Human Lymphoblastoid TK6 Cells Research Articles

Preliminary results of space experiment: Implications for the effects of space radiation and microgravity on survival and mutation induction in human cells

In view of the concern for the health of astronauts that may one day journey to Mars or the Moon, we investigated the effect that space radiation and microgravity might have on DNA damage and repair. We sent frozen human lymphoblastoid TK6 cells to the International Space Station where they were maintained under frozen conditions during a 134-day mission (14 November 2008 to 28 March 2009) except for an incubation period of 8 days under 1G or μG conditions in a CO 2 incubator. The incubation period started after 100 days during which the cells had been exposed to 54 mSv of space radiation. The incubated cells were then refrozen, returned to Earth, and compared to ground control samples for the determination of the influence of microgravity on cell survival and mutation induction. The results for both varied from experiment to experiment, yielding a large SD, but the μG sample results differed significantly from the 1G sample results for each of 2 experiments, with the mean ratio of μG to 1G being 0.55 for the concentration of viable cells and 0.59 for the fraction of thymidine kinase deficient (TK −) mutants. Among the mutants, non-loss of zygosity events (point mutations) were less frequent (31%) after μG incubation than after 1G incubation, which might be explained by the influence of μG on cellular metabolic or physiological function. Additional experiments are needed to clarify the effect of μG interferes on DNA repair.

Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels

Nowadays, there is clear progress in using the threshold concept in genetic toxicology, but its demonstration and acceptance in risk assessment is still under debate. Although it has been accepted for some non-DNA-reactive agents for which mechanisms of action were demonstrated, there is a growing weight of evidence to also support the existence of thresholded dose-responses for DNA-reactive agents. In this context, we have recently shown in human TK6 lymphoblastoid cells, that DNA-oxidizing agents [potassium bromate, bleomycin and hydrogen peroxide (via glucose oxidase)] produced non-linear dose-responses in the in vitro micronucleus test, thus allowing the determination of No-Observed-Genotoxic-Effect-Levels (NOGELs). Therefore, the aim of the present study was to focus on the analysis of thresholded dose–response curves in order to further investigate the existence of NOGELs for these same directly DNA-damaging agents, by use of other genotoxicity endpoints. Mutation frequency was determined after a 1-h treatment in the thymidine kinase (TK) gene-mutation assay. Primary DNA damage, especially oxidative DNA damage, was also assessed after 1h of treatment, followed – or not – by a 23-h recovery period, with the modified version of the comet assay (i.e. with the glycosylases Fpg and hOgg1). Overall, our analysis demonstrates that there is convincing evidence to support the existence of thresholded dose-responses for DNA-oxidizing agents. The determination of NOGELs depends on the genotoxic endpoint studied and consequently requires different genotoxicity assays performed concurrently. NOGELs could only be defined for the induction of chromosomal aberrations and gene mutations, i.e. for an effect-endpoint but not for primary DNA damage, i.e. for an exposure-endpoint. Further statistical analyses of these data are now required in order to draw conclusions on the exact level of the thresholds.

Effects of Simulated Microgravity on Expression Profile of MicroRNA in Human Lymphoblastoid Cells

This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison with static 1 × g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a high aspect ratio vessel (bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNAs was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22, miR-141, miR-618, and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using quantitative RT-PCR. Expression of several transcription factors including EGR2, ETS1, and c-REL was altered in simulated microgravity conditions. Taken together, the results reported here indicate that simulated microgravity alters the expression of miRNAs and genes in TK6 cells. To our knowledge, this study is the first to report the effects of simulated microgravity on the expression of miRNA and related genes.

