Hprt Mutant Research Articles

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N-ethyl-N-nitrosourea.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

In Vitro Effects of Zirconia Nanoparticles: Uptake, Genotoxicity, and Mutagenicity in V-79 cells.

Zirconia nanoparticles are used in various industrial and biomedical applications such as dental implants, thermal barrier sprays, and fuel cells. The interaction of nanoparticles with the environment and humans is inevitable. Despite the enormous application potential of these nanoparticles, there are still some gaps in the literature regarding potential toxicological mechanisms and the genotoxicity of zirconia nanoparticles. The lung is one of the main exposure routes to nanomaterials; therefore, the present study was designed to determine the genotoxic and mutagenic effect of zirconia NPs in V-79 lung cells. Zirconia nanoparticles showed significant internalization in cells at 100μg/mL and 150μg/mL concentrations. Zirconia nanoparticles showed low cytotoxicity and were found to generate ROS in V-79 cells. In alkaline comet assay, zirconia nanoparticles (10μg/mL, 50μg/mL, and 100μg/mL) exposed cells exhibited significant DNA strand breaks, while the neutral comet assay, which was used for double-strand break assessment, only revealed significant damage at 100μg/mL. Chromosomal aberration induced by zirconia nanoparticles mainly resulted in the generation of gaps, few fragments, and breaks which signifies the low clastogenic activity of these nanoparticles in the V-79 cell line. In MN assay, zirconia nanoparticles resulted in no significant micronuclei induction at any given concentration. In the HPRT mutation assay, the particle shows a dose-dependent increase in the mutant frequency. It is evident from the result that zirconia nanoparticles cause dose-dependent cytotoxicity and genotoxicity, but still, more studies are needed to evaluate the clastogenic potential and the possible mechanism involved.

Structural Changes in HPRT Gene of V79 Cells After Irradiation With Heavy Ions—Immediate and Delayed Effects

The radiobiological effects of accelerated ions with high charge and high energy (HZE) on mammalian cells and their propagation in time are still not sufficiently explained and attract great deal of attention. This work aims to compare the immediate and delayed effects with emphasis on the latter. As shown by our group, the dependence of mutant fraction on expression time after irradiation may have interesting, non-monotonic, character depending on LET (linear energy transfer) of the used heavy ions. We speculate that this phenomenon may occur due to the induced genomic instability. Another area of our research is the study of the DNA structural changes in these mutants induced at different expression times. Chinese hamster V79 cells were irradiated with accelerated ions 11B, 18O, 20Ne, and gamma radiation. The LET was ranging from 0.23 keV/μm of 60Co gamma rays up to 136 keV/μm of 20Ne ions. DNA of unique HPRT mutants was isolated, concentration measured, HPRT exons amplified, and analyzed at several different time points, up to about 40 days, after exposure. Over 1200 HPRT mutants were analyzed for deletions of exons and sorted into three main categories: partial deletion, PD—with deletion of one to eight exons; total deletions, TD—with all nine exons deleted; and no deletions—no change in the HPRT structure observed. In general, the number of samples with partial deletion was increasing with LET of the used radiation, suggesting that higher energy deposition to the cell nucleus is more likely to cause larger structural changes. In the case of total deletions, increase in their number with LET was observed up to LET ∼115 keV/μm followed by a sharp decrease. The samples were also analyzed for the distribution of deletions, in particular exons at various expression times, the so-called mutational patterns. Hypothesis of the mechanisms behind observed phenomena is given, and possible implications for further research are discussed.

In vitro analysis reveals necroptotic signaling does not provoke DNA damage or HPRT mutations

