Differential Response to Gamma Radiation of Human Stomach Cancer Cells in Vitro

Summary

In vitro effects of radiation were studied in two permanent cell lines (AGS and SII) from two patients with adenocarcinoma of the stomach and three permanent sublines from each cell line. Radiation survival parameters for AGS and SII parent cell lines and sublines were determined after in vitro irradiation of their cells with 0·5 to 10 Gy of ⁶⁰Co gamma rays. The AGS and SII cell lines had different growth properties, DNA contents and radiation survival curves. Surviving fractions of SII parent cells (76 chromosomes) after 2·0 and 10 Gy were 1·22 and 17·8 times greater, respectively, than values for AGS parent cells (47 chromosomes). Sensitivities (D₀) were 1·08 and 1·45 Gy for AGS and SII parent lines, respectively. The D₀ values for AGS parent cells and sublines were similar (1·01 to 1·08 Gy), but SII parent cells and sublines had D₀ values of 1·45, 1·36, 1·37 and 1·12 Gy (for SII-A). Also, the SII parent cells had survival fractions after 2·0 and 10 Gy that were 1·3 and 11·3 times greater, respectively, than values for the SII-A cells. These data show differences in radiation responses among stomach cancer cell lines and sublines that may relate to DNA content, but there was no consistent correlation between radiation response and a particular cell characteristic.     

Synergistic anti-oral cancer effects of UVC and methanolic extracts of Cryptocarya concinna roots via apoptosis,oxidative stress and DNA damage

Purpose Radiation combined with natural products may improve the radiosensitivity of cancer cells. This study investigated the potential of a combined modality treatment with Ultraviolet C (UVC; wavelength range 200–280?nm) and our previously identified anti-oral cancer agent (methanolic extracts of Cryptocarya concinna roots; MECCrt) in oral cancer cells.

Materials and methods The mechanism of the possible synergy of UVC and MECCrt was explored in terms of cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MitoMP), and DNA damage analyses.

Results In cell viability (%) at 24?h treatment, the low doses of UVC (14 J/m²) and MECCrt (10 μg/ml) resulted in slight damage to human oral cancer Ca9-22 cells (83.2 and 80.4) but was less harmful to human oral normal HGF-1 cells (93.4 and 91.8, respectively). The combined treatment of UVC and MECCrt (UVC/MECCrt) had a lower viability (54.5%) than UVC or MECCrt alone in Ca9-22 cells but no showed significant change in HGF-1 cells. In Ca9-22 cells, the expression of flow cytometry-based apoptosis (sub-G1 phase, annexin V, and pancaspase assays) was significantly higher in UVC/MECCrt than in UVC or MECCrt alone (p < 0.0001). Using flow cytometry, intracellular ROS levels of UVC/MECCrt and MECCrt alone were higher than for UVC alone. MitoMP change and H2A histone family member X (γH2AX; H2AFX)-based DNA damage were synergistically inhibited and induced by MECCrt/UVC compared to its single treatment in Ca9-22 cells, respectively.

Conclusion UVC plus MECCrt treatment had selective killing and synergistic anti-proliferative effects against oral cancer cells involving apoptosis, oxidative stress, and DNA damage. This combination therapy appears to have a great clinical potential against oral cancer cells.     

Effect of γ-irradiation on gene expression of heat shock proteins in the foodborne pathogen Escherichia coli O157:H7

Abstract

Purpose: The expression levels of seven genes (clpB, dnaK, groES, grpE, htpG, htpX and ibpB) encoding heat shock proteins (HSP) in Escherichia coli O157:H7 (E. coli) gamma irradiated was investigated. Timing impact of post-irradiated RNA extraction on the expression levels of these seven genes was also studied at a dose damaging the bacterial cells (0.4 kGy).

Methods: Bacterial samples were γ-irradiated at 0.4 kGy and at a lethal dose of 1.3 kGy. RNA was extracted at 0 min post irradiation for both irradiation doses and at 15, 30, 60, 90 or 120 min post-irradiation at the dose damaging the cells. Quantification of the gene expression was performed using quantitative real-time polymerase chain reaction (q-RT-PCR).

Results: The expression of genes encoding HSP was a very dynamic process evolving rapidly when E. coli cells were irradiated at 0.4 kGy. Notably, groES, grpE and ibpB were more up- regulated at 1.3 kGy than those at 0.4 kGy.

Conclusions: For the seven genes studied there were more damaged proteins during irradiation at the lethal dose and this dose causes increased expression in HSP which contributes to damage reparation. Expression patterns of genes encoding HSP in E. coli treated by γ-irradiation are different from those treated by heat shock.     

Static electric fields interfere in the viability of cells exposed to ionising radiation

Purpose: The interference of electric fields (EF) with biological processes is an issue of considerable interest. No studies have as yet been reported on the combined effect of EF plus ionising radiation. Here we report studies on this combined effect using the prokaryote Microcystis panniformis, the eukaryote Candida albicans and human cells.

Materials and methods: Cultures of Microcystis panniformis (Cyanobacteria) in glass tubes were irradiated with doses in the interval 0.5–5 kGy, using a ⁶⁰Co gamma source facility. Samples irradiated with 3 kGy were exposed for 2 h to a 20 V · cm^?1 static electric field and viable cells were enumerated. Cultures of Candida albicans were incubated at 36°C for 20 h, gamma-irradiated with doses from 1–4 kGy, and submitted to an electric field of 180 V · cm^?1. Samples were examined under a fluorescence microscope and the number of unviable (red) and viable (apple green fluorescence) cells was determined. For crossing-check purposes, MRC5 strain of lung cells were irradiated with 2 Gy, exposed to an electric field of 1250 V/cm, incubated overnight with the anti-body anti-phospho-histone H2AX and examined under a fluorescence microscope to quantify nuclei with γ-H2AX foci.

