TK6 Lymphoblasts Research Articles

Nano-fenretinide demonstrates remarkable activity in acute promyeloid leukemia cells

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the retinoic acid receptor, RARα, which makes all-trans retinoic acid (ATRA) highly effective in the treatment of this disease, inducing promyelocytes differentiation. Current therapy, based on ATRA in combination with arsenic trioxide, with or without chemotherapy, provides high rates of event-free survival and overall survival. However, a decline in the drug activity, due to increased ATRA metabolism and RARα mutations, is often observed over long-term treatments. Furthermore, dedifferentiation can occur providing relapse of the disease. In this study we evaluated fenretinide, a semisynthetic ATRA derivative, encapsulated in nanomicelles (nano-fenretinide) as an alternative treatment to ATRA in APL. Nano-fenretinide was prepared by fenretinide encapsulation in a self-assembling phospholipid mixture. Physico-chemical characterization was carried out by dinamic light scattering and spectrophotometry. The biological activity was evaluated by MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Nano-fenretinide induced apoptosis in acute promyelocytic leukemia cells (HL60) by an early increase of reactive oxygen species and a mitochondrial potential decrease. The fenretinide concentration that induced 90–100% decrease in cell viability was about 2.0 µM at 24 h, a concentration easily achievable in vivo when nano-fenretinide is administered by oral or intravenous route, as demonstrated in previous studies. Nano-fenretinide was effective, albeit at slightly higher concentrations, also in doxorubicin-resistant HL60 cells, while a comparison with TK6 lymphoblasts indicated a lack of toxicity on normal cells. The results indicate that nano-fenretinide can be considered an alternative therapy to ATRA in acute promyelocytic leukemia when decreased efficacy, resistance or recurrence of disease emerge after protracted treatments with ATRA.

IL-6 knockdown in a model of the human bone marrow, abrogates DNA damage induction in bystander cells post-chemotherapy induced cytokine release syndrome

Following infection or exposure to therapeutic agents, an aggressive immune response may result, termed cytokine storm (CS) or cytokine release syndrome. Here the innate immune system becomes uncontrolled, leading to serious consequences including possible death. Patients surviving CS are at greater risk for de novo tumorigenesis, but it is unclear if any specific cytokines are directly responsible for this outcome. De novo tumorigenesis has been observed in donated cells exposed to CS following haematopoietic stem cell transplant (HSCT).Modelling HSCT, we firstly demonstrated the release of CS levels from the HS-5 human bone marrow stromal cell line, post-exposure to chemotherapy. We then exposed the TK6 lymphoblast cell line to healthy and storm doses of IL-6 and measured increased genotoxicity via the micronucleus assay. During HSCT, haematopoietic cells are exposed to a complex mix of cytokines, so to determine if IL-6 was integral in a chemotherapy-induced bystander effect, we attempted to inhibit IL-6 from HS-5 cells using resatorvid or siRNA, treated with chlorambucil or mitoxantrone, and then co-cultured with bystander TK6 cells. Whilst resatorvid did not reduce IL-6 and did not reduce micronuclei in the bystander TK6 cells, siRNA inhibition reduced IL-6 to healthy in vivo levels, and micronuclei aligned with untreated controls.Our data suggests that exposure to high IL-6 (in the absence of inflammatory cells) has potential to induce genetic damage and may contribute to de novo tumorigenesis post-CS. We suggest that for individuals with a pro-inflammatory profile, anti-IL-6 therapy may be an appropriate intervention to prevent complications post-CS.

