blood sampling ,Mutagens - Abstract
The rodent Pig‐a assay is a flow cytometric, phenotype‐based method used to measure in vivo somatic cell mutation. An Organization for Economic Co‐operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig‐a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described.
- Published
- 2021
15. EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
- Author
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Andriy Khobta, Daria Kim, and Marta Rodriguez-Alvarez
- Subjects
DNA Repair ,Transcription, Genetic ,DNA damage ,Mutant ,Genetic Vectors ,Green Fluorescent Proteins ,lcsh:QR1-502 ,host cell reactivation (HCR) ,Biochemistry ,Article ,lcsh:Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,mutation assay ,0302 clinical medicine ,translesion synthesis (TLS) ,transcriptional mutagenesis ,Transcription (biology) ,Genes, Reporter ,Humans ,Cloning, Molecular ,Molecular Biology ,enhanced green fluorescent protein (EGFP) ,Polymerase ,Cells, Cultured ,DNA damage tolerance ,030304 developmental biology ,0303 health sciences ,biology ,DNA synthesis ,Chemistry ,Point mutation ,reporter assay ,RNA ,Cell biology ,Amino Acid Substitution ,Mutagenesis ,030220 oncology & carcinogenesis ,Mutation ,biology.protein ,DNA ,HeLa Cells ,damage bypass - Abstract
The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress, however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers, whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed &ldquo, transcriptional mutagenesis&rdquo, (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system.
- Published
- 2020
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16. A Pilot Study for the Mutation Assay Using a High-throughput DNA Sequencer.
- Author
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Tomonari Matsuda, Makiko Takamune, Yoko Matsuda, and Masami Yamada
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GENETIC mutation , *NUCLEOTIDE sequence , *COLONIES (Biology) , *SALMONELLA typhimurium , *ACRYLAMIDE , *CLONING - Abstract
We present here a mutation assay with little bias which incorporates high-throughput DNA sequencing technology. Our strategy is simple: 1) expose cells to a test compound, 2) isolate colonies, and 3) carry out whole-genome sequencing of the clones. In this pilot study, we used Salmonella typhimurium TA100 as a tester strain and successfully detected mutations induced by the mutagen 2- (2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2). We believe that this new mutation assay will be a very useful tool in hazard assessment of chemicals. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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17. Linking environmental carcinogen exposure to TP53 mutations in human tumours using the human TP53 knock-in (Hupki) mouse model.
- Author
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Kucab, Jill E., Phillips, David H., and Arlt, Volker M.
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TUMORS , *CARCINOGENS , *P53 protein , *MUTAGENS , *FIBROBLASTS - Abstract
TP53 is one of the most commonly mutated genes in human tumours. Variations in the types and frequencies of mutations at different tumour sites suggest that they may provide clues to the identity of the causative mutagenic agent. A useful model for studying human TP53 mutagenesis is the partial human TP53 knock-in (Hupki) mouse containing exons 4–9 of human TP53 in place of the corresponding mouse exons. For an in vitro assay, embryo fibroblasts from the Hupki mouse can be examined for the generation and selection of TP53 mutations because mouse cells can be immortalized by mutation of Tp53 alone. Thus far, four environmental carcinogens have been examined using the Hupki embryo fibroblast immortalization assay: (a) UV light, which is linked to human skin cancer; (b) benzo[ a]pyrene, which is associated with tobacco smoke-induced lung cancer; (c) 3-nitrobenzanthrone, a suspected human lung carcinogen linked to diesel exposure; and (d) aristolochic acid, which is linked to Balkan endemic nephropathy-associated urothelial cancer. In each case, a unique TP53 mutation pattern was generated that corresponded to the pattern found in human tumours where exposure to these agents has been documented. Therefore, the Hupki embryo fibroblast immortalization assay has sufficient specificity to make it applicable to other environmental mutagens that putatively play a role in cancer aetiology. Despite the utility of the current Hupki embryo fibroblast immortalization assay, it has several limitations that could be addressed by future developments, in order to improve its sensitivity and selectivity. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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18. Novel Mutation Assay with High Sensitivity based on Direct Measurement of Genomic DNA Alterations: Comparable Results to the Ames Test.
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Futakami, Masae, Salimullah, Md, Miura, Takashi, Tokita, Sumio, and Nishigaki, Koichi
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- *
GENETIC mutation , *GENOMES , *DNA , *GENES , *MUTAGENS - Abstract
Almost all of the methodologies developed to date to assay the potential mutagenicity of chemical substances are based on detection of altered phenotypic traits. The alternative approach of directly screening the whole genome for mutations is not feasible because of the logistics of carrying out mass sequencing of genes. Here we describe a novel and highly sensitive mutation assay, which we term the ‘genome profiling-based mutation assay’ (GPMA) that directly detects mutations generated in genomic DNA. We used GPMA to detect mutations caused by known mutagens such as AF2 and ethidium bromide even at concentrations of 30 ppb. The number of mutations detected was dependent on the number of generations in culture and the concentrations of the mutagens. Almost complete agreement was observed between GPMA and the Ames test in the discrimination of mutagens (63 out of 64). Owing to the high sensitivity of GPMA, the effects of long-term and low-dose exposures and the influence of chemicals of low solubility can also be screened. Thus, genotype-based GPMA can complement the Ames test, which is the standard technology in this field and is based on phenotypic traits. [ABSTRACT FROM PUBLISHER]
- Published
- 2007
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19. Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay
- Author
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French, C. Tenley, Ross, Carley D., Keysar, Stephen B., Joshi, Dhanashree D., Lim, Chang-Uk, and Fox, Michael H.