Involvement of p53 function in different magnitude of genotoxic and cytotoxic responses in in vitro micronucleus assays

In in vitro micronucleus (MN) assays the sensitivity to MN induction or cytotoxicity can vary depending on the kind of cells employed. This study was conducted to examine the involvement of the p53 function in the different sensitivities between Chinese hamster lung (CHL) cells and human lymphoblastoid TK6 cells in MN assays. MN induction and cytotoxicity were compared using MN-inducing chemicals reported as DNA reactive clastogens, non-DNA reactive clastogens or aneugens. The study revealed that the maximum levels of MN induction in p53-compromised CHL cells were higher than those in p53-competent TK6 cells, but MN were significantly induced in TK6 cells at lower concentrations than in CHL cells. Most of the test chemicals produced a more severe cytotoxicity in TK6 cells, suggesting TK6 cells are more sensitive for cytotoxicity than CHL cells. An additional experiment with 9 MN inducers revealed that the magnitude of MN induction and cytotoxicity were comparable between p53-competent TK6 cells and its p53-null mutant NH32 cells at the same concentrations. Furthermore, the MN frequencies induced by methylmethane sulfonate, aphidicolin and hydroxyurea in NH32 cells were identical to those in TK6 cells at different recovery times. From these results, it is suggested that the p53 abrogation does not explain the difference in sensitivity to MN induction or cytotoxicity between CHL and TK6 cells. In this regard, p53 abrogated NH32 cells can be an option for the in vitro MN assay.

Induction of TK mutations in human lymphoblastoid TK6 cells by the rat carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX)

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water, is carcinogenic in rats and genotoxic in mammalian cells in vitro. In the current study, the mechanism of genotoxicity of MX in human lymphoblastoid TK6 cells was investigated by use of the Comet assay, the micronucleus test, and the thymidine kinase (TK) gene-mutation assay. MX induced a concentration-dependent increase in micronuclei and TK mutations. The lowest effective concentrations in the MN test and the TK gene-mutation assay were 37.5μM and 25μM, respectively. In the Comet assay, a slight although not statistically significant increase was observed in the level of DNA damage induced by MX in the concentration range of 25–62.5μM. Molecular analysis of the TK mutants revealed that MX induced primarily point mutations or other small intragenic mutations (61%), while most of the remaining TK mutants (32%) were large deletions at the TK locus, leading to the hemizygous-type loss-of-heterozygosity (LOH) mutations. These findings show that aside from inducing point mutations, MX also generates LOH at the TK locus in human cells and may thus cause the inactivation of tumour-suppressor genes by LOH.

Involvement of mismatch repair proteins in adaptive responses induced by N-methyl- N′-nitro- N-nitrosoguanidine against γ-induced genotoxicity in human cells

As humans are exposed to a variety of chemical agents as well as radiation, health effects of radiation should be evaluated in combination with chemicals. To explore combined genotoxic effects of radiation and chemicals, we examined modulating effects of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), a direct-acting methylating agent, against genotoxicity of γ-radiation. Human lymphoblastoid TK6 cells and its mismatch-deficient derivative, i.e., MT1 cells, were treated with MNNG for 24 h before they were exposed to γ-irradiation at a dose of 1.0 Gy, and the resulting genotoxicity was examined. In TK6 cells, the pretreatments with MNNG at low doses suppressed frequencies of the thymidine kinase (TK) gene mutation and micronucleus (MN) formation induced by γ-irradiation and thus the dose responses of TK and MN assays were U-shaped along with the pretreatment doses of MNNG. In contrast, the genotoxic effects of MNNG and γ-irradiation were additive in MT1 cells and the frequencies of TK mutations and MN induction increased along with the doses of MNNG. Apoptosis induced by γ-radiation was suppressed by the pretreatments in TK6 cells, but not in MT1 cells. The expression of p53 was induced and cell cycle was delayed at G2/M phase in TK6, but not in MT1 cells, by the treatments with MNNG. These results suggest that pretreatments of MNNG at low doses suppress genotoxicity of γ-radiation in human cells and also that mismatch repair proteins are involved in the apparent adaptive responses.

Comparative analysis of micronuclei and DNA damage induced by Ochratoxin A in two mammalian cell lines