Industrial-relevant TiO2 types do not promote cytotoxicity in the A549 or TK6 cell lines regardless of cell specific interaction

Due to the expansive application of TiO2 and its variance in physico-chemical characteristics, the toxicological profile of TiO2, in all its various forms, requires evaluation. This study aimed to assess the hazard of five TiO2 particle-types in relation to their cytotoxic profile correlated to their cellular interaction, specifically in human lymphoblast (TK6) and type-II alveolar epithelial (A549) cells. Treatment with the test materials was undertaken at a concentration range of 1–100 μg/cm2 over 24 and 72 h exposure. TiO2 interaction with both cell types was visualised by transmission electron microscopy, supported by energy-dispersive X-ray. None of the TiO2 materials tested promoted cytotoxicity in either cell type over the concentration and time range studied. All materials were observed to interact with the A549 cells and were further noted to be internalised following 24 h exposure. In contrast, only the pigmentary rutile was internalised by TK6 lymphoblasts after 24 h exposure. Where uptake was observed there was no evidence, as determined by 2D microscopy techniques, of particle localisation within the nucleus of either cell type. This study indicates that industrially relevant TiO2 particles demonstrate cell interactions that are cell-type dependent and do not induce cytotoxicity at the applied dose range.

Biological effects, including oxidative stress and genotoxic damage, of polystyrene nanoparticles in different human hematopoietic cell lines

In recent years the terms "micro-/nanoplastics" (MNPLs) have caught special attention due to the increasing levels by which humans are exposed. Among MNPLs, polystyrene nanoparticles (PSNPs) are one of the most represented MNPLs in the environment. These tiny particles may enter into the human body, translocate through human barriers, interacting with blood and lymphatic immune cells, and reaching secondary organs. By using three different human leukocytic cell lines: Raji-B (B-lymphocytes), TK6 (lymphoblasts) and THP-1 (monocytes), we pursued to determine the effects of these PSNPs on the immune cell population. With this aim, the three cell lines were exposed to spherical PSNPs of about 50 nm of diameter and cytotoxicity, cellular uptake, reactive oxygen species (ROS) production, and genotoxicity were assessed at different time-points. Results show differences in all the measured endpoints, among the selected cell lines. Thus, whilst the monocytic THP-1 cells showed the highest particle internalization, no adverse effects were observed in such cells. On the other side, although Raji-B and TK6 showed lesser PSNPs uptake, mild toxicity, ROS production and genotoxicity were detected. These results highlight the importance of the cell line selection when the biological effects of PSNPs are evaluated.

Diepoxybutane induces the expression of a novel p53-target gene XCL1 that mediates apoptosis in exposed human lymphoblasts.

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a human carcinogen that exhibits multiorgan systems toxicity. Our previous studies demonstrated that the X-C motif chemokine ligand 1 (XCL1) gene expression was upregulated 3.3-fold in a p53-dependent manner in TK6 lymphoblasts undergoing DEB-induced apoptosis. The tumor-suppressor p53 protein is a transcription factor that regulates a wide variety of cellular processes, including apoptosis, through its various target genes. Thus, the objective of this study was to determine whether XCL1 is a novel direct p53 transcriptional target gene and deduce its role in DEB-induced toxicity in human lymphoblasts. We utilized the bioinformatics tool p53scan to search for known p53 consensus sequences within the XCL1 promoter region. The XCL1 gene promoter region was found to contain the p53 consensus sequences 5'-AGACATGCCTAGACATGCCT-3' at three positions relative to the transcription start site (TSS). Furthermore, the XCL1 promoter region was found, through reporter gene assays, to be transactivated at least threefold by wild-type p53 promoter in DEB-exposed human lymphoblasts. Inactivation of the XCL1 promoter p53-binding motif located at -2.579 kb relative to TSS reduced the transactivation function of p53 on this promoter in DEB-exposed cells by 97%. Finally, knockdown of XCL1 messenger RNA with specific small interfering RNA inhibited DEB-induced apoptosis in human lymphoblasts by 50%. These observations demonstrate, for the first time, that XCL1 is a novel DEB-induced direct p53 transcriptional target gene that mediates apoptosis in DEB-exposed human lymphoblasts.

