scholarly journals A Modern Genotoxicity Testing Paradigm: Integration of the High-Throughput CometChip® and the TGx-DDI Transcriptomic Biomarker in Human HepaRG™ Cell Cultures

2021 ◽  
Vol 9 ◽  
Author(s):  
Julie K. Buick ◽  
Andrew Williams ◽  
Matthew J. Meier ◽  
Carol D. Swartz ◽  
Leslie Recio ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Cultures ◽  
High Throughput ◽  
Aflatoxin B1 ◽  
Human Health Risk ◽  
Damage Sensing ◽  
Dna Damaging Agents ◽  
Chemical Evaluation ◽  
High Concentrations ◽  
Genotoxicity Testing

Higher-throughput, mode-of-action-based assays provide a valuable approach to expedite chemical evaluation for human health risk assessment. In this study, we combined the high-throughput alkaline DNA damage-sensing CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq®, as an integrated genotoxicity testing approach. We used metabolically competent differentiated human HepaRG™ cell cultures to enable the identification of chemicals that require bioactivation to cause genotoxicity. We studied 12 chemicals (nine DDI, three non-DDI) in increasing concentrations to measure and classify chemicals based on their ability to damage DNA. The CometChip® classified 10/12 test chemicals correctly, missing a positive DDI call for aflatoxin B1 and propyl gallate. The poor detection of aflatoxin B1 adducts is consistent with the insensitivity of the standard alkaline comet assay to bulky lesions (a shortcoming that can be overcome by trapping repair intermediates). The TGx-DDI biomarker accurately classified 10/12 agents. TGx-DDI correctly identified aflatoxin B1 as DDI, demonstrating efficacy for combined used of these complementary methodologies. Zidovudine, a known DDI chemical, was misclassified as it inhibits transcription, which prevents measurable changes in gene expression. Eugenol, a non-DDI chemical known to render misleading positive results at high concentrations, was classified as DDI at the highest concentration tested. When combined, the CometChip® assay and the TGx-DDI biomarker were 100% accurate in identifying chemicals that induce DNA damage. Quantitative benchmark concentration (BMC) modeling was applied to evaluate chemical potencies for both assays. The BMCs for the CometChip® assay and the TGx-DDI biomarker were highly concordant (within 4-fold) and resulted in identical potency rankings. These results demonstrate that these two assays can be integrated for efficient identification and potency ranking of DNA damaging agents in HepaRG™ cell cultures.

scholarly journals Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

AMB Express ◽  
2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Josué Daniel Mora-Garduño ◽  
Jessica Tamayo-Nuñez ◽  
Felipe Padilla-Vaca ◽  
Fátima Berenice Ramírez-Montiel ◽  
Ángeles Rangel-Serrano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Water Samples ◽  
Membrane Integrity ◽  
Analytical Techniques ◽  
Bacterial Cells ◽  
Reporter Strain ◽  
Dna Damaging Agents ◽  
Reporter Strains ◽  
High Concentrations

AbstractThe presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

scholarly journals Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast

2021 ◽  
Vol 1 (2) ◽  
pp. 225-238
Author(s):  
Mohsen Hooshyar ◽  
Daniel Burnside ◽  
Maryam Hajikarimlou ◽  
Katayoun Omidi ◽  
Alexander Jesso ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Remodeling ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Repair Efficiency ◽  
Chromosomal Breaks

DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.

scholarly journals Recombinogenic Effects of DNA-Damaging Agents Are Synergistically Increased by Transcription inSaccharomyces cerevisiae: New Insights Into Transcription-Associated Recombination

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 457-466 ◽  
Author(s):  
M García-Rubio ◽  
P Huertas ◽  
S González-Barrera ◽  
A Aguilera
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Dna Sequence ◽  
Direct Repeat ◽  
Methyl Methanesulfonate ◽  
Immunoglobulin Genes ◽  
Developmentally Regulated ◽  
Dna Damaging Agents ◽  
Regulated Promoters ◽  
Synergistic Increase

