Potent mutagenicity of an azide, 3-azido-1,2-propanediol, in human TK6 cells.

Sodium azide is a strong mutagen that has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolically converted to the proximate mutagen azidoalanine, which requires further bioactivation to a putative ultimate mutagen that remains elusive. The nature of the DNA modifications induced by azides leading to mutations is also unknown.

Comparison of the frequencies of ENU-induced point mutations in male germ cells and inherited germline mutations in their offspring.

Background: Gene mutations induced in germ cells may be transmitted to the next generation and cause adverse effects such as genetic diseases. Certain mutations may result in infertility or death in early development. Thus, the mutations may not be inheritable.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species.

New homozygous gpt delta transgenic rat strain improves an efficiency of the in vivo mutagenicity assay.

Background: Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

Background: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies).

Comparative study of cytotoxic effects induced by environmental genotoxins using XPC- and CSB-deficient human lymphoblastoid TK6 cells.

Background: The human genome is constantly exposed to numerous environmental genotoxicants. To prevent the detrimental consequences induced by the expansion of damaged cells, cellular protective systems such as nucleotide excision repair (NER) exist and serve as a primary pathway for repairing the various helix-distorting DNA adducts induced by genotoxic agents. NER is further divided into two sub-pathways, namely, global genomic NER (GG-NER) and transcription-coupled NER (TC-NER).

Absence of mutagenicity of multi-walled carbon nanotubes single intratracheal instillation study using F344 delta rats.

Introduction: It is known that fibrous particles of micrometer length, such as carbon nanotubes, which have same dimensions as asbestos, are carcinogenic. Carcinogenicity of nanomaterials is strongly related to inflammatory reactions; however, the genotoxicity mechanism(s) is unclear. Indeed, inconsistent results on genotoxicity of multi-walled carbon nanotubes (MWCNTs) have been shown in several reports.

Evaluation of mutagenicity of acrylamide using RBC Pig-a and PIGRET assays by single peroral dose in rats.

The Pig-a gene mutation assay, a powerful tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs), which are deficient in glycosylphosphatidylinositol anchored proteins caused by mutation(s) in the Pig-a gene. Various approaches for measuring cells with mutated Pig-a gene have been developed. The Pig-a assay targeting concentrated reticulocytes - the PIGRET assay - has the potential to detect genotoxicity in early stages of the study.

The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×10 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis.

Dose-dependent de novo germline mutations detected by whole-exome sequencing in progeny of ENU-treated male gpt delta mice.

Germline mutations are an important component of genetic toxicology; however, mutagenicity tests of germline cells are limited. Recent advances in sequencing technology can be used to detect mutations by direct sequencing of genomic DNA (gDNA). We previously reported induced de novo mutations detected using whole-exome sequencing in the offspring of N-ethyl-N-nitrosourea (ENU)-treated mice in a single-dose experiment (85mg/kg, i.

Monitoring genotoxicity in patients receiving chemotherapy for cancer: application of the PIG-A assay.

The recently introduced Pig-a in vivo gene mutation assay measures endogeneous mutations of Pig-a (human, PIG-A), an X-linked gene that is conserved across species from rodents to humans. Flow cytometric analysis enables the enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient erythrocytes, resulting from a mutation in Pig-a/PIG-A, in only a few microliters of peripheral blood. Pig-a/PIG-A mutations appear to function in a neutral manner, allowing evaluation of the accumulated genotoxic effects of repeated exposures.

Evaluation of in vivo genotoxicity induced by N-ethyl-N-nitrosourea, benzo[a]pyrene, and 4-nitroquinoline-1-oxide in the Pig-a and gpt assays.

The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear.

Restoration of mismatch repair functions in human cell line Nalm-6, which has high efficiency for gene targeting.

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields.

Mutations in UVSSA cause UV-sensitive syndrome and destabilize ERCC6 in transcription-coupled DNA repair.

UV-sensitive syndrome (UV(S)S) is an autosomal recessive disorder characterized by photosensitivity and deficiency in transcription-coupled repair (TCR), a subpathway of nucleotide-excision repair that rapidly removes transcription-blocking DNA damage. Cockayne syndrome is a related disorder with defective TCR and consists of two complementation groups, Cockayne syndrome (CS)-A and CS-B, which are caused by mutations in ERCC8 (CSA) and ERCC6 (CSB), respectively. UV(S)S comprises three groups, UV(S)S/CS-A, UV(S)S/CS-B and UV(S)S-A, caused by mutations in ERCC8, ERCC6 and an unidentified gene, respectively.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.

Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.

Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH).

Genetic analysis reveals an intrinsic property of the germinal center B cells to generate A:T mutations.

The immunoglobulin genes undergo a high frequency of point mutations at both C:G and A:T pairs in the germinal center (GC) B cells. This hypermutation process is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. Replication of this lesion, or its repair intermediate the abasic site, could introduce C:G mutations but the mechanisms leading to mutations at non-damaged A:T pairs remain elusive.

Role of DNA polymerase theta in tolerance of endogenous and exogenous DNA damage in mouse B cells.

DNA polymerase theta (Poltheta) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Poltheta in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Poltheta polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death.