- Subjects
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GENETIC mutation , *BIOLOGICAL variation , *GENETIC load , *CHEMICAL warfare agents - Abstract
Abstract: Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO AL). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59− and CD59+ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents. [Copyright &y& Elsevier]
- Published
- 2006
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20. Induced mutation spectra at the upp locus in Salmonella typhimurium: Response of the target gene in the FU assay to mechanistically different mutagens
- Author
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Vlasakova, Katerina, Skopek, Thomas R., and Glaab, Warren E.
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GENETIC mutation , *ENTEROBACTERIACEAE , *SALMONELLA typhimurium , *ANTINEOPLASTIC agents - Abstract
Abstract: A novel forward mutation assay has been developed in Salmonella typhimurium based on resistance to 5-fluorouracil (FU). The mutational target in the FU assay was determined to be the uracil phosphoribosyl transferase (upp) gene. To validate the upp gene as a suitable target for monitoring a variety of induced mutations, the mutational specificity was determined for five mechanistically different mutagens. The mutagens included a polycyclic hydrocarbon (benzo[a]pyrene, B[a]P), SN1 and SN2 alkylating agents (N-nitroso-N-methylurea, MNU, and methyl methanesulfonate, MMS, respectively), a frameshift mutagen (ICR-191), and an oxidative-damaging agent (hydrogen peroxide, H2O2). Induced mutation frequencies were measured in the presence and absence of the plasmid pKM101 (strain FU100 and FU1535, respectively). pKM101 renders FU100 more susceptible to induced mutation by providing error-prone replicative bypass of DNA adducts. B[a]P, MMS, and H2O2 failed to induce the mutant frequency in FU1535, demonstrating the dependence of pKM101 on induced mutations with these agents. ICR-191 and MNU were not dependent on pKM101, and did significantly induce mutations in FU1535. In contrast to FU1535, all agents significantly induced mutations in FU100. Approximately 60 independent mutants were sequenced for each agent that significantly induced the mutant frequency above background. The resulting mutational spectra illustrated predictable molecular fingerprints based on known mutagenic mechanisms for each agent. The predominant mutations observed were G:C to T:A transversions for B[a]P, A:T to T:A and G:C to T:A transversions for MMS, G:C to T:A transversions and A:T frameshifts for H2O2, G:C frameshifts for ICR-191, and G:C to A:T transitions for MNU. It can be concluded that the upp gene in the FU assay is a sensitive and suitable target to monitor a variety of induced mutations in Salmonella. [Copyright &y& Elsevier]
- Published
- 2005
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21. 5-Fluorouracil forward mutation assay in Salmonella: Determination of mutational target and spontaneous mutational spectra
- Author
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Glaab, Warren E., Mitchell, Laurie S., Miller, Judith E., Vlasakova, Katerina, and Skopek, Thomas R.
- Subjects
- *
GENETIC mutation , *SPECTRUM analysis , *SALMONELLA , *MOBILE genetic elements - Abstract
Abstract: A forward mutation assay in Salmonella typhimurium that selects for 5-fluoruracil (FU) resistance has been developed. The two genes possibly involved in FU resistance, the uracil phosphoribosyl transferase gene (upp) and the uracil transport protein (uraA), have been cloned from S. typhimurium and sequenced. One hundred percent of FU-resistant clones display sequence changes in the upp gene, indicating that its loss is the major mechanism involved in FU resistance. The spontaneous mutational spectra at the upp locus were then determined in two S. typhimurium strains, FU100 and FU1535, that differ only in the presence of pKM101 plasmid. The pKM101 plasmid provides error-prone replicative bypass of DNA lesions and renders FU100 more susceptible to induced mutagenesis. Fluctuation analysis of FU-resistant clones demonstrated a 10-fold higher spontaneous mutation rate at the upp locus in FU100 relative to FU1535. Over 300 independent FU-resistant clones were then used to generate the spectra at the upp locus in both the strains. Approximately 40% of all the mutations were base substitutions, present at the same relative percentage in both the strains. Frameshift mutations also accounted for approximately 40% of the total; however, their incidence was slightly elevated in FU100. The remaining mutations were larger insertions and deletions, which were both slightly elevated in FU1535. pKM101 significantly elevated the rate of all classes of mutations at the upp locus, with profound effects on A:T to T:A transversions and −2-base frameshift mutations. These initial mutational spectra at the upp locus reveal 147 mutable sites, or 23% of the total 627-base coding sequence and suggest that the target can detect a diverse spectrum of mutagenic events. [Copyright &y& Elsevier]
- Published
- 2005
- Full Text
- View/download PDF
22. Detailed review of transgenic rodent mutation assays
- Author
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Lambert, Iain B., Singer, Timothy M., Boucher, Sherri E., and Douglas, George R.
