Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.     

Acute exposure of human lung cells to 1,3-butadiene diepoxide results in G1 and G2 cell cycle arrest

1,3-Butadiene (BD) causes genetic damage, including adduct formation, sister chomatid exchange, and point mutations. Previous studies have focused on the types of genetic damage and tumors found after long-term exposure of rodents to butadiene. This study examined the effect of the most active BD metabolite, butadiene diepoxide (BDO2), on cell cycle entry and progression in human lung fibroblasts (LU cells) with a normal diploid karyotype. Serum-arrested (G0) LU cells were exposed to BDO2 for 1 hr and stimulated to divide with medium containing 10% fetal bovine serum. The BDO2-treated LU cells were evaluated for cell cycle progression, nuclear localization of arrest mediators, mitotic index, and cellular proliferation. The BDO2-treated cells demonstrated a substantial inhibition of cell proliferation when treated with 100 microM BDO2 for 1 hr. No appreciable levels of apoptosis or mitotic figures were observed in the BDO2-treated cells through 96 hr posttreatment. Flow cytometric analysis revealed that the lack of proliferation in BDO2-treated LU cells was related to G1 arrest in about half of the cells and a delayed progression through S and G2 arrest in nearly all of the remaining cells. Both G1 and G2 arrest were prolonged and only a very small percentage of BDO2-treated cells were eventually able to replicate. Increased nuclear localization of both p53 and p21(cip1) was observed in BDO2-treated cells, suggesting that the cell cycle arrest was p21(cip1)-mediated. These results demonstrate that BDO2 induces cell cycle perturbation and arrest even with short-term exposure that does not produce other pathologic cellular effects.     

Transplacental genotoxicity of triethylenemelamine, benzene, and vinblastine in mice.

Transplacental cytogenetic effects of triethylenemelamine (TEM), benzene, and vinblastine on maternal mice and their fetuses have been investigated using micronucleus and sister chromatid exchange (SCE) as genetic endpoints. CD-1 mice were treated on day 14 and 15 of gestation with TEM (0.125, 0.25, and 0.5 mg/kg), benzene (439,878, and 1,318 mg/kg), and vinblastine (0.5, 1, and 2 mg/kg) by intraperitoneal injection at 24 hr intervals, and sacrificed 40 hr after the first injection. Erythrocytic precursor cells in maternal bone marrow and fetal livers (2-4) from each pregnant mouse were used for the micronucleus and/or the SCE analyses. Significant dose-related increases in both micronuclei and SCE were found in maternal bone marrow and fetal liver following TEM treatment. Benzene at the highest dose (1,318 mg/kg) also caused a significant increase in micronuclei and SCE in both maternal bone marrow and fetal liver cells. The embryonic genotoxic effect of TEM was much higher than that of benzene for both genetic endpoints, and the frequency of micronuclei induced by benzene was higher in fetal liver than in maternal bone marrow cells. Vinblastine, a spindle poison, induced micronuclei but not SCE. Micronuclei induction by vinblastine was 7 fold greater in maternal bone marrow than in fetal liver cells. All three chemicals were cytotoxic in maternal bone marrow cells, but not in fetal liver cells except for TEM, which showed a weak cytotoxicity in fetal liver cells in the micronucleus assay. These results indicate that TEM, benzene, and vinblastine are transplacental genotoxicants in mice.     

Dose- and time-dependent responses for micronucleus induction by X-rays and fast neutrons in gill cells of medaka (Oryzias latipes)

Medaka fish (Oryzias latipes) were exposed to various doses of X-rays or fast neutrons, and the frequency of micronucleated cells (MNCs) was measured in gills sampled at 12- or 24-hr intervals from 12 to 96 hr after exposure. The resulting time course of MNC frequency was biphasic, with a clear peak 24 hr after exposure, irrespective of the kind of radiation applied and the dose used. The half-life of MNCs induced in the gill tissues by the two exposures fluctuated around 28 hr, with no significant dose-dependent trend for either X-ray- or neutron-exposed fish. As assayed 24 hr after exposure, the MNC frequency increased linearly over the control level with increasing doses of both X-rays and fast neutrons. The relative biological effectiveness (RBE) of fast neutrons to X-rays for MNC induction was estimated to be 4.3 +/- 0.6. This value is close to the RBE value of 5.1 +/- 0.3 reported for fast neutron induction of somatic crossing-over mutations in Drosophila melanogaster that arise from recombination repair of DNA double-strand breaks. These results and other data support our conclusion that the medaka gill cell micronucleus assay is a reliable short-term test for detecting potential inducers of DNA double-strand breaks.     