Development of ambient storable meal for calamity victims and other targets employing radiation processing and evaluation of its nutritional, organoleptic, and safety parameters

During natural calamities nutritious, safe, and ambient storable meal is issue of immense concern. Recently Joint FAO/IAEA programme of nuclear techniques in Food and Agriculture has undertaken Coordinated Research Project (CRP) where a product ‘Stuffed Baked Food (SBF)’ was developed in India. SBF consists of partially fermented multigrain dough enriched with 5% saturated fat and stuffed with flour of roasted chick pea; boiled and peeled potato (mashed); and cooked chick pea split (mashed) with spices and salt. Stuffed lobe was convection baked, vacuum packaged and gamma irradiated (15 kGy). The product was well acceptable after prolonged storage at ambient temperature while retaining its quality attributes. SBF can also be useful for other targets like defence personnel, school lunch programme, expeditions, and astronauts. Mutation analyses in models including human TK6 lymphoblast cell line at genes tk+/− and hprt+; and bacterial systems [Escherichia coli MG1655 cells (rpoB gene); and Ames strains (TA 100 and TA 102)] endorsed its genotoxic safety.

Analysis of the benzene oxide-DNA adduct 7-phenylguanine by liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry-parallel reaction monitoring: Application to DNA from exposed mice and humans

Benzene oxide, the initial metabolite of the human carcinogen benzene, reacts with DNA producing 7-phenylguanine (7-PhG) and other products. We developed a highly sensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry-parallel reaction monitoring method for the analysis of 7-PhG in DNA. Accuracy and precision of the method were established and the detection limit was about 8amol of 7-PhG injected on the column and less than 1 adduct per 109 nucleotides in DNA. 7-PhG was detected in calf thymus DNA reacted with 1μM to 10mM benzene oxide. The method was applied for the analysis of DNA isolated from bone marrow, lung, and liver of B6C3F1 mice treated by gavage with 50mg/kg benzene in corn oil 5 times weekly for 4weeks. 7-PhG was not detected in any of these DNA samples. The method was applied to DNA from mouse hepatocytes exposed to 100μM benzene oxide and human TK-6 lymphoblasts exposed to 100μM, 1, and 10mM benzene oxide. 7-PhG was only detected in TK-6 cell DNA from the 10mM exposure. The method was also applied to leukocyte DNA from 10 smokers and 10 nonsmokers. 7-PhG was detected in only one DNA sample, from a nonsmoker. The results of this study do not support the hypothesis that the benzene oxide-DNA adduct 7-PhG is involved in carcinogenesis by benzene.

Predictive Toxicology of cobalt ferrite nanoparticles: comparative in-vitro study of different cellular models using methods of knowledge discovery from data

BackgroundCobalt-ferrite nanoparticles (Co-Fe NPs) are attractive for nanotechnology-based therapies. Thus, exploring their effect on viability of seven different cell lines representing different organs of the human body is highly important.MethodsThe toxicological effects of Co-Fe NPs were studied by in-vitro exposure of A549 and NCIH441 cell-lines (lung), precision-cut lung slices from rat, HepG2 cell-line (liver), MDCK cell-line (kidney), Caco-2 TC7 cell-line (intestine), TK6 (lymphoblasts) and primary mouse dendritic-cells. Toxicity was examined following exposure to Co-Fe NPs in the concentration range of 0.05 -1.2 mM for 24 and 72 h, using Alamar blue, MTT and neutral red assays. Changes in oxidative stress were determined by a dichlorodihydrofluorescein diacetate based assay. Data analysis and predictive modeling of the obtained data sets were executed by employing methods of Knowledge Discovery from Data with emphasis on a decision tree model (J48).ResultsDifferent dose–response curves of cell viability were obtained for each of the seven cell lines upon exposure to Co-Fe NPs. Increase of oxidative stress was induced by Co-Fe NPs and found to be dependent on the cell type. A high linear correlation (R2=0.97) was found between the toxicity of Co-Fe NPs and the extent of ROS generation following their exposure to Co-Fe NPs. The algorithm we applied to model the observed toxicity belongs to a type of supervised classifier. The decision tree model yielded the following order with decrease of the ranking parameter: NP concentrations (as the most influencing parameter), cell type (possessing the following hierarchy of cell sensitivity towards viability decrease: TK6 > Lung slices > NCIH441 > Caco-2 = MDCK > A549 > HepG2 = Dendritic) and time of exposure, where the highest-ranking parameter (NP concentration) provides the highest information gain with respect to toxicity. The validity of the chosen decision tree model J48 was established by yielding a higher accuracy than that of the well-known “naive bayes” classifier.ConclusionsThe observed correlation between the oxidative stress, caused by the presence of the Co-Fe NPs, with the hierarchy of sensitivity of the different cell types towards toxicity, suggests that oxidative stress is one possible mechanism for the toxicity of Co-Fe NPs.