The fungal toxin, Ochratoxin A (OTA), is a common contaminant in human food and animal feed. The present study evaluated micronucleus (MN) induction by OTA in comparison with its ability to induce cytotoxicity and DNA damage in two mammalian cell lines, CHO-K1-BH 4 Chinese hamster ovary cells and TK6 human lymphoblastoid cells. Micronuclei were evaluated by flow cytometry, cytotoxicity was estimated by relative population doubling (RPD), while direct DNA damage and oxidative DNA damage were measured with the Comet assay, performed without and with digestion by formamidopyrimidine-DNA glycosylase (fpg). For the MN and cytotoxicity measurements, the cell lines were treated for 24 h (CHO cells) or 27 h (TK6 cells) with 5–25 μM OTA in the absence of exogenous metabolic activation. The OTA treatments resulted in concentration–responsive increases in cytotoxicity, with higher concentrations of the agent being more cytotoxic in CHO cells than TK6 cells. 15 μM OTA produced positive responses for MN induction and hypodiploid events (a measure of aneugenicity) in both cell lines; this concentration of OTA also produced cytotoxicity near to the recommended limit for the assay (45 ± 5% RPD). A time course assay with TK6 cells indicated that at least 4 h of OTA treatment were required to produce a positive MN response. For the Comet assay DNA damage assessments, the cell lines were treated with 5–50 μM OTA for 4 h. Direct DNA damage was detected in TK6 cells, but not CHO cells, while concentration-related increases in fpg-sensitive sites were detected for both cell lines. The consistent association of oxidative DNA damage with OTA exposure suggests its involvement in producing OTA-induced clastogenicity and aneugenicity; however, based on its detection in TK6 cells direct DNA damage could be involved in any human risk posed by OTA exposure.

Induction of apoptosis in human lymphoblastoid cells by kaempferol 3-O-β-isorhamninoside and rhamnocitrin 3-O-β-isorhamninoside from Rhamnus alaternus L. (Rhamnaceae).

Kaempferol 3-O-β-isorhamninoside (K3O-ir) and rhamnocitrin 3-O-β-isorhamninoside (R3O-ir) from Rhamnus alaternus L leaves are investigated for their ability to induce apoptosis in human lymphoblastoid cells. We have attempted to characterize apoptotic pathway activated by these two flavonoids. Apoptosis of the human TK6 lymphoblastoid cell line was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activity. Apoptosis was observed after 24- and 48-h incubation of the cells with the tested compounds. DNA fragmentation was observed after treatment with flavonoids; this was confirmed by demonstration of PARP cleavage. Caspase-3 and caspase-8 activities were induced by both K3O-ir and R3O-ir flavonoids showing highest activity with compound concentration of 400 μg/ml. We have demonstrated that K3O-ir and R3O-ir induce apoptosis in human lymphoblastoid cells by the extrinsic pathway of apoptosis.

Abstract 5413: Is a non-conjugated triple-helix forming oligonucleotide targeting the genomic HPRT locus capable of sequence specific mutagenesis

Abstract Oligonucleotide strands have been shown to be capable of binding to duplex DNA in a sequence specific manner to form a triple-helix or triplex structure. Such phenomenons have been shown to induce mutation, as demonstrated using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). These studies have engendered concern from the European Medicines Agency (EMEA) in regard to biotechnology derived pharmaceuticals such that antisense oligonucleotides may also form triplex structures in a sequence specific manner at genomic DNA with potentially mutagenic effects. In this study, we have examined the mutagenic potential of a non-conjugated triplex forming oligonucleotide (TFO27) targeting the genomic hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mutagenicity of TFO27 was assessed using the human lymphoblastoid TK6 cell line through 6-thioguanine resistance (6TGr). Electrophoretic mobility shift assays were used to demonstrate triplex formation by TFO27 at the target motif at sub-nanomolar concentrations. As expected, a control oligonucleotide, SCR27, failed to form a triplex at the target motif. Subsequently, a range of transfection facilitators were evaluated for optimal delivery of a fluorescently labelled oligonucleotide probe. In preliminary experiments treating 3×106 cells, TFO27 appeared to produce a dose dependent increase in 6TGr mutants. Furthermore, TFO27 failed to induce mutation at the non-targeted thymidine kinase (TK) locus suggesting locus specificity for its mode of action. Moreover, SCR27 failed to induce mutation at both loci. However, TFO27 failed to induce mutation at the HPRT locus in experiments treating 10×106 cells. Increasing the oligonucleotide concentration had no effect on the number of 6TGr recovered. These contradictory findings are difficult to explain, but emphasise the importance of using appropriate target cell numbers in mutation assays. Overall, our data would suggest that triplex formation could occur in cells but chromatin structure, intranuclear pH, ion concentrations and nucleases are all potential barriers that probably influence the dynamics of triplex formation and stability. In view of these factors, we suggest that studies of the mutagenic potential of triplex formation are better performed at genomic loci rather than extra-chromosomal test systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2011-5413

The influence of scoring method on variability in results obtained with the comet assay

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.

