ABSTRACT
The pore-forming α-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channel-mediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney.NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+.
Subject(s)
Alternative Splicing , Kidney , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits , Potassium, Dietary , Animals , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/genetics , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/metabolism , Potassium, Dietary/metabolism , Kidney/metabolism , Mice, Inbred C57BL , Mice , Male , Gene Expression Regulation, Developmental , Exons , FemaleABSTRACT
The New South Wales Biocontainment Centre is a statewide referral facility for patients with high-consequence infectious disease (HCID). The facility collaborates with researchers to adapt existing HCID procedures such as donning and doffing of personal protective equipment (PPE). However, information on how to respond safely to collapse of a healthcare provider in full PPE within a contaminated zone is scarce. To address this gap, we adapted Nebraska Medicine's "provider down" protocol on paper and then simulated and video recorded the process, iteratively, in the facility. Clinicians analyzed the recordings collaboratively in researcher-facilitated reflexive discussions. Our primary aim was to ascertain how to maintain optimal infection prevention and control while providing urgent care for the healthcare provider. We tested participants' suggested modifications, in repeated video recorded simulations, until consensus on optimal practice was achieved. Our secondary aim was to assess the utility of video-reflexive methods to enhance clinicians' awareness and understanding of infection prevention and control in a rare and complex scenario. Six adaptations and simulations were discussed in video-reflexive sessions before consensus was reached; the final version of the protocol differed considerably from the first. Viewing footage of simulations in situ enabled participants to (1) identify infection and occupational risks not identified on paper or during verbal postsimulation debriefs and (2) test alternative perspectives on safe procedure. Video-reflexivity enables context-sensitive and consensus-building codesign of policies and procedures, critical to protocol development in a new unit. It contributes to a culture of teamwork, preparedness, and confidence before, rather than in the heat of, a crisis.
Subject(s)
Personal Protective Equipment , Humans , New South Wales , Video Recording , Infection Control/methods , Health Personnel , Containment of Biohazards/methodsABSTRACT
Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.
Subject(s)
DNA/genetics , Gene Targeting/methods , Hypoxanthine Phosphoribosyltransferase/genetics , Mutation , Oligonucleotides/metabolism , Purines/chemistry , Cell Line , DNA/chemistry , DNA Damage , Electrophoretic Mobility Shift Assay , Genetic Loci , Humans , Hypoxanthine Phosphoribosyltransferase/metabolism , Oligonucleotides/genetics , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
There is some evidence that the mouse lymphoma TK assay (MLA) can detect aneugens, and this is accepted in the current International Conference on Harmonisation guidance for testing pharmaceuticals. However, whether or not it can be used as a reliable screen for aneugenicity has been the subject of debate. Consequently, aneugens with diverse mechanisms of action were tested in the MLA using 24-h exposure. No evidence of increased mutant frequency was seen with noscapine, diazepam or colchicine and increases were seen with taxol, carbendazim, econazole and chloral hydrate only at high levels of toxicity (for all but one taxol concentration survival reduced to ≤10% of control). None of these agents would be unequivocally classified as positive using currently accepted criteria. The largest increases in mutant number were seen with taxol and carbendazim; therefore, trifluorothymidine (TFT)-resistant clones resulting from treatment with them were cultured and analysed for chromosome 11 copy number using fluorescent in situ hybridisation (FISH) and loss of heterozygosity (LOH). High concentrations of these aneugens induced LOH at all loci examined indicating only one chromosome 11 was present but, perhaps surprisingly, all were found to have two copies of chromosome 11 using FISH. This would be consistent with loss of the tk(+) chromosome 11b with concomitant duplication of chromosome 11a, which has been proposed as a likely mechanism for induction of TFT-resistant clones. However, it was also surprising that analysis of centromere size showed that almost all the clones had both small and large centromeres, i.e. suggesting the presence of both chromosomes 11a and 11b. In conclusion, it appears that the TFT-resistant mutants resulting from treatment with toxic concentrations of some aneugens such as taxol and carbendazim have undergone complex genetic changes. However, these data show that the MLA cannot be used as a routine screen to detect aneugens.