Telomere loss, not average telomere length, confers radiosensitivity to TK6-irradiated cells

Many and varied are the proposed mechanisms that lead to resistance to ionizing radiation treatment. Among them, an inverse relationship between telomere length and radioresistance has been recently advanced. Investigating such a relationship in TK6 lymphoblasts, we found that clones originating from cells survived to 4Gy of X-rays showed a significantly higher telomere length when compared with clones grown from untreated cells. The lengthening observed was not attributable to a radiation-induced increase in telomerase activity, as demonstrated by TRAP assay performed in the dose range of 1–10Gy. Given the evidence that TK6 whole population was characterized by heterogeneity in cellular mean telomere length and telomere loss, we tested the hypothesis that a process of selection may favour cells with longer telomeres (more radioresistant cells) following exposure to irradiation. In order to do this 15 independent TK6 clones were selected and characterized for telomere length and loss on the basis of q-FISH and flow-FISH analysis. Among the screened clones four characterized by long telomeres and four characterized by short telomeres were tested for their radiosensitivity by means of clonogenic assay. The results obtained showed that, in our experimental conditions (cellular model, radiation doses) no significant correlation was observed between radiosensitivity and mean telomere lengths, whereas a positive correlation was observed with respect to telomere loss. Overall, these results indicate that telomere loss and not mean telomere length plays a critical role in the phenomenon of radiosensitivity/radioresistance.

Mitochondrial permeability and toxicity of diethylhexyl and monoethylhexyl phthalates on TK6 human lymphoblasts cells

Phthalates are ubiquitous compounds used in the manufacturing industry. Some are known endocrine disruptors, acting as xenoestrogens, others induce reproductive toxicity and damage to DNA among other effects. Studies on apoptosis induction and mitochondrial damage capacity of phthalates on the immune system are limited. This study aims to determine cell viability inhibition and apoptosis induction of diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) on the human TK6 lymphoblast cell line at concentrations found in the environment. Key hallmark events, such as mitochondrial membrane permeability, generation of reactive oxygen species (ROS) and activation of caspase 3 and 7 were measured. Concentrations that inhibit viability of 50% (IC50) of the cells were determined at 24, 48 and 72h with doses ranging from 10 to 500μM. Changes in mitochondrial membrane permeability, ROS generation and activation of caspases 3 and 7, were measured as part of the cell death mechanism. The IC50 at 24h was approximately 250μM for both phthalates; at 48h were 234 and 196μM for DEHP and MEHP, respectively and at 72h IC50s were 100 and 80μM for DEHP and MEHP, respectively. Overall the longer the time of exposure the lower the IC50’s for both compounds. Both compounds affected mitochondrial membrane potential, promoted ROS generation and activated caspases 3 and 7. MEHP is more toxic, promotes higher level of ROS production and caspases activation. Our findings suggest that DEHP and MEHP have the capacity to induce apoptosis in cells of the immune system at concentrations found in the environment.