Prior bleeding enhances the sensitivity of peripheral blood and bone marrow micronucleus tests in rats

The rat peripheral blood micronucleus (MN) assay is not considered to be a sufficient biomarker of genotoxin exposure due to the selective elimination of micronucleated cells from peripheral circulation by the spleen. However, several recent reports suggest that peripheral blood reticulocytes of rats may represent a suitable cell population for use in the MN assay. The MN assay in rats with prior bleeding was thus conducted to determine the sensitivity of the bioassay. Hirai et al. reported that prior bleeding enhances the sensitivity of the in vivo MN test in mice. Based on these findings, the rat was used as a model to see the effect of prior bleeding on the sensitivity of the peripheral blood and bone marrow MN assays. In the present experiment, young male Sprague-Dawley (SD) rats ranging in age from 21 to 24 days were used. However, for the comparison of strain-specific induction of MN, Wistar rats were used. The kinetics of MN formation were investigated in adult, young bled and non-bled SD rats treated with cyclophosphamide (CP). For the MN kinetic study, CP was administered intraperitoneally 2 h after bleeding and sampling was done at intervals of 12, 24, 36, 48 and 96 h after chemical administration. Significant increases in MN induction activity in both bone marrow and peripheral blood were observed with prior bleeding. To further validate the influence of prior bleeding in the induction of MN frequency, two other known genotoxins (chlorambucil and mitomycin C) were used. It was concluded that prior bleeding can significantly increase the sensitivity of MN induction in both bone marrow and peripheral blood of rats compared with non-bled animals. Once validated, this model may be suitable for detecting different genotoxins, especially weak and marginally active clastogens.     

Different responses to 5-fluoraouracil in mutagenicity and gene expression between two human lymphoblastoid cell lines with or without TP53 mutation

Human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. TK6 cells have wild-type TP53 alleles, while WTK-1 cells have one allele of mutated TP53. Both cells were treated with 5-fluorouracil (5-FU), and gene mutation assay and micronucleus assay were performed to clarify the differential response related to the TP53 gene status. The effects of 5-FU on gene expression were assessed by microarray and quantitative RT-PCR analyses. In WTK-1 cells, 5-FU increased the frequency of cells with micronucleus and mutation. In TK6 cells, frequency of cells with micronucleus was increased but the mutation frequency was not. The cytotoxicity induced by 5-FU was more prominent in TK6 cells than in WTK-1 cells. Analysis of gene expression showed that the genes involved in the TP53 pathway were up-regulated in TK6 cells but not in WTK-1 cells. The differential responses to 5-FU between these cell lines appeared to be due to the difference in the TP53 gene status, thus providing a molecular basis for the bioassays using these cell lines in the toxicology field. Our results indicate that the clinical efficacy of 5-FU chemotherapy may depend on the TP53 genotype.     