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GENETIC mutation , *RODENTS , *CARCINOGENESIS , *GENETIC toxicology - Abstract
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. [1.] The TGR mutation assay is based on transgenic rats and mice that contain multiple copies of chromosomally integrated plasmid and phage shuttle vectors that harbour reporter genes for detection of mutation. Mutagenic events arising in the rodent are scored by recovering the shuttle vector and analyzing the phenotype of the reporter gene in a bacterial host. TGR gene mutation assays allow mutations induced in a genetically neutral transgene to be scored in any tissue of the rodent, and therefore circumvent many of the existing limitations to the study of in vivo gene mutation. TGR models for which sufficient data are available to permit evaluation include Muta™ mouse, Big Blue® mouse and rat, LacZ plasmid mouse, and the gpt delta mouse. Mutagenesis in the TGR models is normally assessed as a mutant frequency (MF); however, if required, molecular analysis can provide additional information. [2.] OECD guidelines exist for a range of in vitro mutation assays that are capable of detecting both chromosomal and gene mutations. In vivo assays are required components of a thorough genetic toxicity testing programme. For somatic cells, those assays that are most commonly conducted, for which OECD guidelines are currently available, assess induced chromosomal mutation. In addition there are non-transgenic assays that can be used for analysis of gene mutation; none of these have an OECD test guideline. Existing in vivo assays are limited by a range of different factors, including cost of the assay, the number of tissues in which genotoxicity may be measured, the state of understanding of the endpoint, and the nature of the chemicals that will be detected. [3.] As of July 2004, 163 agents have been evaluated using TGR assays. The majority of experimental records have assessed a subset of these chemicals, most of which are strong mutagens and carcinogens. Of the 103 agents whose carcinogenicity has been evaluated 90 are carcinogens and only 13 are noncarcinogens. The following conclusions may be drawn from the existing TGR mutation data. [•] The ability to use all routes of administration has been demonstrated. Experiments can be tailored to use the most relevant route of administration. [•] The ability to examine mutation in virtually all tissues has been demonstrated. TGR assays have most commonly examined mutagenicity in the liver and bone marrow. [•] The majority of the experiments have used shorter administration times than is currently recommended by the International Workshops on Genotoxicity Testing (IWGT); there are limited data available to assess the effects of longer sampling time except at extremely short administration times. [•] Although it is recognized that a number of factors may influence the tissue specificity of mutation, including cell turnover, DNA repair, toxicokinetics, and the nature of the genetic target, there are currently limited experimental data specific to transgenes that are available to inform the discussion. [•] Limited data are available to evaluate the results of TGR assays in known target tissues for carcinogenicity. A case-by-case analysis of instances in which discrepancies are apparent suggests that in the majority of cases, factors such as nongenotoxic mechanism of action, inappropriate mode of administration, or inadequate study design may account for the observed negative result in the tissue of interest. [•] Qualitatively similar results have been obtained in the majority of experiments that have assayed different transgenes using similar experimental parameters. [•] The spontaneous mutant frequency (SMF) in most somatic tissues of TGR animals is 5–10-fold higher than observed in available endogenous loci using the same animals. Factors such as the age of the animal, the tissue, and the animal model influence the absolute value of the SMF. In most somatic tissues, with the exception of brain, there is an age related increase in mutation frequency throughout the life of the animal. Most, but not all, studies suggest that the SMF in male germline tissues remains low and constant throughout the life of the animal. [•] Multiple treatments of a mutagen appear to increase mutant frequencies in neutral transgenes in an approximately additive manner. However, extremely long treatment times of 12 weeks or longer may produce an apparent increase in MF through clonal expansion, genomic instability in developing preneoplastic foci or tumours, or through oxidation damage of DNA resulting from chronic induction of cytochrome P-450 monooxygenases. [•] The time required to reach the maximum mutant frequency is tissue-specific, and appears to be related to the turnover time of the cell population: the optimal sampling time differs according to tissue, with liver and bone marrow at opposite extremes among proliferating somatic tissues: in bone marrow, the mutant frequency appears to reach a maximum at extremely short sampling times and then decreases over 28 days following an acute treatment; in liver the induced mutation frequency increases over the month following exposure, reaches a maximum, and remains relatively constant thereafter. There are insufficient data available for other tissues to support any conclusion regarding optimal sampling time. [•] The results of studies carried out on a given chemical using similar experimental protocols suggest that the TGR assays show good qualitative reproducibility in both somatic and germ cells, and quantitative reproducibility over a limited range of conditions and laboratories. The data are insufficient to draw conclusions regarding the quantitative reproducibility of the assays over a wider range of conditions. [•] Although there exists a theoretical possibility that ex vivo and in vitro mutations may arise during the course of a TGR experiment, these types of mutations are expected to be extremely rare in a properly conducted experiment using the major TGR models. For positive selection systems, any such mutations will not be detected. [•] The weight of evidence suggests that transgenes and endogenous genes respond in approximately the same manner to mutagens in the few instances where direct comparisons are possible. Sensitivity is determined in large part by the SMF: the higher SMF in transgenes, as compared to testable endogenous genes, appears to reduce their sensitivity, especially when acute treatments are used. The sensitivity of transgenes can be enhanced by increasing the administration time. [•] Mutagens that induce deletions are likely to be detected more easily in certain endogenous genes than in transgenes due to phenotypic selection issues. [•] A very high proportion of the TGR experiments carried out to date have examined the activity of compounds that are known to be strong mutagens. A limited number of noncarcinogens have been evaluated with TGRs. The specificity of the TGR assay for predicting carcinogenicity is generally higher than other assays evaluated in this paper. However, additional data from TGR assays on noncarcinogens is required. [•] Molecular analysis of induced mutations in transgenic targets is possible and provides additional information in situations where high interindividual