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 (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

Study of gene expression profiles in TK6 human cells exposed to DNA-oxidizing agents

During the last decade, there has been clear progress in using threshold in risk assessment but its acceptance by scientists is still under debate. Contrary to indirect DNA-damaging agents, DNA-reactive agents have been assumed to have a non-threshold mode of action, as they directly induce DNA lesions that potentially can be converted into mutations. However, in recent years there is a growing number of data establishing threshold doses even for these DNA-reactive compounds. Indeed, there are several defence and repair mechanisms that provide protection and that may be responsible for genotoxic thresholds. In this context, we recently showed that DNA-oxidizing agents exhibit a thresholded dose–response in vitro with respect to chromosomal alterations. We have hypothesized the involvement of different cellular responses whose nature and efficiency depend on the stress level. The aim of this study was to develop a more complete understanding of these underlying mechanisms. We investigated global gene expression profiles of human lymphoblastoid TK6 cells after exposure to potassium bromate and hydrogen peroxide ( via glucose oxidase). Cells were treated for 1 h and mRNAs were isolated either immediately at the end of the treatment or after a 23-h recovery period. Our results showed that cells have developed elaborate cellular responses to oxidative stress in order to maintain genomic integrity. Many of altered genes were redox-sensitive transcription factors such as p53, NF-κB, AP-1 and Nrf2. Their downstream target genes and signalling pathways were subsequently activated leading mainly to the induction of antioxidant defenses, inflammation, cell cycle arrest, DNA repair and cell death. Overall, our study allowed the identification of key events involved in the thresholded response observed after DNA-oxidizing agents exposure and shows the usefulness of the combination of standard in vitro genotoxicity assays with gene expression profiling technology to determine modes of action, particularly for critical risk assessment.

Evaluation of phenolphthalein, diazepam and quinacrine dihydrochloride in the in vitro mammalian cell micronucleus test in Chinese hamster ovary (CHO) and TK6 cells

The in vitro micronucleus assay has been extensively used as an in vitro screening tool to identify test articles that might have aneugenic or clastogenic potential. Currently, the Organization for Economic Co-operation and Development (OECD) is working towards a final version of the guideline for the conduct of the in vitro mammalian cell micronucleus Test (MNvit). A few questions regarding appropriate cytotoxicity measurements and cytotoxicity limits to use remain to be answered. In order to resolve the remaining questions, we compared the induction of micronuclei at the top dose (50–60% cytotoxicity) determined by either Relative Cell Counts (RCC), Relative Increase in Cell Counts (RICC), Relative Population Doublings (RPD), or Cytokinesis-Blocked Proliferating Index (CBPI) using weak and strong inducers of micronuclei in both the presence and absence of cytochalasin B (CYB) in Chinese hamster ovary (CHO) and human lymphoblastoid TK6 cells. In order to assess extensive dose–response relationships, we selected expected weak (diazepam, phenolphthalein, quinacrine dihydrochloride dihydrate) and strong (cytosine arabinoside, mitomycin C, vinblastine sulphate) inducers of micronuclei with a variety of different mechanisms of action for testing. The results clearly demonstrated that all six compounds produced positive responses using either cytotoxicity measurement. The outcome from these studies further supports the cytotoxicity measurements and cytotoxicity limits proposed in the draft OECD guideline.

Abstract 6: A triple-helix forming oligonucleotide targeting a genomic locus is capable of sequence specific mutagenesis in human lymphoblastoid TK6 cells