Subject(s)
Aneugens/analysis , Enzyme Assays/methods , Lymphoma/metabolism , Thymidine Kinase/metabolism , Aneugens/toxicity , Animals , Cell Line, Tumor , Centromere/drug effects , Centromere/metabolism , Chromosomes, Mammalian/genetics , Gene Dosage/drug effects , Gene Dosage/genetics , In Situ Hybridization, Fluorescence , Karyotyping , Loss of Heterozygosity/drug effects , Loss of Heterozygosity/genetics , Mice , Microsatellite Repeats/genetics , Polymerase Chain ReactionABSTRACT
As part of a project to develop high throughput versions of the comet assay (single cell gel electrophoresis), with a consequent need for more efficient scoring, we have compared the performance of visual scoring, automated and semi-automated image analysis when assessing comets in the same set of gels from dose-response experiments with typical DNA-damaging agents. Human lymphoblastoid TK-6 cells were treated with concentrations of methylmethanesulphonate between 0.04 and 0.6 mM, and peripheral human lymphocytes were incubated, after embedding in agarose, with H(2)O(2) concentrations from 2.5 to 160 µM. All three scoring methods proved capable of detecting a significant level of damage at the lowest concentration of each agent. Visual scoring systematically overestimates low levels of damage compared with computerised image analysis; on the other hand, heavily damaged comets are less efficiently detected with image analysis. Overall, the degree of agreement between the scoring methods is within acceptable limits according to a Bland-Altman analysis.
Subject(s)
Comet Assay/methods , Comet Assay/standards , Image Processing, Computer-Assisted/methods , Research Design/statistics & numerical data , Cell Line , Humans , Hydrogen Peroxide/toxicity , Lymphocytes/drug effects , Methyl Methanesulfonate/toxicityABSTRACT
An improved protocol for the radiolabel DNA-binding assay, which gives a high yield of highly pure DNA has been developed by use of mouse lymphoma cells. The critical difference from previously published methods is the use of enzymatic degradation of proteins in the later DNA purification steps rather than during the homogenisation procedure. Different DNA-purification methodologies were first compared and the protocol of choice was optimized later on; both steps were performed with [(35)S]-labelled amino acids for labelling of cellular protein, which enabled both the quantification of cellular protein contaminating the DNA sample and the distinction between cellular and enzyme-derived protein. The assay was later evaluated and shown to give reproducible results based on the data obtained with benzo[a]pyrene (B[a]P) and doxorubicin in two different laboratories. In addition, two further reference compounds, dopamine and diazepam and one proprietary AstraZeneca compound were also tested in mouse lymphoma cells in one laboratory. The two compounds B[a]P and doxorubicin were identified as suitable positive controls for routine testing in the presence and absence of S9, respectively. Exposing 90-100×10(6) cells to (14)C-labelled compound with a molar radioactivity of 2MBq/µmol, yields approximately 500µg DNA with <3% total protein contamination, of which approximately 7% is of cellular origin (<0.2%). The detection level is approximately 2adducts/10(8) dNTP.
Subject(s)
DNA Adducts/biosynthesis , DNA/isolation & purification , DNA/metabolism , Mutagenicity Tests/methods , Mutagens/metabolism , Animals , Biotransformation , Cell Line, Tumor , Isotope Labeling , Leukemia L5178 , Mice , Nucleic Acid Denaturation , Protein Binding , Research DesignABSTRACT
A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.