variation is observed and clonal expansion is suspected, when weak responses are obtained, or when mechanistic information is desired. However, DNA sequence analysis of mutants is laborious and adds to the cost of the experiment; sequencing would not normally be required when testing drugs or chemicals for regulatory applications, particularly where a clear positive or negative result is obtained. [4.] Analysis of the predictivity of TGR assays for carcinogenicity is hindered somewhat by the fact that TGR data are available for only a small number of noncarcinogens. Of the 90 carcinogens and 13 noncarcinogens that have been assessed using TGR assays, the following conclusions can be drawn regarding the predictivity and complementarity of TGR assays in comparison to a range of other OECD in vitro and in vivo genotoxicity tests. [•] The TGR assay has high sensitivity and positive predictivity, meaning that most carcinogens have positive results in TGR and there is a high probability that a chemical with a positive result in TGR is a carcinogen. [•] As is the case with most genotoxicity assays, the TGR assay exhibits low specificity and negative predictivity, meaning that relatively few noncarcinogens were negative in TGR and there is a low probability that a chemical with a negative result in TGR is a noncarcinogen; however, it was no worse than the Salmonella mutagenicity assay in this regard. [•] Considering all the test batteries and single assays examined using the current dataset, best positive and negative predictivity was obtained from the TGR assay alone, the Salmonella mutagenicity assay alone, and a battery in which a positive result in TGR or Salmonella was considered positive and negative results in both assays was considerd negative. Despite the lack of substantial increases in predictive values of the test batteries compared with the component assays alone, the test batteries had a much lower false negative rate. [•] TGR and the in vivo micronucleus (MN) assay exhibited significant complementary – i.e. they offered greater predictivity for the detection of mutagens when combined than when alone – consistent with the fact that these two assays measure different genotoxic endpoints. [•] TGR was usually positive for those carcinogens that were positive in Salmonella and the in vitro chromosomal aberration (CA) assay. In contrast, in vivo MN had a much higher false negative rate for the same chemicals. If in vivo confirmation of positive results from both Salmonella and in vitro CA is warranted, TGR is likely a better choice than in vivo MN. [•] For chemicals having positive Salmonella and negative in vitro CA results (presumptive gene mutagens), selecting either TGR or in vivo MN as the in vivo confirmation assay did not markedly affect the proportion of correct carcinogenicity predictions. [•] For chemicals having positive in vitro CA and negative Salmonella results (presumptive clastogens), selecting in vivo MN as the in vivo confirmation assay led to a slightly higher proportion of correct carcinogenicity predictions than did selecting TGR. [•] For those carcinogens with negative results in both Salmonella and in vitro CA, adding either TGR or in vivo MN to the test battery did not improve the overall predictivity, since neither assay identified the carcinogens missed by the in vitro assays. [5.] Recommendations, based on internationally harmonized criteria, are made regarding the proper conduct of a TGR assay. These recommendations relate to accepted characteristics of a transgenic rodent mutation assay, treatment protocols, and post treatment sampling procedures. Of particular importance in optimizing TGR protocols are two experimental variables—the administration time and the sampling time. Based on observations that mutations accumulate with each treatment, a repeated-dose regimen for a period of 28 days is strongly encouraged, with sampling at 3 days following the final treatment. If slowly proliferating tissues are of particular importance, then a longer sampling time may be more appropriate. Additional confidence in the recommended test protocol will be provided by research that examines the following: [•] The influence of the administration time on the observed mutation frequency for weak mutagens. It has not conclusively been determined if data (especially negative results) from experiments using an administration time of less than 28 days should be discounted, if a 28 day treatment period is sufficiently long to permit the detection of weak mutagen-induced mutations in all tissues, or if weak mutagens could in fact be detected using treatment times shorter than 28 days. [•] The influence of the frequency of treatment on the observed mutation frequency. The difference between weekly and daily administrations on mutant frequency and on the ultimate conclusions of transgenic rodent experiments has not yet been thoroughly investigated. [•] The influence of sampling time following repeat administrations on the mutant frequency in both slowly and rapidly dividing tissue, particularly when examining weak mutagens. At the current time there are insufficient comparative data available for a range of tissues. Recommendations are made regarding how a TGR assay might be used within a short-term test battery for assessing new compounds. The test battery consists of various combinations of four assays—Salmonella, in vitro CA, in vivo MN and TGR. This proposed strategy is based on the conclusions obtained from the predictivity analysis, and the relative costs of the in vivo assays. TGR assays may also be used to resolve conflicts between in vitro and in vivo tests that are currently components of the standard genotoxicity test battery—Salmonella, in vitro CA and in vivo MN. In situations where the standard test battery has been conducted and there are conflicting results—particularly in situations where Salmonella has a positive result but in vivo MN is negative—TGR may be conducted as an additional test to resolve the conflict. Recommended test strategies are based on an analysis of the existing data. Confidence in these recommendations would be enhanced by additional experimental data in the following areas. [•] TGR data for additional non-carcinogens to increase the proportion of non-carcinogens in the data set. [•] Additional testing to fill data gaps for chemicals having known TGR assay but missing data from the Salmonella, in vitro CA, or in vivo MN assays. [•] The testing of additional chemicals using an accepted test guideline for TGR mutation assays. Based on the information and analyses in this review, there is sufficient evidence to support the recommendation that the OECD undertake the development of a Test Guideline on Transgenic Rodent Gene Mutation Assays. Accordingly, it is recommended that the OECD establish an Expert Working Group to develop such a Test Guideline, and serve as an international forum for undertaking any additional research that would lead to the development of a fuller understanding of the variables surrounding the conduct of TGR mutation assays. [Copyright &y& Elsevier]
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- 2005
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23. Time Course of cII Gene Mutant Manifestation in the Liver, Spleen, and Bone Marrow of N-Ethyl-N-Nitrosourea-Treated Big Blue Transgenic Mice.