AbstractHomologous recombination of a particular DNA sequence is strongly stimulated by transcription, a phenomenon observed from bacteria to mammals, which we refer to as transcription-associated recombination (TAR). TAR might be an accidental feature of DNA chemistry with important consequences for genetic stability. However, it is also essential for developmentally regulated processes such as class switching of immunoglobulin genes. Consequently, it is likely that TAR embraces more than one mechanism. In this study we tested the possibility that transcription induces recombination by making DNA more susceptible to recombinogenic DNA damage. Using different plasmid-chromosome and direct-repeat recombination constructs in which transcription is driven from either the PGAL1- or the Ptet-regulated promoters, we haveshown that either 4-nitroquinoline-N-oxide (4-NQO) or methyl methanesulfonate (MMS) produces a synergistic increase of recombination when combined with transcription. 4-NQO and MMS stimulated recombination of a transcriptionally active DNA sequence up to 12,800- and 130-fold above the spontaneous levels observed in the absence of transcription, whereas 4-NQO and MMS alone increased recombination 193- and 4.5-fold, respectively. Our results provide evidence that TAR is due, at least in part, to the ability of transcription to enhance the accessibility of DNA to exogenous chemicals and internal metabolites responsible for recombinogenic lesions. We discuss possible parallelisms between the mechanisms of induction of recombination and mutation by transcription.

Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents

1989 ◽  
Vol 9 (10) ◽  
pp. 4196-4203
Author(s):  
A J Fornace ◽  
D W Nebert ◽  
M C Hollander ◽  
J D Luethy ◽  
M Papathanasiou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Growth ◽  
Growth Arrest ◽  
Homozygous Deletion ◽  
Negative Control ◽  
Cdna Clones ◽  
Growth Cessation ◽  
Dna Damaging Agents ◽  
Mammalian Cell Growth ◽  
Serum Reduction

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Exposure of mammalian cell cultures to benzo[a] and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanosine formation

1995 ◽  
Vol 16 (1) ◽  
pp. 133-137 ◽  
Author(s):  
Robert J. Mauthe ◽  
Vanessa M. Cook ◽  
Stephanie L. Coffing ◽  
William M. Baird
Keyword(s):  
Dna Damage ◽  
Mammalian Cell ◽  
Cell Cultures ◽  
Oxidative Dna Damage ◽  
Mammalian Cell Cultures

scholarly journals Arsenic Biotransformation as a Cancer Promoting Factor by Inducing DNA Damage and Disruption of Repair Mechanisms

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Victor D. Martinez ◽  
Emily A. Vucic ◽  
Marta Adonis ◽  
Lionel Gil ◽  
Wan L. Lam
Keyword(s):  
Dna Damage ◽  
Drinking Water ◽  
Repair Process ◽  
Dna Methyltransferases ◽  
Health Concern ◽  
Dna Damage And Repair ◽  
Repair Mechanisms ◽  
Contaminated Drinking Water ◽  
High Concentrations ◽  
Epigenetic Patterns

Chronic exposure to arsenic in drinking water poses a major global health concern. Populations exposed to high concentrations of arsenic-contaminated drinking water suffer serious health consequences, including alarming cancer incidence and death rates. Arsenic is biotransformed through sequential addition of methyl groups, acquired from s-adenosylmethionine (SAM). Metabolism of arsenic generates a variety of genotoxic and cytotoxic species, damaging DNA directly and indirectly, through the generation of reactive oxidative species and induction of DNA adducts, strand breaks and cross links, and inhibition of the DNA repair process itself. Since SAM is the methyl group donor used by DNA methyltransferases to maintain normal epigenetic patterns in all human cells, arsenic is also postulated to affect maintenance of normal DNA methylation patterns, chromatin structure, and genomic stability. The biological processes underlying the cancer promoting factors of arsenic metabolism, related to DNA damage and repair, will be discussed here.

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