Abstract 4474: Cellular effects of fluorescent quinolinium drugs on normal lymphoblast and A431 carcinoma cells

Abstract This study aims to evaluate the cellular effects of amino benzazolo [3,2- a] quinolinium drugs on normal lymphoblast cells and compare them to effects on A431 epitheloid carcinoma cells. The drugs under study are amino containing heterocyclic compounds possessing a positive charge that could facilitate their interaction with cell organelles specially mitochondria. The fluorescent properties could also facilitate observation of their cellular binding location. These compounds have also been demonstrated to have mutagenic and DNA binding capacity on A431 cancer cells. A431 and TK6 cells in culture where exposed for 48 hours to determine the IC50 dose, mitochondrial membrane permeability, ROS generation and fluorescent microscopy analysis of intracellular binding. IC50 was determined by trypan blue exclusion and mitochondrial membrane permeability applying the Nucleo counter 3000, JC-1 assay. For ROS determination the fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA) was applied. Determination of intracellular binding was performed applying confocal microscopy. In the TK6 normal lymphoblast cells the observed IC50 concentrations were as follow: ABQ38=310µM, and ABQ95=270µM. On A431 IC50s were as follows: ABQ38 = 32 µM and ABQ95 = 36 µM. Overall, normal TK6 demonstrated higher tolerance to the tested ABQS than A 431. Preliminary results on ROS generation indicated a stronger ROS release on A431 than on TK6 lymphoblast. Analysis of the effects of ABQS on the mitochondrial permeability where also consistent with the cytotoxicity observed, demonstrating higher levels of membrane permeability on treated tumor cells than on normal. Microscopy images of ABQS treated cells revealed location of these fluorescent compounds in organelles such as mitochondria and DNA. DNA fragmentation analyses are being implemented. This study presents preliminary evidence of an overall higher tolerance of normal TK6 lymphoblast to the tested fluorescent drugs in comparison to treated A431. 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 4474. doi:10.1158/1538-7445.AM2011-4474

Mechanism of diepoxybutane‐induced p53 regulation in human cells

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a known mutagen and human carcinogen and possesses multisystems organ toxicity. We previously reported the elevation of p53 in human TK6 lymphoblasts undergoing DEB-induced apoptosis. In this study, we have characterized the DEB-induced p53 accumulation and investigated the mechanisms by which DEB regulates this p53 accumulation. The elevation of p53 levels in DEB-exposed TK6 lymphoblasts and human embryonic lung (HEL) human fibroblasts was found to be largely due to the stabilization of the p53 protein. DEB increased the acetylation of p53 at lys-382, dramatically reduced complex formation between p53 and its regulator protein mdm2 and induced the phosphorylation of p53 at serines 15, 20, 37, 46, and 392 in human lymphoblasts. A dramatic increase in phosphorylation of p53 at serine 15 in correlation to total p53 levels was observed in DEB-exposed Ataxia Telangiectasia Mutated (ATM) proficient human lymphoblasts as compared to DEB-exposed ATM-deficient human lymphoblasts; this implicates the ATM kinase in the elevation of p53 levels in DEB-exposed cells. Collectively, these findings explain for the first time the mechanism by which p53 accumulates in DEB-exposed cells and contributes to the understanding of the molecular toxicity of DEB and BD.

Diepoxybutane activates the mitochondrial apoptotic pathway and mediates apoptosis in human lymphoblasts through oxidative stress

Diepoxybutane (DEB) is the most potent metabolite of the environmental chemical 1,3-butadiene (BD), which is prevalent in petrochemical industrial areas. BD is a known mutagen and human carcinogen, and possesses multi-systems organ toxicity. We recently reported that DEB-induced cell death in TK6 lymphoblasts was due to the occurrence of apoptosis, and not necrosis. In this study, we investigated the molecular mechanisms responsible for DEB-induced apoptosis in these cells. Bax and Bak were found to be over-expressed and activated, and the mitochondrial trans-membrane potential was attenuated in cells undergoing DEB-induced apoptosis. Cytochrome c was depleted from the mitochondria of TK6 cells undergoing apoptosis, and was released into the cytosol in Jurkat T-lymphoblasts exposed to the same concentrations of DEB. Executioner caspase 3 was deduced to be activated by initiator caspase 9. DEB-induced reactive oxygen species (ROS) formation, and the ROS scavenger N-acetyl- l-cysteine effectively blocked DEB-induced apoptosis in TK6 cells. Collectively, these results demonstrate that the mitochondrial apoptotic pathway is activated to mediate DEB-induced apoptosis in human TK6 lymphoblasts. These results further demonstrate that DEB-induced apoptosis is also mediated by the DEB-induced generation of ROS. This is the first report to examine the mechanism of DEB-induced apoptosis in human lymphoblasts.