- Author
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Jianyong Wang, Xiaoli Liu, Heflich, Robert H., and Tao Chen
- Subjects
GENETIC mutation ,TRANSGENIC animals ,NITROSOUREAS ,MUTAGENESIS ,LIVER cells ,BONE marrow - Abstract
The time between treatment and the appearance of mutants (mutant manifestation time) is a critical variable for in vivo transgenic mutation assays. There are, however, limited data describing the optimal sampling time for detecting mutations in various tissues of mutagen-treated animals. In this study, we investigated the time course of cII gene mutant induction in the liver, spleen, and bone marrow of Big Blue transgenic mice treated with N-ethyl-N-nitrosourea (ENU). Six-month-old female mice were treated with a single dose (120 mg/kg) of ENU, and the animals were sacrificed, and the cII mutant frequencies (MFs) were determined at 1, 3, 7, 15, 30, and 120 days after the treatment. The MFs in the liver cII gene of ENU-treated mice increased with time after the treatment, while the MFs for concurrent controls remained constant. The liver cII MFs in ENU-treated mice were significantly increased at day 30 and 120 (p < 0.01), with the largest increase at day 120. The spleen cII MFs in ENU-treated mice were increased significantly at day 7 and later (p < 0.01), and reached a plateau at day 30. In the bone marrow, the cII MFs in ENU-treated mice were increased significantly at all sampling times (p < 0.01), with the maximum MF at day 3. These results confirm that the time after treatment required to reach the maximum MF is tissue specific, with the approximate time for the maximum ENU-induced cII MF response being: bone marrow, 3 days; spleen, 14–30 days; and liver, more than 30 days. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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24. ROLE OF HUMAN CYTOCHROME P450 (CYP) IN THE METABOLIC ACTIVATION OF NITROSAMINE DERIVATIVES: APPLICATION OF GENETICALLY ENGINEERED SALMONELLA EXPRESSING HUMAN CYP.
- Author
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Kamataki, Tetsuya, Fujita, Ken-ichi, Nakayama, Kazuo, Yamazaki, Yoshiyuki, Miyamoto, Masami, and Ariyoshi, Noritaka
- Subjects
- *
CYTOCHROMES , *NITROSOAMINES , *SALMONELLA typhimurium - Abstract
The role of human cytochrome P450 (CYP) in the metabolic activation of tobacco-related N-nitrosamines was examined by Salmonella mutation test using a series of genetically engineered Salmonella typhimurium YG7108 strains each co-expressing a form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) together with human NADPH–cytochrome P450 reductase. Seven tobacco-related N-nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosonornicotine, N-nitrosoanabasine, and N-nitrosoanatabine were used. The CYP2A6 was found to be responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines examined. On the basis of the evidence, genetic polymorphism of the CYP2A6 gene appeared to be one of the factors determining cancer susceptibility caused by smoking. Previously, we found the whole deletion of the CYP2A6 gene (CYP2A6*4C) as a type of genetic polymorphism in Japanese. We hypothesized that individuals possessing the gene homozygous for CYP2A6*4C were incapable of activating tobacco-related N-nitrosamines and showed lower susceptibility to lung cancer induced by tobacco smoke. Thus, the relationship between the CYP2A6*4C and the susceptibility to the lung cancer was evaluated. The frequency of the CYP2A6*4C was significantly lower in the lung cancer patients than healthy volunteers, suggesting that the subjects carrying the CYP2A6*4C alleles are resistant to carcinogenesis caused by N-nitrosamines because of the poor metabolic activation capacity. Taking these results into account, CYP2A6 is an enzyme enhancing lung cancer risk. [ABSTRACT FROM AUTHOR]
- Published
- 2002
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25. Antimutagenic and anticarcinogenic effects of Phyllanthus amarus.
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Sripanidkulchai, B., Tattawasart, U., Laupatarakasem, P., Vinitketkumneun, U., Sripanidkulchai, K., Furihata, C., and Matsushima, T.
- Abstract
Summary: This study aimed to examine the antimutagenic and anticarcinogenic potential of Phyllanthus amarus Schum. et Thonn. using the bacterial preincubation mutation assay and an in-vivo alkaline elution method for DNA single-strand breaks in hamster liver cells. The aqueous extract of the entire plant showed an antimutagenic effect against induction by 2-aminofluorene (AF2), 2-aminoanthracene (2AA) and 4-nitroquinolone-1-oxide (4-NQO) in Salmonella typhimurium strains TA98 and TA100, and in Escherichia coli WP2 uvrA/pKM101. All the results were dose-dependent; however, inhibition of N-ethyl-N-nitrosoguanidine (ENNG)-induced mutagenesis was observed only with S. typhimurium TA100. The extract also exhibited activity against 2-nitrofluorene (2NF) and sodium azide-induced mutagenesis with S. typhimurium TA98 and TA100, respectively. Based on the alkaline elution method, the plant extract prevented in vivo DNA single-strand breaks caused by dimethylnitrosamine (DMN) in hamster liver cells. When the extract was administered 30 min prior to the administration of DMN, the elution rate constant decreased more than 2.5 times, compared to that of control. These results indicate that P. amarus possesses antimutagenic and antigenotoxic properties. [Copyright &y& Elsevier]
- Published
- 2002
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26. Testing of acetaminophen in support of the international multilaboratory in vivo rat Pig-a assay validation trial
- Author
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Kathleen De Vlieger, Ann Schuermans, Bas-jan van der Leede, Helena Geys, Marjolein van Heerden, Sandra De Jonghe, Terry Van Doninck, Fetene Tekle, Jacky Van Gompel, and Sandy Weiner
- Subjects