Comparison of gene expression changes induced in mouse and human cells treated with direct-acting mutagens

Exposure to DNA-damaging agents can elicit a variety of stress-related responses that may alter the gene expression of numerous biological pathways. We used Affymetrix microarrays to detect gene expression changes in mouse lymphoma (L5178Y) and human lymphoblastoid (TK6) cells in response to methyl methanesulfonate (MMS; a prototypical alkylating agent) and bleomycin (a prototypical oxidative mutagen). Cells were treated for 4 hr, and RNA was isolated either at the end of the treatment or after a 20-hr recovery period. Two concentrations of each agent were used based on cytotoxicity levels and Tk mutant frequencies. Our microarray data analysis indicated that MMS and bleomycin gene expression responses were considerably different in mouse cells versus human cells. The results also suggested that more comprehensive cellular responses to MMS and bleomycin occurred in TK6 cells than in L5178Y cells. In contrast to L5178Y cells, the response of TK6 cells to MMS and bleomycin was characterized by the induction of p53-dependent genes that are involved in DNA repair, cell cycle regulation, and apoptosis. It appears that the induction of DNA damage by MMS in human TK6 cells mediated cytotoxicity and led to decreased cell survival. This may explain the greater sensitivity of TK6 cells to cytotoxic effects of MMS compared to L5178Y cells. Bleomycin exerted comparable cytotoxic effects in the two cell lines. Overall, these studies were unable to identify distinctive gene expression changes that differentiated bleomycin from MMS in either TK6 cells or mouse lymphoma cells.     

A micromethod for the in vitro micronucleus assay.

A micromethod for the in vitro micronucleus assay was developed using L5178Y cells to enable the rapid screening of a large number of molecules. The method is quick, simple to perform and needs very small amounts of compound, i.e. <10 mg. In this methodology, three types of treatment were carried out in parallel, enabling an optimal detection of both aneugenic and clastogenic compounds: two treatments without metabolic activation with or without a recovery period after a 24 h continuous treatment and one treatment with metabolic activation by Aroclor 1254-induced rat or hamster liver S9 mix. Seventeen known genotoxins (12 clastogens and five aneugens) and seven known non-genotoxins were tested. The in vitro micronucleus micromethod using L5178Y cells exhibited good sensitivity (16 positive/17 known genotoxins tested) and specificity (7 negative/7 known non-genotoxins tested) for the 24 test compounds studied with or without metabolic activation. Furthermore, this test showed a good correlation with other in vitro micronucleus tests performed using macromethods with various mammalian cell cultures. We conclude that the in vitro micronucleus micromethod with L5178Y cells could be used in the earliest stages of development of new molecules as a preliminary short-term screening assay before starting regulatory tests.     

Effect of erythropoietin on the micronucleus test

The micronucleus test is used widely as an in vivo short-term assay for potential carcinogens. In the present study, results of the micronucleus test were affected by the rate of erythropoiesis in the bone marrow erythropoietin, a growth factor for the erythroblast, which was used to induce erythropoiesis. The highest frequency of micronucleated polychromatic erythrocytes (MPCE) and a dose-response relationship between erythropoietin doses and MPCE frequency were seen 30 hr after injection of 1,1-dimethylhydrazine (DMH) to mice administered 24 hr previously with erythropoietin. The effect of erythropoietin was maximal when erythropoietin was given 24 hr before DMH, indicating that accelerating the multiplication of erythroblasts will increase the frequency of micronuclei induced by mutagens. Induction of MPCE in the bone marrow by four other compounds--benzo(a)pyrene, 2-naphthylamine, mitomycin C, and vincristine--was also increased by pretreatment with erythropoietin.     

Chromosome aberrations in mouse bone marrow cells following treatment in vivo with vinblastine and Colcemid

The present study was undertaken to determine if chemicals that are capable of inducing mitotic arrest and aneuploidy can also induce chromosomal breakage. Two chemicals, vinblastine and Colcemid, were selected to be studied. Their potentially clastogenic effects were investigated in mouse bone marrow cells in vivo. Two doses of vinblastine, 10(-5) M (9 mg/kg) and 10(-6) M (0.9 mg/kg), and one dose of Colcemid 10(-4) M (37 mg/kg), were administered to mice as single intraperitoneal injections. Bone marrow preparations were made at multiple time periods after injections, ie, 17, 24, 48, 72, and 96 hr. Both these agents, at the concentrations tested, induced mitotic arrest of bone marrow cells within 5 hr after injection. In recovering bone marrow cell populations, 2-3 days after drug administration, significantly elevated levels of chromosomal breakage (mainly the chromatid type) were observed. Vinblastine was found to be much more potent than Colcemid. Since both of these agents affect mainly microtubules, their actions to cause chromosomal breakage are likely to be indirect. Several possible clastogenic mechanisms such as interference with DNA synthesis, active metabolites, and cytoplasmic endonucleases are discussed.     

Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons

Non‐DNA binding genotoxins (e.g., aneugens), unlike DNA‐binding genotoxins, are theoretically expected to show thresholded concentration‐effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose‐response curves for tubulin interacting agents, a specific class of aneugens. All experiments with aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non‐linear dose‐dependent increase in micronuclei frequencies for all tested aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg^?1, respectively. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Cell cycle dependence of simian virus 40 induction from transformed hamster cells by ultraviolet irradiation.

The induction of infectious simian virus 40 (SV40) was studied in synchronized cultures of SV40-transformed hamster cells. Cells were synchronized with a double excess thymidine block and induced by mitomycin C treatment or uv irradiation at hourly intervals after release from the block. Infectious virus was measured in cell-free extracts at 72 hr after initiating induction. The results indicated that virus activation by these agents was restricted to certain phases of the cell cycle. Mitomycin C was most effective if it was applied to cells 4 hr after the peak of DNA synthesis. With uv radiation, virus production was maximal if cells were irradiated 1–2 hr after the peak of DNA synthesis during the late S-G₂ phase of the cell cycle. The most sensitive period of the cell cycle for virus activation by uv is the one in which eukaryotic cells are most resistant to uv radiation, as measured by enhanced survival after irradiation. Therefore, it is possible that cellular DNA repair processes are more active during this interval, promoting DNA strand break formation and viral genome excision.     

Modulation of cell proliferation in the embryonic retina of zebrafish (Danio rerio).

We describe light-microscopically the development of the embryonic zebrafish eye with particular attention to cell number, cell proliferation, and cell death. The period from 16 to 36 hr post fertilization (hpf) comprises two phases; during the first (16-27 hpf) the optic vesicle becomes the eye cup, and during the second (27-36 hpf) the eye cup begins to differentiate into the neural retina and pigmented epithelium. All cells in the eye primordium are proliferative prior to 28 hpf, and the length of the cell cycle has been estimated to be 10 hr at 24-28 hpf (Nawrocki, 1985). Our cell counts are consistent with that estimate at that age, but not at earlier ages. A 10-hr cell cycle predicts that the cell number should increase by 7% per hr, but during 16-24 hpf the cell number increased by only 1.5% per hr. Despite the low rate of increase, all cells labeled with bromo-deoxyuridine, so all were proliferative. We considered three possible explanations for the nearly-constant cell number in the first phase: proliferation balanced by cell emigration from the eye, proliferation balanced by cell death, and low proliferation caused by a transient prolongation of the cell cycle. We excluded the first two, and found direct support for the third. Previous examinations of the cell cycle length in vertebrate central nervous system have concluded that it increases monotonically, in contrast to the modulation that we have shown. Modulation of the cell cycle length is well-known in flies, but it is generally effected by a prolonged arrest at one phase, in contrast to the general deceleration that we have shown.     

Caffeine effect on the mitotic delay induced by G2 treatment with UVC or mitomycin C