Induction of Micronuclei in Human TK6 Lymphoblasts by 2‐Dodecylcyclobutanone, a Unique Radiolytic Product of Palmitic Acid

ABSTRACT: Palmitic acid, the most abundant fatty acid in the human diet, causes oxidative DNA damage, DNA strand breakage, necrosis, and apoptosis in human and rodent cells in vitro, while excess fatty acids in the diet increase the risk of colon cancer in vivo. Irradiation of palmitic acid leads to the formation of 2‐dodecylcyclobutanone (2‐DCB), a unique radiolytic product formed at part‐per‐million levels in fats. Recent research has raised the possibility that, like palmitic acid, 2‐DCB can also cause DNA strand breaks in human and rodent cells. In order to more fully understand the clastogenic potential of 2‐DCB, it was tested for the ability to induce the formation of micronuclei (MN) in human TK6 lymphoblasts. TK6 lymphoblasts were exposed to 13.3, 26.5, and 53 μM of 2‐DCB for 24 h, both with and without exogenous metabolic activation (EMA). The number of MN per 1000 binucleated cells was induced 2.65‐fold without EMA, and 2.85‐fold in the presence of EMA at the highest 2‐DCB concentration of 53 μM. Cytotoxicity of the TK6 lymphoblasts treated with 53 μM 2‐DCB was 51.7% in the absence of EMA, and 61.7% in the presence of EMA. Thus, like palmitic acid, 2‐DCB may be weakly clastogenic against human cells in vitro. Given the amount of 2‐DCB that would be consumed when compared with that of its clastogenic parent fatty acid, 2‐DCB is unlikely to have any significant impact on human health.

Cell Growth Modulation of Human Cells Irradiatedin Vitrowith Low-Level Laser Therapy

The aim of this study was to investigate the effects of different wavelengths and doses of laser radiation on in vitro cell proliferation. We evaluated the biological effects of low-level laser therapy (LLLT) on two human cancer cell lines: HeLa (epithelial adenocarcinoma) and TK6 (lymphoblast). Our attention was focused on the combination of the two laser emissions as it could have a synergic effect greater than the single emission applied separately. The effects of LLLT on human cells are still poorly understood and unexplained. Several cell types were found non responsive to laser bio stimulation; in other cases, only a partial activation was observed. A laser device was used for cell irradiation with a continuous wave diode (lambda = 808 nm), a pulsed wave diode (lambda = 905 nm), and a combined wave diodes (lambda = 808 nm + 905 nm), in the dose range of 1-60 J/cm(2). The effect of the combined low-level 808-905-nm diode laser irradiation were slightly superior to those achieved with either laser alone in HeLa cells. TK6 cellular proliferation was not found to be significantly affected by any of the energy levels and varying exposure doses investigated. Our results are a confirmation of previous observations carried out on human cells, where only the proliferation of slowly growing cell populations appeared to be stimulated by laser light.

Apoptosis signals in lymphoblasts induced by focused ultrasound.