Male ,medicine.medical_specialty ,Internationality ,reticulocytes ,Epidemiology ,DNA damage ,Health, Toxicology and Mutagenesis ,Cmax ,010501 environmental sciences ,Pharmacology ,01 natural sciences ,Flow cytometry ,Rats, Sprague-Dawley ,03 medical and health sciences ,mutation assay ,In vivo ,medicine ,Animals ,Genetics (clinical) ,Research Articles ,030304 developmental biology ,0105 earth and related environmental sciences ,Acetaminophen ,0303 health sciences ,Micronucleus Tests ,medicine.diagnostic_test ,business.industry ,Mutagenicity Tests ,flow cytometry ,Reproducibility of Results ,In vitro ,glycosyl phosphatidylinositol ,in vivo ,Histopathology ,Biological Assay ,Comet Assay ,Micronucleus ,business ,Laboratories ,medicine.drug ,Mutagens ,Research Article ,red blood cells - Abstract
Acetaminophen, a nonmutagenic compound as previously concluded from bacteria, in vitro mammalian cell, and in vivo transgenic rat assays, presented a good profile as a nonmutagenic reference compound for use in the international multilaboratory Pig‐a assay validation. Acetaminophen was administered at 250, 500, 1,000, and 2,000 mg·kg−1·day−1 to male Sprague Dawley rats once daily in 3 studies (3 days, 2 weeks, and 1 month with a 1‐month recovery group). The 3‐Day and 1‐Month Studies included assessments of the micronucleus endpoint in peripheral blood erythrocytes and the comet endpoint in liver cells and peripheral blood cells in addition to the Pig‐a assay; appropriate positive controls were included for each assay. Within these studies, potential toxicity of acetaminophen was evaluated and confirmed by inclusion of liver damage biomarkers and histopathology. Blood was sampled pre‐treatment and at multiple time points up to Day 57. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as CD59‐negative RBC and CD59‐negative RET frequencies, respectively. No increases in DNA damage as indicated through Pig‐a, micronucleus, or comet endpoints were seen in treated rats. All positive controls responded as appropriate. Data from this series of studies demonstrate that acetaminophen is not mutagenic in the rat Pig‐a model. These data are consistent with multiple studies in other nonclinical models, which have shown that acetaminophen is not mutagenic. At 1,000 mg·kg−1·day−1, Cmax values of acetaminophen on Day 28 were 153,600 ng/ml and 131,500 ng/ml after single and repeat dosing, respectively, which were multiples over that of clinical therapeutic exposures (2.6–6.1 fold for single doses of 4,000 mg and 1,000 mg, respectively, and 11.5 fold for multiple dose of 4,000 mg) (FDA 2002). Data generated were of high quality and valid for contribution to the international multilaboratory validation of the in vivo Rat Pig‐a Mutation Assay.
- Published
- 2019
27. EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions.
- Author
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Rodriguez-Alvarez, Marta, Kim, Daria, and Khobta, Andriy
- Subjects
- *
DNA damage , *GREEN fluorescent protein , *RNA synthesis , *DNA synthesis , *MUTAGENS , *NUCLEOTIDE sequence , *DNA replication - Abstract
The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress; however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers; whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed "transcriptional mutagenesis" (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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28. Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup.
- Author
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Dertinger SD, Bhalli JA, Roberts DJ, Stankowski LF Jr, Gollapudi BB, Lovell DP, Recio L, Kimoto T, Miura D, and Heflich RH
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- Animals, Biological Assay, Flow Cytometry, Male, Mutagens toxicity, Rats, Reticulocytes pathology, Rodentia genetics, Mutagenicity Tests, Mutagens pharmacology, Mutation genetics, Reticulocytes drug effects
- Abstract
The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig-a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described., (© 2021 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagen Society.)
- Published
- 2021
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29. The Mutagenicity of Benzidine, 4,4″-Diaminoterphenyl, N,N-Dimethyl-4-aminoazobenzene and 4-Cyano-N,N-dimethylaniline in the L51787 Mouse Lymphoma Assay
- Author
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Kennelly, James C., Garner, R. Colin, Parry, J. M., editor, and Arlett, C. F., editor
- Published
- 1985
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30. Mammalian Cell Mutation Assays with Benzidine, 4,4″-Diaminoterphenyl, 4-Dimethylaminoazobenzene and 4-Cyanodimethylaniline
- Author
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Arlett, Colin F., Parry, J. M., editor, and Arlett, C. F., editor
- Published
- 1985
- Full Text
- View/download PDF
31. The Absence of Mutagenic Activity of Benzidine and 4,4″-Diaminoterphenyl in Tk6 Human Lymphoblast Cells
- Author
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Cole, J., Arlett, C. F., Muriel, W. J., Parry, J. M., editor, and Arlett, C. F., editor
- Published
- 1985
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32. Mutagenicity of Benzidine and 4,4″-Diamenoterphenyl in Chinese Hamster V79 Cells
- Author
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O’Donovan, M. R., Parry, J. M., editor, and Arlett, C. F., editor
- Published
- 1985
- Full Text
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33. Chinese Hamster Ovary Mutation Assays
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Brusick, David J. and Douglas, J. F., editor
- Published
- 1984
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34. Bacterial Reverse and Forward Mutation Assays
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Haworth, S. R. and Douglas, J. F., editor
- Published
- 1984
- Full Text
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35. Mutagenicity Testing of Complex Environmental Mixtures with Chinese Hamster Ovary Cells
- Author