Most anticancer drugs provoke apoptotic signaling by damaging DNA or other means. Genotoxic therapies may enhance a patient’s risk of developing “therapy-related cancers” due to the accumulation of oncogenic mutations that may occur in noncancerous cells. Mutations can also form upon apoptotic signaling due to sublethal caspase activity, implying that apoptosis activating drugs may also be oncogenic. Necroptosis is a different way of killing cancer cells: this version of caspase-independent cell death is characterized by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase-like domain protein (MLKL) activation, leading to cell membrane rupture and controlled cell lysis. The mutagenic potential of sublethal necroptotic signaling has not yet been directly investigated. Smac mimetics drugs, which activate apoptotic or necroptotic cell death, do not induce mutations but the mechanistic basis for this lack of mutagenic activity has not been determined. In this study, we compared the mutagenic potential of these two cell death pathways by engineering cells to activate either apoptotic or necroptotic signaling by exposing them to Smac mimetics with or without TNFα, and/or enforcing or preventing expression of apoptotic or necroptotic regulators. We discovered that sublethal concentrations of Smac mimetics in contexts that activated apoptotic signaling provoked DNA damage and mutations in surviving cells. Mutagenesis was dependent on executioner caspase activation of the nuclease CAD. In contrast, RIPK3- and MLKL-dependent necroptotic signaling following Smac mimetic treatment was not mutagenic. Likewise, DNA damage was not provoked in cells expressing a lethal constitutively active MLKL mutant. These data reveal that cells surviving sublethal necroptotic signaling do not sustain genomic damage and provide hope for a reduced risk of therapy-related malignancies in patients treated with necroptosis-inducing drugs.

Evaluation of HPRT Gene mutation and Comet Assay in Some Breast Cancer Patients Undergoing Radiotherapy

Introduction: Radiotherapy is a toxic cellular treatment that destroys rapidly dividing cells such as cancer cells. The strategy for HPRT mutation examination in human’s cells was commonly used to establish in vivo history along with mutation rates in children and adults inhabitants subjected to experienced and unfamiliar external mutagens. As just a practical approach to human biosurveillance studies, the comet assay has been introduced over the past years as biomarker to identify ionizing radiation Consequences with Patients undergoing radiotherapy in breast cancer. Materials and Method: This study was carried out on thirty Iraqi women patients with BC patients undergoing radiotherapy about 20-30 Gy locally gamma cells at Al-Amel National Hospital for cancer Management in Baghdad during time 2-13 years, non-smokers and non- alcoholic, aged (35-55 year), with stage (grade) I-III, as well as thirty apparently healthy individuals females collected randomly from population living Baghdad, Old age (35-55) Which are non-alcoholic non-smokers as group of control. Around the ongoing research comet-test and hprt mutation test could be applied to study damages in DNA for genetic two endpoints For patients with cancer throughout radiation therapy. Results: The present study showed significant increase (p<0.05) in the HPRT gene mutation assay for the in breast cancer patients undergoing radiotherapy as compared with the control group. Also there were found that the values of comet tail moment and tail length was increased significantly (p<0.05) in the human lymphocyte in these for breast cancer patients undergoing radiotherapy as compared with the control group. Conclusions: Present results revealed that there is a probability of utilizing human lymphocytes changes as useful biodosimetric markers for the detection of human exposure to ionizing radiation, the data gathered also demonstrated the utility of the HPRT gene mutation and (Alkaline) comet test as just a precise alternative biological marker for routine preventative care of cancer patients receiving radiation therapy.

Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

Gene (HPRT) and chromosomal (MN) mutations of nickel metal powder in V79 Chinese hamster cells

Nickel metal is a naturally occurring element used in many industrial and consumer applications. Human epidemiological data and animal cancer bioassays indicate that nickel metal is not likely to be a human carcinogen. Yet, nickel metal is classified as a suspected human carcinogen (CLP) and possibly carcinogenic to humans (IARC). There are no reliable studies on the potential for nickel metal to induce gene and micronucleus (MN) mutations. To fill these datagaps and increase our understanding of the mechanisms underlying the lack of nickel metal carcinogenicity, gene and micronucleus mutation studies were conducted with nickel metal powder (N36F) in V79 Chinese Hamster cells following OECD 476 and 487 guidelines, respectively, under GLP. Gene mutation at the hprt locus was tested, with and without metabolic activation, after 4-h treatment with 0.05–2.5 mM nickel metal powder. Cytokinesis-block MN frequency following exposure to 0.25–1.5 mM nickel metal was tested after 4-h treatment, with and without metabolic activation, followed by a 24-h treatment without metabolic activation. In the gene mutation assay, there were modest increases in hprt mutants observed at some test concentrations, not exceeding 2.2-fold, which were either within the historical control values and/or showed no concentration-response trend. The positive controls showed increases of at least 7-fold. Likewise, no increases in the MN frequency exceeding 1.5-fold were observed with nickel metal, with no concentration-response trends. Taking these results together, it can be concluded that nickel metal is non-mutagenic and does not cause gene nor chromosomal mutations.