We investigated the effects of focused ultrasound (FUS) on specific molecular signaling and cellular response in three closely related human Tk6 lymphoblast cell lines that differed only in their p53 status. The applied ultrasound parameters fell between the physical dose range, which is safely used in medical diagnostics (peak pressure<0.1 MPa) and that used for high-energy FUS thermal ablation therapy (peak pressure>10 MPa). Based on cDNA microarrays and protein analysis, we found that FUS at the intermediate peak pressure of 1.5 MPa induced a complex signaling cascade with upregulation of proapoptotic genes [e.g., p53, p21, Thy1 (CD 90)]. Simultaneously, FUS downregulated cellular survival components (e.g., bcl-2, SOD). The p53 status was important for the reaction of the cells to ultrasound. Apoptosis and G1 arrest were induced primarily in p53+ cells, while p53- cells showed less apoptosis but exhibited G2 arrest. Likewise, the proliferation of lymphoblasts was much more strongly inhibited in p53+ than in p53- cells. Microarray analysis further demonstrated an upregulation of genes involved in oxidative stress (e.g., ferritin), suggesting that indirect sonochemical effects via reactive oxygen species play a causative role in the interaction of ultrasound with lymphoblasts. An important characteristic of FUS in therapeutic ultrasound applications is its ability to be administered to the human body in a targeted manner while sparing intermediate tissues. Therefore, our data indicate that this noninvasive, mechanical wave transmission, which is free of ionizing radiation, has the potential to specifically induce localized cell signals and apoptosis.

Diepoxybutane induces caspase and p53-mediated apoptosis in human lymphoblasts

Diepoxybutane (DEB) is the most potent metabolite of the environmental chemical 1,3-butadiene (BD), which is prevalent in petrochemical industrial areas. BD is a known mutagen and human carcinogen, and possesses multiorgan systems toxicity that includes bone marrow depletion, spleen, and thymus atrophy. Toxic effects of BD are mediated through its epoxy metabolites. In working towards elucidating the cellular and molecular mechanisms of BD toxicity, we investigated the ability of DEB to induce apoptosis in human lymphoblasts. DEB induced a concentration and exposure time-dependent apoptosis, which accounted for the DEB-induced loss of cell viability observed in TK6 lymphoblasts. The DEB-induced apoptosis was inhibited by inhibitors of caspases 3 and 9. The role of p53 in mediating the DEB-induced apoptosis was also investigated. DEB induced elevated p53 levels in direct correlation to the extent of DEB-induced apoptosis, as the concentration of DEB increased up to 5 μM. The extent of DEB-induced apoptosis was dramatically higher in TK6 lymphoblasts as compared to the genetically paired p53-deficient NH32 lymphoblasts under the same experimental conditions. Our results confirm and extend observations on the occurrence of apoptosis in DEB exposed cells, and demonstrate for the first time the elevation of p53 levels in human lymphoblasts in response to DEB exposure. In addition, our results demonstrate for the first time that DEB-induced apoptosis is mediated by caspases 3 and 9, as well as the p53 protein. It is possible that DEB-induced apoptosis may explain BD-induced bone marrow depletion, spleen and thymus atrophy in BD-exposed animals.

Semiquinone radical intermediate in catecholic estrogen-mediated cytotoxicity and mutagenesis: chemoprevention strategies with antioxidants.

Modulation of the cytotoxicity and mutagenicity of 4-hydroxyestradiol (4-OHE(2)), an oxidative metabolite of estrogen, by antioxidants was assessed in human MCF7 cells and TK-6 lymphoblast cells. The cytotoxicity of the catecholic estrogens was potentiated by depletion of intracellular glutathione and was independent of oxygen concentration. Agents such as the nitroxide Tempol can facilitate the oxidation of the semiquinone to the Q and enhanced 4-OHE(2) cytotoxicity. Conversely, reducing agents such as ascorbate, cysteine, and 1,4-dihydroxytetramethylpiperidine (THP) protected against cytotoxicity and decreased mutation induction, presumably by reducing the semiquinone to the hydroquinone. Our results support the proposition that oxidation of the semiquinone to the corresponding Q is crucial in eliciting the deleterious effects of catecholic estrogens. Furthermore, because the deleterious effects of 4-OHE(2) were abrogated by dietary and synthetic antioxidants, our results would support the chemopreventive use of diets rich in reducing substances (vitamins and added synthetic antioxidants) as a means of decreasing the risks associated with estrogen exposure and developing of breast cancer.