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Li, A. P., Brooks, A. L., Clark, C. R., Shimizu, R. W., Hanson, R. L., Dutcher, J. S., Waters, Michael D., editor, Sandhu, Shahbeg S., editor, Lewtas, Joellen, editor, Claxton, Larry, editor, Chernoff, Neil, editor, and Nesnow, Stephen, editor
- Published
- 1983
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36. Mutation in Single-Cell Systems Induced by Low-Level Mutagen Exposure
- Author
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Liber, Howard L., Danheiser, Susan L., Thilly, William G., Hollaender, Alexander, editor, Woodhead, Avril D., editor, Shellabarger, Claire J., editor, and Pond, Virginia, editor
- Published
- 1985
- Full Text
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37. Use of Fluorescence-Activated Cell Sorter for Screening Mutant Cells
- Author
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Bigbee, William L., Branscomb, Elbert W., de Serres, Frederick J., editor, and Ansari, Aftab A., editor
- Published
- 1984
- Full Text
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38. An Escherichia coli Differential Killing Test for Carcinogens Based on a uvrA recA lexA Triple Mutant
- Author
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Green, M. H. L., Tweats, D. J., Schaefer, Karl E., editor, Stich, H. F., editor, and San, R. H. C., editor
- Published
- 1981
- Full Text
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39. SOP/DTX/147 Mouse Lymphoma Mutation Assay
- Author
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Sword, I. P., Thomson, Ritchie, Sword, I. P., editor, and Thomson, Ritchie, editor
- Published
- 1980
- Full Text
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40. Descriptions and Evaluation of Genetic Toxicology Assays
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Brusick, David and Brusick, David
- Published
- 1987
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41. Parvoviral Probe for Assessing the Mutagenic Risk of Low Doses of Radiation and Chemicals Administered to Human Cells
- Author
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Cornelis, J. J., Dinsart, C., Su, Z. Z., Rommelaere, J., and Castellani, Amleto, editor
- Published
- 1983
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42. Gene-Locus Mutation Assays in Diploid Human Lymphoblast Lines
- Author
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Thilly, W. G., DeLuca, J. G., Furth, E. E., Hoppe, H., IV, Kaden, D. A., Krolewski, J. J., Liber, H. L., Skopek, T. R., Slapikoff, S. A., Tizard, R. J., Penman, B. W., de Serres, Frederick J., editor, and Hollaender, Alexander, editor
- Published
- 1980
- Full Text
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43. Mutation assay using single-molecule real-time (SMRT™) sequencing technology
- Author
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Matsuda, Tomonari, Matsuda, Shun, and Yamada, Masami
- Subjects
PacBio RSII DNA sequencer ,Mutation assay ,Single-molecule real-time (SMRT) sequencing technology - Abstract
Introduction: We present here a simple, phenotype-independent mutation assay using a PacBio RSII DNA sequencer employing single-molecule real-time (SMRT) sequencing technology. Salmonella typhimurium YG7108 was treated with the alkylating agent N-ethyl-N-nitrosourea (ENU) and grown though several generations to fix the induced mutations, the DNA was extracted and the mutations were analyzed by using the SMRT DNA sequencer. Results: The ENU-induced base-substitution frequency was 15.4 per Megabase pair, which is highly consistent with our previous results based on colony isolation and next-generation sequencing. The induced mutation spectrum (95% G:C→A:T, 5% A:T→G:C) is also consistent with the known ENU signature. The base-substitution frequency of the control was calculated to be less than 0.12 per Megabase pair. A current limitation of the approach is the high frequency of artifactual insertion and deletion mutations it detects. Conclusions: Ultra-low frequency base-substitution mutations can be detected directly by using the SMRT DNA sequencer, and this technology provides a phenotype-independent mutation assay.
- Published
- 2015
44. Mutation assay using single-molecule real-time (SMRT(TM)) sequencing technology
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Tomonari Matsuda, Masami Yamada, and Shun Matsuda
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Genetics ,PacBio RSII DNA sequencer ,Mutation ,Social Psychology ,Mutation assay ,Environmental Science (miscellaneous) ,Biology ,medicine.disease_cause ,Molecular biology ,Human genetics ,DNA sequencer ,chemistry.chemical_compound ,chemistry ,Deletion mutation ,medicine ,Single-molecule real-time (SMRT) sequencing technology ,DNA ,Induced mutation ,Research Article - Abstract
Introduction We present here a simple, phenotype-independent mutation assay using a PacBio RSII DNA sequencer employing single-molecule real-time (SMRT) sequencing technology. Salmonella typhimurium YG7108 was treated with the alkylating agent N-ethyl-N-nitrosourea (ENU) and grown though several generations to fix the induced mutations, the DNA was extracted and the mutations were analyzed by using the SMRT DNA sequencer. Results The ENU-induced base-substitution frequency was 15.4 per Megabase pair, which is highly consistent with our previous results based on colony isolation and next-generation sequencing. The induced mutation spectrum (95% G:C → A:T, 5% A:T → G:C) is also consistent with the known ENU signature. The base-substitution frequency of the control was calculated to be less than 0.12 per Megabase pair. A current limitation of the approach is the high frequency of artifactual insertion and deletion mutations it detects. Conclusions Ultra-low frequency base-substitution mutations can be detected directly by using the SMRT DNA sequencer, and this technology provides a phenotype-independent mutation assay.
- Published
- 2015
45. Development of a Fast and Accurate Mutation Assay in Human Cell Lines
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Robeson, Kalen Z.