Radiation-Induced Mutagenesis in Mammalian Cells after Exposure to Accelerated Ions with Different LET

The mutagenic effect of accelerated heavy charged particles on the Chinese hamster V79 cell line is studied. The induction of HPRT mutations in irradiated cells for a long expression time (up to 45 days) after exposure to radiation with different LET are analyzed. The maximum yield of mutant clones is observed at different expression times, depending on the characteristics of ionizing radiation; in this case, the position of the maximum is shifted toward later time intervals with increasing LET of radiation.

Analysis of In Vivo Mutation in the Hprt and Tk Genes of Mouse Lymphocytes.

Determining mutant frequencies in endogenous reporter genes is a tool for identifying potentially genotoxic environmental agents, and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe a high-throughput method for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation at both the Hprt and Tk genes.

223 Lesch-Nyhan syndrome: from patient to mouse model

Abstract Introduction. HPRT1 is a Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) coding gene, mutations of which cause, among other diseases, Lesch-Nyhan syndrome. LNS is a severe X-linked recessive neurological disorder. Alignment shows 98.6% homology of human and murine protein sequences. Objectives. We assume that murine model of this human disease should be developed. So, our purpose is to create a transgenic mouse model of Lesch-Nyhan syndrome by CRISPR/Cas9 genome editing system. Methods. The BLAST was used to find homologous mutation in murine HPRT1 gene comparing to Human one. X-ray crystallographic structural model of the HPRT1 was used as template for M4T Raptor algorithm to generate predicted structure of mutated protein. Models were visualized in PyMol (Schrodinger, Portland, OR). Lack of enzymatic activity of the HGPRT could be caused via troubled homodimerization. Absence of the aliphatic Valine could be the reason of the hindered monomers interaction. Genetic construction based on the px330 plasmid was brought using microinjection method in mouse fertilized ovum for producing primary transgenic organisms. Results. The CRISPR/Cas9 system was specifically designed to carry out mutations in HPRT. The resulting genetic construct was introduced into the fertilized mouse ovum to obtain primary transgenic organisms. This mouse was obtained at the Institute of Biology of the Russian Academy of Sciences. Conclusion. Homologous mutation in human and murine HPRT1 gene resulting into comparable conformational change in the protein model structure was revealed, so murine personalized model of the Lesch-Nyhan syndrome could be developed. Structural changes can be further studied to provide treatment strategy for people suffering from Lesch-Nyhan syndrome. This study was supported by Russian Science Foundation (Grant #17-75-20249).

Role of Human N-Acetyltransferase 2 Genetic Polymorphism on Aromatic Amine Carcinogen-Induced DNA Damage and Mutagenicity in a Chinese Hamster Ovary Cell Mutation Assay.

Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235-245, 2020. © 2019 Wiley Periodicals, Inc.