Mutational spectrum of 1,3-butadiene and metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane to assess mutagenic mechanisms

1,3-Butadiene (BD) is a multisite carcinogen and is mutagenic in multiple tissues of B6C3F1 mice. BD is bioactivated to at least three directly mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). However, the contribution of these individual metabolites to the carcinogenicity and in vivo mutatidnal spectrum of BD is uncertain. To assess the role of two BD metabolites EB and DEB in the in vivo mutagenicity of the parent compound BD, we examined the in vitro mutational spectra of EB and DEB in human and rodent cells. We also examined the in vivo mutagenicity and mutational spectrum of inhaled EB in the lung. In the bone marrow and spleen of B6C3F1 laci transgenic mice, BD-induced an increased frequency of the identical class of point mutations at A:T base pairs: AT→GC transitions and AT→TA transversions. BD exposure also induced an increased frequency of GC→AT transitions in the spleen that was not observed in bone marrow, demonstrating tissue-specific differences in mutation spectrum. Exposure of Rat2 laci transgenic cells and human TK6 lymphoblasts to EB-induced an increased frequency of AT→TA transversions. DEB exposure induced an increased frequency of AT→TA transversions and partial deletions at hprt in human cells. In Rat laci transgenic cells, DEB was not mutagenic at laci but induced an increased frequency of micronuclei. In contrast to inhaled BD, inhaled DEB and EB were not mutagenic in the bone marrow or spleen. However, EB was mutagenic in the lungs. In the lung of mice, EB-induced specific increases in GC→AT transitions, AT→TA transversions, and deletion events. AT→TA transversions are the most consistent mutation observed across biological systems following in vivo exposure to BD or in vitro exposures to EB and DEB. Although, BD exposure in mice induces chromosomal alterations and single base substitutions, the specific BD metabolite that induces the genetic events leading to tumors is uncertain. At present, it appears that only DEB can effectively induce this range of mutagenic events at levels of this metabolite that occur in the blood of mice exposed to BD. Detailed investigations to identify relevant biomarkers of BD exposure and response, particularly DNA adducts or lesions, that can be biologically linked to the range of genotoxic events known to occur in mice exposed to BD are needed.

RPA foci are associated with cell death after irradiation.

MacPhail, S. H. and Olive, P. L. RPA Foci are Associated with Cell Death after Irradiation. Radiat. Res. 155, 672-679 (2001). Complexes containing replication protein A (RPA) were observed in human TK6 and WIL-2NS lymphoblast cells and SiHa cervical carcinoma cells exposed to 250 kV X rays. Image analysis of individual cells with fluorescence-tagged anti-RPA antibodies was used to measure numbers of discrete foci per cell. RPA foci formed in S-phase cells in response to radiation doses as low as 0.5 Gy, and the number of foci/nucleus was linearly related to dose up to 50 Gy. The maximum number of cells with foci occurred 4-8 h after exposure to 4 Gy, and subsequently declined. However, the number of RPA foci per nucleus (in those cells with foci) reached a maximum after 2-4 h. Apoptotic nuclei from irradiated TK6 and WIL-2NS cells initially contained foci, but these were lost as degradation continued. Radiation-induced micronuclei in SiHa cells were greatly enriched for RPA foci, and cells with nuclei without foci often contained micronuclei with multiple RPA foci. In SiHa cells examined up to 7 days after 4 Gy, RPA foci reappeared in one or more cells in up to 90% of the surviving colonies, and some cells contained 150 or more distinct foci. Reappearance of these complexes could be indicative of radiation-induced genomic instability. These results are consistent with the idea that RPA foci observed several hours after irradiation represent irreparable lesions and as such might be useful in identifying radiosensitive cells.