Subject(s)
Mutagenicity Tests/methods , Animals , Comet Assay/methods , Humans , Micronucleus Tests/methods , Rats , Toxicity Tests/standardsABSTRACT
A collaborative trial was conducted to evaluate the possibility of integrating the rat-liver Comet assay into repeat-dose toxicity studies. Fourteen laboratories from Europe, Japan and the USA tested fifteen chemicals. Two chemicals had been previously shown to induce micronuclei in an acute protocol, but were found negative in a 4-week Micronucleus (MN) Assay (benzo[a]pyrene and 1,2-dimethylhydrazine; Hamada et al., 2001); four genotoxic rat-liver carcinogens that were negative in the MN assay in bone marrow or blood (2,6-dinitrotoluene, dimethylnitrosamine, 1,2-dibromomethane, and 2-amino-3-methylimidazo[4,5-f]quinoline); three compounds used in the ongoing JaCVAM (Japanese Center for the Validation of Alternative Methods) validation study of the acute liver Comet assay (2,4-diaminotoluene, 2,6-diaminotoluene and acrylamide); three pharmaceutical-like compounds (chlordiazepoxide, pyrimethamine and gemifloxacin), and three non-genotoxic rodent liver carcinogens (methapyrilene, clofibrate and phenobarbital). Male rats received oral administrations of the test compounds, daily for two or four weeks. The top dose was meant to be the highest dose producing clinical signs or histopathological effects without causing mortality, i.e. the 28-day maximum tolerated dose. The liver Comet assay was performed according to published recommendations and following the protocol for the ongoing JaCVAM validation trial. Laboratories provided liver Comet assay data obtained at the end of the long-term (2- or 4-week) studies together with an evaluation of liver histology. Most of the test compounds were also investigated in the liver Comet assay after short-term (1-3 daily) administration to compare the sensitivity of the two study designs. MN analyses were conducted in bone marrow or peripheral blood for most of the compounds to determine whether the liver Comet assay could complement the MN assay for the detection of genotoxins after long-term treatment. Most of the liver genotoxins were positive and the three non-genotoxic carcinogens gave negative result in the liver Comet assay after long-term administration. There was a high concordance between short- and long-term Comet assay results. Most compounds when tested up to the maximum tolerated dose were correctly detected in both short- and long-term studies. Discrepant results were obtained with 2,6 diaminotoluene (negative in the short-term, but positive in the long-term study), phenobarbital (positive in the short-term, but negative in the long-term study) and gemifloxacin (positive in the short-term, but negative in the long-term study). The overall results indicate that the liver Comet assay can be integrated within repeat-dose toxicity studies and efficiently complements the MN assay in detecting genotoxins. Practical aspects of integrating genotoxicity endpoints into repeat-dose studies were evaluated, e.g. by investigating the effect of blood sampling, as typically performed during toxicity studies, on the Comet and MN assays. The bleeding protocols used here did not affect the conclusions of the Comet assay or of the MN assays in blood and bone marrow. Although bleeding generally increased reticulocyte frequencies, the sensitivity of the response in the MN assay was not altered. These findings indicate that all animals in a toxicity study (main-study animals as well as toxicokinetic (TK) satellite animals) could be used for evaluating genotoxicity. However, possible logistical issues with scheduling of the necropsies and the need to conduct electrophoresis promptly after tissue sampling suggest that the use of TK animals could be simpler. The data so far do not indicate that liver proliferation or toxicity confound the results of the liver Comet assay. As was also true for other genotoxicity assays, criteria for evaluation of Comet assay results and statistical analyses differed among laboratories. Whereas comprehensive advice on statistical analysis is available in the literature, agreement is needed on applying consistent criteria.
Subject(s)
Mutagens/toxicity , Animals , Carcinogens/toxicity , Comet Assay/methods , Dose-Response Relationship, Drug , Drug Administration Schedule , Liver/drug effects , Male , Micronucleus Tests/methods , Rats , Rats, Wistar , Toxicity TestsABSTRACT
Nucleoside analogs with 2'-modified sugar moieties are often used to improve the RNA target affinity and nuclease resistance of therapeutic oligonucleotides in preclinical and clinical development. Despite their enhanced nuclease resistance, oligonucleotides could slowly degrade releasing nucleoside analogs that have the potential to become phosphorylated and incorporated into cellular DNA and RNA. For the first time, the phosphorylation and DNA/RNA incorporation of 2'-O-(2-methoxyethyl) (2'-O-MOE) nucleoside analogs have been investigated. Using liquid chromatography/tandem mass spectrometry, we showed that enzymes in the nucleotide salvage pathway including deoxycytidine kinase (dCK) and thymidine kinase (TK1) displayed poor reactivity toward 2'-O-MOE nucleoside analogs. On the other hand, 2'-fluoro (F) nucleosides, regardless of the nucleobase, were efficiently phosphorylated to their monophosphate forms by dCK and TK1. Consistent with their efficient phosphorylation by dCK and TK1, 2'-F nucleoside analogs were incorporated into cellular DNA and RNA while no incorporation was detected with 2'-O-MOE nucleoside analogs. In conclusion, these data suggest that the inability of dCK and TK1 to create the monophosphates of 2'-O-MOE nucleoside analogs reduces the risk of their incorporation into cellular DNA and RNA.