Abstract 3509: An unexpected effect of low-dose arsenic on genomic integrity

Abstract Background: Arsenic trioxide is currently used to treat acute promyelocytic leukemia and is known as a cytotoxic agent. Arsenic is also known as a carcinogen. However, a cytoprotective effect of low-dose arsenic (LDA) has also been observed in some studies. Our previous studies have found that pretreatment with LDA before DNA damaging agents such as chemotherapy and radiation therapy protects normal cells without impairing the cancer killing effects. We have demonstrated that intact p53 function is required in this process, and therefore, LDA does not protect cancer cells as most of them have mutated or dysfunctional p53 pathway. We have also reported that LDA helps protect the telomere from enhanced erosion by DNA damaging agents in ConA-activated normal human lymphocytes. As enhanced telomere shortening is associated with genomic instability, we decided to investigate the effect of LDA in genomic integrity. Methods: Mouse embryonic stem cells (mESCs) with a mutator phenotype in DNA repair pathways (MSH2-/-, Rad51KA and Ku70null) and their wildtype counterparts were utilized for cell viability studies and HPRT minigene mutation assay. Cell viability under different doses of LDA (0-1000nM) and radiation (0-4Gy) was firstly examined by colony formation assay. DNA damage and DNA repair capacity were also measured by comet assay in MSH2-/- mESCs. TailDNA% was analyzed by OpenComet and used to describe the comet feature. Results: Arsenic had little effect on the cell viability at doses ≤200nM in all tested cell lines. In MSH2-/- mESCs, there was no significant difference between the tailDNA% of cells treated with 200nM LDA (11.41±0.52%) and the control (10.82±0.49%) immediately after radiation. However, LDA treatment significantly decreased the tailDNA% 30 minutes after radiation (4.68±0.35% vs. 7.29±0.51%, Student’s test P&amp;lt;0.0014). While HPRT mutation frequency was not detectable in radiation-treated Rad51KA, Rad51wt and MSH2wt mESCs, LDA decreased the fraction of 6-Thioguanine resistant cells after radiation in MSH2-/-, Ku70null and Ku70res mESCs, suggesting fewer radiation-induced mutants were formed after LDA treatment. Discussion: Our data suggest that brief use of LDA enhances DNA repair activities and provides protection from radiation-induced mutations in mESCs. The inability to correctly repair DNA damage from chemotherapy and radiation therapy has been attributed to the development of therapy related secondary malignancy. Helping maintain genomic integrity with LDA during chemotherapy and radiation therapy may have a role in reducing therapy related secondary malignancy. Further work is in progress to test the potential use of LDA as a genome protector in vivo. Citation Format: Hang Su, Meijun Long, Mi Young Son, Teresa C Marple, Paul Hasty, Chul Soo Ha. An unexpected effect of low-dose arsenic on genomic integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3509.

A review of the genotoxic potential of 1,4-naphthoquinone

1,4-Naphthoquinone (1,4-NQ; CAS RN 130-15-4), a derivative of naphthalene, is a commonly used pre-cursor in industrial processes. Since the early 1980’s 1,4-NQ has been tested in a number of genotoxicity assays, both in vitro and in vivo. There is strong evidence that 1,4-NQ does not induce gene mutations in bacteria or mammalian cells in vitro with predominantly negative Ames tests and negative Hprt and tk mutation studies. However, there is clear evidence of a clastogenic response in vitro from positive micronucleus, sister chromatid exchange and chromosome aberration assays. 1,4-NQ-treated mice and hamsters were, however, negative for micronucleus or chromosomal aberration induction in GLP-compliant studies with clear evidence of target tissue exposure, suggesting an in vitro only effect. Evidence indicates that the mechanism of in vitro clastogenicity is predominantly via ROS generation, and since in vitro mammalian cell tests systems have poor anti-oxidant defence mechanisms, they are particularly sensitive to oxidative DNA damage. On the other hand, healthy mammalian tissues have more efficient anti-oxidant defence mechanisms, and therefore it is not surprising that 1,4-NQ is not genotoxic in vivo.

Carbon Disulfide (CS2) Induced Chromosomal Alterations and Apoptosis in Circulated Blood Lymphocytes of Personnel Working in Viscose Industry

Background and objective: Carbon disulfide (CS2) is a naturally occurring chemical substance in the environment and also an important endogenous substance in the human body. This study was done to collect data on the effects and to find a possible relationship between in-vivo CS2 induced apoptosis and genotoxic effects.Material and method: Circulated blood lymphocytes (PBL) of 41 workers occupationally exposed to Carbon disulfide (CS2) in viscose industry were investigated. The participants involved three groups. The first group included 41 participants exposed to CS2 together with various confounding factors; the second group comprised of 41 participants who were inhabitant of viscose industry and were partially exposed to CS2 in long periods; and third group consisting 41 participants as control group who were not exposed to any kind of chemicals and radiation hazards. Ambient air concentrations of CS2 were measured in different workplaces. Measures of genotoxicity included the determination of the frequencies of chromosomal aberrations (CA), sister-chromatid exchange (SCE), HPRT mutations (variant frequency, VF), and measurement of UV-induced unscheduled DNA-repair synthesis (UDS). The percentages of premature centromere division (PCD) and of cells with a high frequency of SCE (HF/SCE) were also scored. Apoptosis and c l proliferation were determined by flow cytometry.Results: In both CS2 exposed groups, the apoptotic activity and the CA levels in PBLs were significantly higher than in controls. CA was mostly breaks of the chromatid type. In group II, CA was slightly lower in comparison with group I, which can be attributed to a different rate of elimination of damaged lymphocytes as a consequence of CS2 induced apoptotic activity.Conclusion: The results demonstrate that exposure to CS2 induced apoptosis and CA, indicating an excess cancer risk among participants occupationally exposed to CS2. The results also emphasized the importance of the measurement of occupationally exposed pollutants such as CS2 in order to avoid genotoxic effects in the workers.