Abstract It has been reported that DNA triplex formation induces mutagenesis as determined using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). Triplex mediated mutagenesis has been shown to include point mutations, deletions, small insertions and homologous recombination. To the best of our knowledge, no study has successfully examined the mutagenic potential of a non-conjugated triplex-forming oligonucleotide (TFO) targeting a genomic sequence. In this study, we have designed a TFO that targets the hemizygous hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, in the human lymphoblastoid TK6 cell line, and assessed mutagenicity through resistance to 6-thioguanine. Our TFO, TFO27, has been designed to form a triplex with a purine tract in exon 3 of the HPRT gene. Triplex formation at the target motif was shown to occur at nanomolar concentrations, confirmed by Electrophoretic Mobility Shift Assays. A scrambled oligonucleotide, SCR27, failed to form a triplex at the target motif. A range of transfection reagents were evaluated for facilitated delivery of TFO27, and resulted in variable cellular toxicity. Transfection of high concentrations of oligonucleotide, with acceptable levels of cytotoxicity, resulted in HPRT mutation with TFO27 but not SCR27. Similar experiments failed to result in mutation at the non-targeted thymidine kinase (TK) locus, suggesting locus specificity for the mode of action of TFO27. The target specificity and sequence context of these mutagenic events is being determined to establish the mechanism of mutation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 6.

Abstract 3930: Radiation-induced dicentrics: Tracking by parallel sequencing

Abstract Radiation and cytotoxic drugs exert much of their lethal effects by creating aberrations, such as dicentrics, that are lethal at a subsequent mitosis. Their accurate enumeration in experimental and clinical settings would provide valuable information on the efficacy of radiation and radiation/chemotherapy combinations. In this study a method was sought that could rapidly and efficiently quantitate this process using next generation sequencing. The use of modern molecular technology for aberration analysis has been restricted by the random nature of the breaks induced in individual chromosome that inhibit their detection. To overcome this problem, a common fragile site at 11q23 that is readily cleaved in response to a broad range of cytotoxic agents was used. Using irradiated human TK6 lymphoblastoid cells and blood samples taken from patients undergoing chemotherapy, partner chromosomes that are ligated to this location were detected using inverse PCR to capture the breakpoint. In order to enhance the efficiency of the process, amplified inverse PCR material was processed for Solexa type parallel sequencing. Here individual amplicons, representing the total content of a treated sample, are attached to a flow cell and their sequence recorded by sequential additions of fluorescent bases. Each aberration was then reconstructed with reference to a standard human genome database. In one specific sample of treated TK6 cells, two translocations involving Chr. 2 and 18 and two dicentrics involving Chr. 3 and 13 were found linked to the Chr 11q23 fragmentation site. In addition, aberrations were found in patients’ blood 6 months after initiation of therapy. Thus translocations that are rarely lethal could be readily discriminated from dicentric type rearrangements that are predominately lethal. The advantage of this analytical process is that all normal sequence is also recorded allowing the fractional content of all aberrations to be recorded in response to dose or time. In order to mitigate concerns over the arbitrary nature of aberrations induced at a common fragile site, a zinc finger nuclease was constructed targeting a region adjacent to the fragile site at 11q23. With this zinc finger system repetitive DNA breaks at the predicted site generated micro rearrangements (insertions/deletions) that were produced at far higher rates than translocations or dicentric type rearrangements measured by parallel processing. In summary, a novel analytical system for detecting a lethal class (dicentrics) of chromosome rearrangements has been described that is suitable for therapy monitoring both in-vitro and in-vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3930.

Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe2+−EDTA

The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

Cytosine arabinoside, vinblastine, diethylstilboestrol and 2-aminoanthracene tested in the in vitro human TK6 cell line micronucleus test (MNvit) at Institut Pasteur de Lille in support of OECD draft test guideline 487

The reference genotoxic agents Cytosine arabinoside, Vinblastine, Diethylstilboestrol and 2-Aminoanthracene were tested in the in vitro micronucleus assay, in human lymphoblastoid TK6 cells, without cytokinesis block, at the laboratories of Institut Pasteur de Lille, France. This was done in support of the toxicity measures recommended in the late 2007 version of the draft OECD Test Guideline 487 for the testing of chemicals. All four reference agents were positive in the assay at concentrations giving approximately 50% toxicity or less as assessed by draft Test Guideline 487 recommended measures, relative population doublings and relative increase in cell counts. Accordingly, this work supports the premise that relative population doublings and relative increase in cell counts are appropriate measures of toxicity for the non-cytokinesis blocked in vitro micronucleus assay.

Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP ‘GreenScreen HC’ genotoxicity assay

The GADD45a-GFP (GreenScreen HC) reporter assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens. In this study, a collection of 75 marketed pharmaceuticals were tested in the assay. Compounds in the collection represent a broad range of chemical structures, pharmacologies and therapeutic indications, including neoplasia and viral infection where positive genotoxicity results are often associated with the pharmacological activity. Based on the results of this study, two main conclusions can be drawn: (i) the GreenScreen HC is more predictive of in vivo genotoxicity (88%) and genotoxic carcinogenicity (93%) data than the any of the other regulatory in vitro genotoxicity assay and (ii) no compounds were uniquely positive in the GADD45a-GFP assay. This analysis therefore provides additional evidence to support the use of the GADD45a-GFP assay as an effective tool either in early genotoxic liability identification or non-clinical safety assessment of candidate pharmaceuticals during development.

Fluoroquinolones lower constitutive H2AX and ATM phosphorylation in TK6 lymphoblastoid cells via modulation of the intracellular redox status

Accumulation of reactive oxygen species (ROS)-induced damage and mutations in the genomic DNA is considered the primary etiology of aging and age-related pathologies including cancer. Strategies aimed at slowing these conditions often involve protecting against oxidative DNA damage via modulation of the intracellular redox state. Recently, a biomarker of DNA double-strand breaks (DSBs), serine 139-phosphorylated histone H2AX (γH2AX), and its upstream mediator, activated PI-3-related kinase, ATM (ATMP1981), were shown to be constitutively expressed in cells and modulated by antioxidant treatment. Thus, both constitutive histone H2AX phosphorylation (CHP) and constitutive ATM activation (CAA) are thought to reflect a cell’s response to endogenous ROS-induced DSBs. In the present study, we investigated the effects of a battery of fluoroquinolone (FQ) compounds, namely ciprofloxacin, enrofloxacin, gatifloxacin, lomefloxacin and ofloxacin, on CHP and CAA in human TK6 lymphoblastoid cells. All FQs tested reduced CHP and CAA compared to controls following 6 and 24h treatment with CAA being more sensitive to their effects at both time points. In addition, intracellular ROS levels and mitochondrial activities were also lowered in FQ-treated cells at 6 and 24h. We presume that FQs mediate this effect via a combination of ROS-scavenging and mitochondrial suppression and therefore may protect against the onset or may slow the progression of numerous oxidative pathophysiological conditions.

Aloe-emodin-induced DNA fragmentation in the mouse in vivo comet assay

Aloe-emodin (AE) and derivatives may be present as undesired components co-extracted during extraction of plants containing anthraquinonic derivatives for preparation of diacetylrhein. AE is a well-known in vitro mutagen, but up to now it failed to induce any clear in vivo genotoxic activity in the chromosome aberration assay in rat bone marrow or the in vivo/ in vitro UDS test in liver. However, the two target organs noted during rodent carcinogenicity studies with danthron and emodin, two other well-known anthraquinone derivatives, are the colon and the kidney. Therefore, the choice of the organs for testing the genotoxicity of AE, i.e. bone marrow and liver, may be considered inadequate to demonstrate a possible in vivo genotoxic activity. In this context, the in vivo mouse comet assay was performed on both isolated kidney and colon cells in order to demonstrate a possible organospecific genotoxicity after oral administration of AE. Concurrently, the Ames test and the in vitro micronucleus assay with TK6 human lymphoblastoid cells were performed in their microscale version both with S9 from Aroclor 1254-induced liver or kidney, and without S9. AE induced primary DNA damage in the liver and in the kidney as observed between 3 and 6 h after two oral administrations at 500, 1000 and 2000 mg/kg bw, underlining an in vivo genotoxic mechanism of action. Furthermore, AE induced a clear genotoxic activity both in the Salmonella typhimurium strains TA1537 and TA98 and in the in vitro micronucleus assay in the absence as well as in the presence of metabolic activation. As no significant variation in the genotoxic activity of AE was noted when using either liver or kidney S9-mix, it seems that no quantitatively and/or qualitatively specific renal metabolism occurs. The kidney may be a target organ of AE as it is the major route of excretion. Under such conditions the separation of AE components should take place and the residual content of undesired AE derivatives should be made as low as reasonably achievable. AE present in plant extracts should be considered as an in vivo genotoxin and this property should be taken into account in the risk assessment for human exposure.