It is well established that DNA lesions trigger cell cycle check-pointscausing a mitotic delay that is required for their repair beforecells enter the mitotic phase. Caffeine, in some cases, canremove this delay and consequently potentiates the yield ofinduced chromosome aberrations. The objective of this studywas to test the effect of a G2 treatment with S-dependent agents(UV light and mitomycin C) on the cell kinetics of a G2 cellpopulation and evaluate whether post-treatments with caffeinecould modulate removal of the expected cell cycle delay. Cellkinetics were monitored by analysing the mitotic index (MI)values in combination with the 5-bromo-2'-deoxyuridine (BrdUrd)labelling technique. Chinese hamster fibroblast cultures (AA8)were treated in G2 phase of the cell cycle with 8 and 15 J/m²UV light or 0.1 and 0.6 µg/ml mitomycin C for 1.5 h. Post-treatmentswith caffeine were performed at dose levels and recovery timeswhere the mitotic indices were substantially reduced. The resultsobtained showed that both UV light and mitomycin C induced aG2 arrest, as indicated by MI values and the absence of Brd-Urdlabelledmetaphases. For UV light the G2 block was observed at lowerand higher dose levels after 1.5 h, while for mitomycin C itwas observed only at the higher dose level after 1 h. However,in both cases the block lasted {small tilde}1 h, after which,even though slowed down, the cell population entered mitosis,as indicated by increased MI values. This block was not removedby caffeine post-treatment. In contrast, caffeine G2 post-treatmentwas able to remove G2 arrest induced by G1–treatments.Accordingly, our results suggest that both UV light- and mitomycinC-induced damage must be processed during S phase to allow caffeineto remove induced G2 blocks.      

Development of an intact hepatocyte activation system for routine use with the mouse lymphoma assay

We have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK+/- -3.7.2C mouse lymphoma cells. This method should provide a means of stimulating more closely in-vivo metabolism compared to metabolism by liver homogenates, while still being useful for routine screening. Hepatocytes were isolated from 200-250 gm adult male Sprague-Dawley rats; 1 X 10(6) viable hepatocytes were seeded per flask. Rapid attachment of the hepatocytes (2 hr) was obtained by using fibronectin-coated 25-cm2 tissue culture flasks. Cocultivated cultures were incubated at 37 degrees C on a platform rocker at 32 oscillations per minute. A 16-hr cocultivated period was selected. With this hepatocyte activation methodology, CP, DMN, DMBA, and B(a)P, genotoxins that require metabolic activation, could be detected as mutagens in L5178Y/TK+/- cells.     

Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens,aneugens, and misleading positive agents

The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, “misleading” or “irrelevant” positives. We set out to develop a rapid and effective follow‐up testing strategy that would predict whether apparent in vitro micronucleus‐inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous‐type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp‐positive chromatin, cleaved caspase 3‐positive chromatin, ethidium monoazide bromide‐positive chromatin, polyploid nuclei, phospho‐histone H3‐positive (metaphase) cells, tetramethylrhodamine ethyl ester‐negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ‐H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave‐one‐out approach indicated that a promising base model includes γ‐H2AX shift and change in phospho‐histone H3‐positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26—30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput‐compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results. Environ. Mol. Mutagen. 55:542–555, 2014. © 2014 Wiley Periodicals, Inc.     

Infection of human B lymphocytes with high multiplicities of Epstein-Barr virus: kinetics of EBNA expression, cellular DNA synthesis, and mitosis

We studied the time course of early events during the transformation of human B lymphocytes by Epstein-Barr virus (EBV). EBV nuclear antigen (EBNA) was first detectable 6–8 hr after infection in 1–10% of the cells. Even though cells were exposed to virus under conditions which would maximize the chances of synchronous infection, the proportion of EBNA (+) cells increased until 32–36 hr when a maximum level was reached. DNA synthesis in infected cultures increased above control levels between 20 and 36 hr after exposure to virus and thereafter increased progressively. The first cell divisions observed in EBNA (+) cells occurred between 36 and 48 hr after infection and the mitotic index of EBNA (+) cells increased with time reaching 25% of the EBNA (+) cells by 96 hr. However, the total cell number and the percentage of EBNA (+) cells remained unchanged after 36 hr, suggesting that the rate of cell death in EBNA (+) cells was equivalent to the rate of proliferation. Adenine arabinoside (Ara A) and cytosine arabinoside (Ara C), at concentrations which inhibit VCA expression in EBV producer cell lines, did not affect EBNA expression during the first 24 hr after infection. These data together with the very early appearance of EBNA after inoculation show that EBNA synthesis is independent of viral or cellular DNA synthesis and support the hypothesis that synthesis of EBNA is one of the crucial earliest events in lymphocyte transformation by EBV.