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- Biology, Cellular Biology, Mutation, Ultraviolet Light, UV, Mutation Assay, Assay, HeLa, Cancer, Carcinogen, Mutation Workflow
- Abstract
While many mutation assays already exist, they are often either extremely costly or ineffective at providing molecular and mechanistic information about a mutagen. These assays are often assembled into a workflow from inexpensive less informative assays to expensive informative assays. This study sought to develop an assay to allow for more detailed information to be generated inexpensively in the early stages of a mutagen assessment workflow.A plasmid designed and constructed for mutation measurement using Ion Torrent Next Generation Sequencing was stably transfected into HeLa cells. To elucidate this assay’s ability to measure mutation, transfected cells were then exposed to three doses of the LD50 for ultraviolet light for a total exposure of 6 mJ/cm2. Finally, genomic DNA from mutated and non-mutated cells was isolated and sequenced to measure induced mutation. Genomic DNA was successfully sequenced from transfected cells, and an increase in percent mutation was seen between mutated and non-mutated cells. No statistically significant sequence specific mutation rate could be determined and this study was unable to show that this induced mutation was consistent with the known mutation signature of ultraviolet light. Nonetheless, these results represent a proof of concept and demonstrate that this assay can be used to measure mutation.
- Published
- 2017
46. Evaluation of the Mutagenic Activity of 4-Dimethylaminoazobenzene (DAB) and 4-Cyanodimethylaniline (CDA) in the Salmonella/Microsome Assay
- Author
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Anderson, Diana, Blowers, S. D., Parry, J. M., editor, and Arlett, C. F., editor
- Published
- 1985
- Full Text
- View/download PDF
47. Assay of mutation induced in human lymphoblastoid cells by combustion-generated soot particles
- Author
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Feitelberg, A. S., Sarofim, A. F., Bolsaitis, P. B., Dekermendjian, V., Elliot, J. F., and Thilly, W. G.
- Subjects
POLYCYCLIC aromatic hydrocarbons - Published
- 1991
48. Comparison of KRAS genotype: therascreen assay vs. LNA-mediated qPCR clamping assay.
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Chang SC, Denne J, Zhao L, Horak C, Green G, Khambata-Ford S, Bray C, Celik I, Van Cutsem E, and Harbison C
- Subjects
- Adult, Aged, Aged, 80 and over, Antibodies, Monoclonal, Humanized administration & dosage, Camptothecin administration & dosage, Camptothecin analogs & derivatives, Cetuximab, Colorectal Neoplasms drug therapy, Colorectal Neoplasms genetics, DNA, Neoplasm genetics, Female, Fluorouracil administration & dosage, Follow-Up Studies, Genotype, Humans, Irinotecan, Leucovorin administration & dosage, Male, Middle Aged, Neoplasm Staging, Prognosis, Proto-Oncogene Proteins p21(ras), Retrospective Studies, Survival Rate, Young Adult, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Colorectal Neoplasms mortality, Mutation genetics, Oligonucleotides, Polymerase Chain Reaction, Proto-Oncogene Proteins genetics, ras Proteins genetics
- Abstract
Background: Kirsten rat sarcoma virus (KRAS) wild-type status determined using a locked nucleic acid (LNA)-mediated quantitative polymerase chain reaction (qPCR) clamping assay (LNA assay) predicted response to therapy in the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) study. A companion KRAS diagnostic tool has been developed for routine clinical use (QIAGEN therascreen kit) (QIAGEN Manchester Ltd, Manchester, UK). We wanted to assess the concordance between the validated US Food and Drug Administration (FDA)-approved therascreen assay and the LNA assay in determining the KRAS status of a subset of patients enrolled in the CRYSTAL study., Patients and Methods: DNA extracted from paraffin-embedded tumor sections was tested for KRAS status using the therascreen assay. Efficacy data from the CRYSTAL study were assessed to determine if the overall survival (OS) hazard ratio for cetuximab in patients identified as having KRAS wild-type status using the therascreen assay was equivalent to that in patients identified as KRAS wild-type using the LNA assay. This was determined by assessing if the concordance between the therascreen assay and the LNA assay met the minimum threshold (prespecified as 0.8) to achieve a significant difference in the OS hazard ratio in favor of the cetuximab + FOLFIRI (5-fluorouracil, leucovorin [folinic acid], irinotecan) arm in the KRAS wild-type population as identified using the therascreen assay., Results: Of the 148 samples determined to be KRAS wild-type (therascreen assay), 141 (95.3%) samples were also KRAS wild-type (LNA assay) and 7 samples (4.7%) were KRAS mutant (LNA assay). The prespecified primary concordance measure p was 141/148 = 0.953 (95% confidence interval [CI], 0.905-0.981). The concordance was statistically significantly higher than the prespecified threshold of 0.8 for concordance between the therascreen assay and the LNA assay. Consistent with the concordance exceeding the prespecified threshold, the OS hazard ratio (cetuximab + FOLFIRI arm vs. FOLFIRI arm) in the KRAS wild-type population, determined by the therascreen assay, supported a significant benefit for cetuximab (ie, the 95% CI excluded 1) and was comparable to the OS hazard ratio observed in the CRYSTAL study KRAS wild-type population (LNA assay) even after adjustment for potentially confounding baseline variables., Conclusion: These results support the utility of the therascreen assay for identifying patients who may benefit from cetuximab therapy for metastatic colorectal cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
49. Mutation in a Reporter Gene Depends on Proximity to and Transcription of Immunoglobulin Variable Transgenes
- Author
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Umar, Asad, Schweitzer, Peter A., Levy, Nina S., Gearhart, John D., and Gearhart, Patricia J.
- Published
- 1991
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