Subject(s)
Cell Nucleus/drug effects , DNA/metabolism , Genome, Human , Nucleosides/pharmacology , RNA/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Deoxycytidine Kinase/metabolism , Humans , Nucleosides/chemistry , Phosphorylation , Substrate Specificity , Thymidine Kinase/metabolismABSTRACT
Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analyzed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures. Subsequent assessment of mutant frequency under physiological conditions, by pre-transfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.
Subject(s)
Guanine/metabolism , Mutagens/pharmacology , Oligonucleotides/pharmacology , Animals , COS Cells , Chlorocebus aethiops , Genetic Vectors , Mutation , Protein BindingABSTRACT
The Oligonucleotide Safety Working Group subcommittee on genotoxicity testing considers therapeutic oligonucleotides (ONs) unlikely to be genotoxic based on their properties and on the negative results for ONs tested to date. Nonetheless, the subcommittee believes that genotoxicity testing of new ONs is warranted because modified monomers could be liberated from a metabolized ON and incorporated into DNA and could hypothetically cause chain termination, miscoding, and/or faulty replication or repair. The standard test battery as described in Option 1 of International Conference on Harmonisation S2(R1) is generally adequate to assess such potential. However, for the in vitro assay for gene mutations, mammalian cells are considered more relevant than bacteria for most ONs due to their known responsiveness to nucleosides and their greater potential for ON uptake; on the other hand, bacterial assays may be more appropriate for ONs containing non-ON components. Testing is not recommended for ONs with only naturally occurring chemistries or for ONs with chemistries for which there is documented lack of genotoxicity in systems with demonstrated cellular uptake. Testing is recommended for ONs that contain non-natural chemical modifications and use of the complete drug product (including linkers, conjugates, and liposomes) is suggested to provide the most clinically relevant assessment. Documentation of uptake into cells comparable to those used for genotoxicity testing is proposed because intracellular exposure cannot be assumed for these large molecules. ONs could also hypothetically cause mutations through triple helix formation with genomic DNA and no tests are available for detection of such sequence-specific mutations across the entire genome. However, because the potential for triplex formation by therapeutic ONs is extremely low, this potential can be assessed adequately by sequence analysis.
Subject(s)
Oligonucleotides/toxicity , Animals , Cells, Cultured , DNA Damage , Drug Evaluation, Preclinical , Humans , Mutagenicity Tests , Oligonucleotides/therapeutic useABSTRACT
As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, 4,4'-diaminodiphenyl ether (DPE), a known rodent genotoxic carcinogen, was tested in this laboratory. Sprague Dawley rats (7-9 weeks of age) were given three oral doses of DPE, 24 and 21 h apart and liver or stomach sampled 3h after the final dose. Under the conditions of the test, no increases in DNA damage in liver and stomach were observed with DPE (up to 200 mg/kg/day). A dose-dependent decrease in DNA migration, compared to vehicle controls, was noted for DPE in rat stomach. Further analysis is required to elucidate fully whether this decrease is a consequence of the mode of action or due to the toxicity of DPE. What is perhaps surprising is the inability of the comet assay to detect a known rat genotoxic carcinogen in liver. Further investigation is needed to clarify whether this apparent lack of response results from limited tissue exposure or metabolic differences between species. This finding highlights a need for careful consideration of study design when evaluating assay performance as a measure of in vivo genotoxicity.