Genotoxic and mutagenic properties of Ni and NiO nanoparticles investigated by comet assay, γ-H2AX staining, Hprt mutation assay and ToxTracker reporter cell lines.

Nickel (Ni) compounds are classified as carcinogenic to humans but the underlying mechanisms are still poorly understood. Furthermore, effects related to nanoparticles (NPs) of Ni have not been fully elucidated. The aim of this study was to investigate genotoxicity and mutagenicity of Ni and NiO NPs and compare the effect to soluble Ni from NiCl2. We employed different models; i.e., exposure of (1) human bronchial epithelial cells (HBEC) followed by DNA strand break analysis (comet assay and γ‐H2AX staining); (2) six different mouse embryonic stem (mES) reporter cell lines (ToxTracker) that are constructed to exhibit fluorescence upon the induction of various pathways of relevance for (geno)toxicity and cancer; and (3) mES cells followed by mutagenicity testing (Hprt assay). The results showed increased DNA strand breaks (comet assay) for the NiO NPs and at higher doses also for the Ni NPs whereas no effects were observed for Ni ions/complexes from NiCl2. By employing the reporter cell lines, oxidative stress was observed as the main toxic mechanism and protein unfolding occurred at cytotoxic doses for all three Ni‐containing materials. Oxidative stress was also detected in the HBEC cells following NP‐exposure. None of these materials induced the reporter related to direct DNA damage and stalled replication forks. A small but statistically significant increase in Hprt mutations was observed for NiO but only at one dose. We conclude that Ni and NiO NPs show more pronounced (geno)toxic effects compared to Ni ions/complexes, indicating more serious health concerns. Environ. Mol. Mutagen. 59:211–222, 2018. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society

PARP1 protects from benzo[a]pyrene diol epoxide-induced replication stress and mutagenicity

Poly(ADP-ribosyl)ation (PARylation) is a complex and reversible posttranslational modification catalyzed by poly(ADP-ribose)polymerases (PARPs), which orchestrates protein function and subcellular localization. The function of PARP1 in genotoxic stress response upon induction of oxidative DNA lesions and strand breaks is firmly established, but its role in the response to chemical-induced, bulky DNA adducts is understood incompletely. To address the role of PARP1 in the response to bulky DNA adducts, we treated human cancer cells with benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE), which represents the active metabolite of the environmental carcinogen benzo[a]pyrene [B(a)P], in nanomolar to low micromolar concentrations. Using a highly sensitive LC-MS/MS method, we revealed that BPDE induces cellular PAR formation in a time- and dose-dependent manner. Consistently, PARP1 activity significantly contributed to BPDE-induced genotoxic stress response. On one hand, PARP1 ablation rescued BPDE-induced NAD+ depletion and protected cells from BPDE-induced short-term toxicity. On the other hand, strong sensitization effects of PARP inhibition and PARP1 ablation were observed in long-term clonogenic survival assays. Furthermore, PARP1 ablation significantly affected BPDE-induced S- and G2-phase transitions. Together, these results point towards unresolved BPDE-DNA lesions triggering replicative stress. In line with this, BPDE exposure resulted in enhanced formation and persistence of DNA double-strand breaks in PARP1-deficient cells as evaluated by microscopic co-localization studies of 53BP1 and γH2A.X foci. Consistently, an HPRT mutation assay revealed that PARP inhibition potentiated the mutagenicity of BPDE. In conclusion, this study demonstrates a profound role of PARylation in BPDE-induced genotoxic stress response with significant functional consequences and potential relevance with regard to B[a]P-induced cancer risks.