Subject(s)
Comet Assay/methods , Phenyl Ethers/toxicity , Administration, Oral , Animals , Carcinogens/toxicity , DNA Damage/drug effects , Dose-Response Relationship, Drug , Liver/drug effects , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Stomach/drug effectsABSTRACT
The degradation of phosphorothioate oligonucleotides (PS-ONDs) and the release of potentially genotoxic modified mononucleotides raise a safety concern for OND-based therapeutics. Deoxyadenosine monophosphorothioate (dAMPαS), a PS nucleotide analog, has been reported to be a potent in vitro mutagen at the thymidine kinase (TK) locus in human TK6 lymphoblastoid cells. This led us to explore the mechanism behind the apparent positive response induced by dAMPαS in the TK gene-mutation assay in TK6 cells. In this work, treatment of TK6 cells with dAMPαS produced a dose-dependent increase in cytotoxicity and mutant frequency at the TK locus. Surprisingly, when the colonies from dAMPαS were re-challenged with the selective agent trifluorothymidine (TFT), the TFT-resistant phenotype was lost. Moreover, dAMPαS-induced colonies displayed distinct growth kinetics and required longer incubation time than 4-nitroquinoline-1-oxide-induced colonies to start growing. Treatment of TK6 cells with dAMPαS induced cell cycle arrest at the G1 phase, enabling cells to grow, and form a colony after the efficacy of TFT in the culture medium was lost. Our findings suggest that a fraction of parental "nonmutant" TK6 cells escaped the toxicity of TFT, possibly via G1 arrest, and resumed growth after the degradation of TFT. We conclude that dAMPαS did not induce real TFT-resistant mutants and caution should be taken with interpretation of mutation data from TK gene-mutation assay in TK6 cells when assessing modified nucleotides.
Subject(s)
Mutagens/toxicity , Nucleotides/toxicity , Organophosphorus Compounds/toxicity , Cell Line , HumansABSTRACT
The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.
Subject(s)
Carcinogens/analysis , Comet Assay/methods , Comet Assay/standards , Animals , DNA Damage , Ethyl Methanesulfonate , Liver/drug effects , Male , Rats , Rats, Sprague-Dawley , Stomach/drug effectsABSTRACT
A serious limitation of the conventional comet assay (single cell gel electrophoresis) is the restriction on the number of samples that can be processed in one experiment, imposed by the size of the electrophoresis platform. One approach to increasing throughput is to reduce the size of gels. We here compare the conventional system of two large gels on a microscope slide, with two recent developments, namely 12 minigels per slide, and a format with 96 minigels on GelBond® film. We used cells treated with X-rays or methylmethanesulphonate (MMS). The level of damage detected (% tail DNA) in X-irradiated or MMS-treated cells was not affected by the format used. Parallel experiments, using all three formats, were performed with MMS-treated cells in two independent laboratories; the difference in results between the two laboratories was of borderline significance. The potential problem of anomalous comets seen at the border of the gel, the so-called 'edge effects', has been addressed. A reliable, high throughput comet assay has applications in genotoxicity testing (particularly for in vivo studies with samples from different organs) as well as ecogenotoxicology and human biomonitoring, where the numbers of samples collected can be considerable.
Subject(s)
Comet Assay/methods , Cell Line , DNA Damage , Gels , High-Throughput Screening Assays , Humans , Methyl Methanesulfonate/toxicity , Mutagens , X-RaysABSTRACT
The European Medicines Agency has expressed concern regarding (1) the potential for antisense oligonucleotide (ASO) therapeutics to induce sequence-specific mutation at genomic DNA and (2) the capability of ASO degradation products (nucleotide analogues) to incorporate into newly synthesized genomic DNA via DNA polymerase and cause mutation if base pairing occurs with reduced fidelity. Treating human lymphoblastoid cells with a biologically active antisense molecule induced sequence-specific mutation within genomic DNA over fourfold, in a system where RAD51 protein expression was induced. This finding has implications for ASO therapeutics with individuals with an induced DNA damage response, such as cancer patients. Furthermore, a phosphorothioate nucleotide analogue potently induced mutation at genomic DNA two orders of magnitude above control. This study shows that a biologically active ASO molecule can induce heritable sequence alterations, and if degraded, its respective analogue may incorporate into genomic DNA with mutagenic consequences.