Delayed effects of accelerated heavy ions on the induction of HPRT mutations in V79 hamster cells

Fundamental research on the harmful effects of ionizing radiation on living cells continues to be of great interest. Recently, priority has been given to the study of high-charge and high-energy (HZE) ions that comprise a substantial part of the galactic cosmic ray (GCR) spectra that would be encountered during long-term space flights. Moreover, predictions of the delayed genetic effects of high linear energy transfer (LET) exposure is becoming more important as heavy ion therapy use is increasing. This work focuses mainly on the basic research on the delayed effects of HZE ions on V79 Chinese hamster cells, with emphasis on the induction of HPRT mutations after prolonged expression times (ET). The research was conducted under various irradiation conditions with accelerated ions 18O (E=35.2MeV/n), 20Ne (E=47.7MeV/n and 51.8MeV/n), and 11B (E=32.4MeV/n), with LET in the range from 49 to 149 keV/μm and with 60Co γ-rays. The HPRT mutant fractions (MF) were detected in irradiated cells in regular intervals during every cell culture recultivation (every 3days) up to approximately 40days (70–80 generations) after irradiation. The MF maximum was reached at different ET depending on ionizing radiation characteristics. The position of the maximum was shifting towards longer ET with increasing LET. We speculate that the delayed mutations are created de novo and that they are the manifestation of genomic instability. Although the exact mechanisms involved in genomic instability initiation are yet to be identified, we hypothesize that differences in induction of delayed mutations by radiations with various LET values are related to variations in energy deposition along the particle track. A dose dependence of mutation yield is discussed as well.

In vitro genotoxicity of airborne Ni-NP in air-liquid interface.

Studies using advanced toxicological methods enabling in vitro conditions that are more realistic are currently needed for understanding the risks of pulmonary exposure to airborne nanoparticles. Owing to the carcinogenicity of certain nickel compounds, the increased production of nickel nanoparticles (Ni‐NPs) raises occupational safety concerns. The aim of this study was to investigate the genotoxicity of airborne Ni‐NPs using a recently developed air–liquid interface exposure system. The wild‐type Chinese hamster lung fibroblast cell line (V79) was used and cytotoxicity, DNA damage and mutagenicity were studied by testing colony forming efficiency, alkaline DNA unwinding and HPRT mutation assays, respectively. Additionally, co‐exposure to a PARP‐1 inhibitor was performed to test possible involvement of base excision repair (BER) in repair of Ni‐induced DNA damage. The results showed that cell viability was reduced significantly (to 45% and 46%) after 48 hours Ni‐NP exposure at concentrations of 0.15 and 0.32 μg cm−2. DNA damage was significantly increased after Ni‐NP exposure in the presence of the BER inhibitor indicating that Ni‐NP‐induced DNA damages are subsequently repaired by BER. Furthermore, there was no increased HPRT mutation frequency following Ni‐NP exposure. In conclusion, this study shows that Ni‐NP treatment of lung fibroblasts in an air–liquid interface system that mimics real‐life exposure, results in increased DNA strand breaks and reduced cellular viability. These DNA lesions were repaired with BER in an error‐free manner without resulting in mutations. This study also underlines the importance of appropriate quantification of the actual exposure concentrations during air–liquid interface exposure studies.

Intracellular and extracellular factors influencing the genotoxicity of nitric oxide and reactive oxygen species.

A number of factors affect cellular responses to nitric oxide (NO•) and reactive oxygen species (ROS), including their source, concentration, cumulative dose, target gene and biological milieu. This limits the extrapolation of data to in vivo pathological states in which NO• and ROS may be important. The present study investigated lethality and mutagenesis in the HPRT and TK1 genes of human lymphoblastoid TK6 cells exposed to NO• and ROS derived from two delivery methods: A reactor system and a Transwell™ co-culture. The delivery of NO• into the medium at controlled steady-state concentrations (given in µM/min) and the production of NO• and ROS by activated macrophages, resulted in a time-dependent decrease in total cell numbers, and an increase in mutation frequency (MF), compared with untreated controls. This increase in MF was effectively suppressed by N-methyl-L-arginine monoacetate. Single base substitutions were the most common type of spontaneous and NO• induced mutations in HPRT, followed by exon exclusions and small deletions in both delivery systems. Among the single base pair substitutions, an equal frequency of four types of single base substitutions were identified in TK6 cells exposed to NO• delivered by the reactor system, whereas G:C to T:A transversions and A:T to G:C transitions were more frequent in the co-culture system. Taken together, these results demonstrate that both the delivery method of NO• and ROS, and the target genes are determinants of observed cytotoxic and mutagenic responses, indicating that these parameters need to be considered in assessing the potential effects of NO• and ROS in vivo.