Subject(s)
Mutagenesis , Mutation , Oligonucleotides, Antisense/toxicity , Phosphorothioate Oligonucleotides/toxicity , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAMTS Proteins , Base Sequence , Biotransformation , Cell Line , DNA Damage , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Hypoxanthine Phosphoribosyltransferase/metabolism , Molecular Sequence Data , Mutagenicity Tests , Oligonucleotides, Antisense/metabolism , Phosphorothioate Oligonucleotides/metabolism , Procollagen N-Endopeptidase/genetics , Procollagen N-Endopeptidase/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Up-RegulationABSTRACT
The genotoxicity of methapyrilne (MP) has been evaluated in a number of assays since it was found to be a rat hepatocarcinogen with subsequent withdrawal as an over-the-counter antihistamine. Whilst it has not been classified as a genotoxin, there are reports of positive findings from mammalian cell gene mutation and transformation assays. To investigate further the genotoxic potential of MP, the alkaline Comet assay was used to evaluate DNA damage both in primary hepatocytes in culture and in vivo in the rat. To confirm bioactivation was required to induce the hepatotoxic mechanism, aminobenzotriazole, a broad spectrum cytochrome P450 enzyme inhibitor was used as a pre-treatment. The levels of glutathione and glutathione disulfide were determined in both hepatocytes in culture and in the liver following in vivo exposure. MP showed significant increases in DNA damage in freshly isolated male rat hepatocyte suspensions that could be significantly reduced by pre-incubation of aminobenzotriazole (ABT). DNA damage showed a marked sex difference, with male hepatocytes being more susceptible, and showing a concurrent depletion of glutathione (GSH) compared with female hepatocytes. Modulation of the GSH levels by diethylmaleate and γ-glutamylcysteinylethyl ester, elevated and reduced the levels of DNA damage, respectively. In the in vivo Comet assay, there was no evidence of DNA damage following MP (150mg/kg p.o) treatment for three consecutive days, although histological and liver enzyme changes were seen. Total protein GSH content was elevated in MP-treated animals and superoxide dismutase levels were increased specifically in periportal regions. Taken together, these data support the potential for MP to induce oxidative stress. The differences in DNA damage detected by the Comet assay in vitro, and in rat liver in vivo, could be attributed to differences in metabolism and response to oxidant insult or the inability of the assay to discriminate damage in a small number of individual cells in the whole liver.
Subject(s)
DNA Damage/drug effects , Hepatocytes/drug effects , Histamine H1 Antagonists/toxicity , Methapyrilene/toxicity , Oxidative Stress/drug effects , Animals , Comet Assay , Female , Glutathione/drug effects , Glutathione/metabolism , Hepatocytes/pathology , Liver/drug effects , Liver/pathology , Male , Mutagenicity Tests , Rats , Rats, Wistar , Sex Factors , Superoxide Dismutase/drug effects , Superoxide Dismutase/metabolism , Triazoles/pharmacologyABSTRACT
Potassium bromate (KBrO3) is a well-established rodent kidney carcinogen and its oxidising activity is considered to be a significant factor in its mechanism of action. Although it has also been shown to be clearly genotoxic in a range of in vivo and in vitro test systems, surprisingly, it is not readily detected in several cell lines using the standard alkaline Comet assay. However, previous results from this laboratory demonstrated huge increases in tail intensity by modifying the method to include incubation with either human 8-oxodeoxyguanosine DNA glycosylase-1 (hOGG1) or bacterial formamidopyrimidine DNA glycosylase (FPG) indicating that, as expected, significant amounts of 8-oxodeoxyguanosine (8-OHdG) were induced. The purpose of this work, therefore, was to investigate why KBrO3, in contrast to other oxidising agents, gives a relatively poor response in the standard Comet assay. Results confirmed that it is a potent genotoxin in mouse lymphoma L5178Y cells inducing micronuclei and mutation at the tk and hprt loci at relatively non-cytotoxic concentrations. Subsequent time-course studies demonstrated that substantial amounts of 8-OHdG appear to remain in cells 24h after treatment with KBrO3 but result in no increase in frank stand breaks (FSB) even though phosphorylated histone H2AX (gamma-H2AX) antibody labelling confirmed the presence of double-strand breaks. Using bromodeoxyuracil (BrdU) incorporation together with measured increases in cell numbers, L5178Y cells also appeared to go through the cell cycle with unrepaired hOGG1-recognisable damage. Since unrepaired 8-OHdG can give rise to point mutations through G:C-->T:A transversions, it was also surprising that mutation could not be detected at the Na+/K+ATPase locus as determined by ouabain resistance. Some increases in strand breakage could be seen in the Comet assay by increasing the unwinding time, but only at highly toxic concentrations and to a much smaller extent than would be expected from the magnitude of the other genotoxic responses. It was considered unlikely that these anomalous observations were due to the inability of L5178Y cells to recognise 8-OHdG because these cells were shown to express mOGG1 and have functional cleavage activity at the adducted site. It appears that the responses of L5178Y cells to KBrO3 are complex and differ from those induced by other oxidising agents.