Results: In cells exposed to EF, death increased substantially compared to irradiation alone. In C. albicans we observed suppression of the DNA repair shoulder. The effect of EF in growth of M. panniformis was substantial; the number of surviving cells on day-2 after irradiation was 12 times greater than when an EF was applied. By the action of a static electric field on the irradiated MRC5 cells the number of nuclei with γ-H2AX foci increased 40%, approximately.

Conclusions: Application of an EF following irradiation greatly increases cell death. The observation that the DNA repair shoulder in the survival curve of C. albicans is suppressed when cells are exposed to irradiation + EF suggests that EF likely inactivate cellular recovering processes. The result for the number of nuclei with γ-H2AX foci in MRC5 cells indicates that an EF interferes mostly in the DNA repair mechanisms. A molecular ad-hoc model is proposed.     

Low dose ionizing irradiation suppresses cellular senescence in normal human fibroblasts

Purpose: Exposure to high dose ionizing radiation leads to premature cell senescence and suppression of cell proliferation. In contrast, low dose and low dose-rate gamma-irradiation can lead to stimulation of cell proliferation. We aimed to examine whether the low dose radiation-induced proliferation of normal human fibroblasts can lead to a progressive depletion of proliferation potential and to an early onset of senescence.

Materials and methods: Normal human embryonic lung fibroblasts (HELF-104) at passage 22–24 were gamma-irradiated with doses of 0 (sham-irradiation), 10, 30, 50, 90, 120, 150, 200, and 500 mGy as well as 1 and 2?Gy. After irradiation, the fraction of cells positively stained for senescence-associated β-galactosidase activity was measured weekly until the cell culture completely ceased to proliferate.

Results: We show that single irradiation of HELF-104 cells with 30 and 50 mGy resulted in deceleration of senescence. The suppression of senescence was observed during almost the entire length of the study up to a complete arrest of cell growth.

Conclusions: Our data, together with the previously published observation of delayed stimulation of proliferation in HELF-104 cells exposed to 30 mGy, suggest that low dose gamma-irradiation can increase the overall proliferative potential of normal human fibroblasts.     

Effect of gamma radiation as a post-harvest disinfestation treatment against life stages of the coffee berry borer,Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae)

Abstract

Purpose: Gamma radiation is mainly used for disinfesting insect pests as an alternative for harmful fumigants. The specific dose of radiation is known to affect different developmental stages of insect pests. The study was conducted to determine the effective irradiation doses for inhibition of developmental stages and adult longevity of the coffee berry borer, Hypothenemus hampei (Ferrari).

Materials and methods: Irradiation was carried with the following doses: five levels between 0.01 and 0.16?kGy for eggs, seven levels between 0.10 and 2.00?kGy for larva and prepupa, six levels between 0.10 and 1.60?kGy for pupa and ten levels between 0.10 and 3.20?kGy for adults.

Results: Egg development was completely arrested at 0.160?kGy. A dose of 2.00?kGy caused 100% mortality in the first and second instar larva and 98.99% mortality in prepupa. The dose of 1.60?kGy prevented adult eclosion from the irradiated pupa. The adult mortality was 100% at 3.20?kGy.

Conclusion: A dose of 3.20 kGy could successfully provide complete security from all developmental stages of H. hampei and prevent yield loss in green coffee as well as the spread of the pest.     

Effects of gamma radiation on fungi infected rice (in vitro)

Abstract

Purpose: This work focuses on the effect of gamma radiation on seed born fungi (in vitro) on Oryza sativa (Swarna, Initial Evaluation Trial-5656).

Materials and methods: The responses of fungi to gamma radiation (0–4.2 kGy; 0.12 kGy/h) were studied in individual cultures of major seed-borne fungi including Alternaria alternata, Aspergillus flavus, Trichoderma viride and Curvularia geniculata.

Results: The inactivation of individual fungal-viability was noted between 1.0–2.0 kGy for A. alternata and A. flavus and 0.5–1.0 kGy for T. viride and C. geniculata. Complete inhibition was observed at <2.5 kGy. Formations of multiple germ tubes were noted in A. alternata and A. flavus at 2 kGy and 2.5 kGy, respectively. A. flavus required a higher dose to reduce viability to 10% (D₁₀) value in comparison to other selected fungi.

Conclusions: Different fungi exhibited different radiosensitivity. The dose range of 2–2.5 kGy was effective in killing all selected fungi. The fungi showing a higher D₁₀ value exhibited multiple germ tubes.     

Lack of induced mutagenesis in E. coli or human lymphoblast cell line upon long-term sub-culturing in medium from irradiated meat

Purpose: Current study was aimed to enhance the confidence of consumers as well as entrepreneurs towards food irradiation program.

Materials and methods: In this work, safety of high dose (25?kGy) irradiated meat samples (HDIMS) was ascertained by scoring mutation frequency through a long-term sub-culturing study in Escherichia coli MG1655 cells (ATCC 700926) up to 1500 generations (at 1%), 250 generations (at 5% and 10%) and human lymphoblast thymidine kinase heterozygote (TK6) cell line (ATCC CRL-8015) [at two gene loci, tk^?/+ (thymidine kinase) and hprt⁺ (Hypoxanthine Phosphoribosyltransferase)] up to 156 generations using goat meat sample. Also these samples were assayed at further radiation doses of 10, 45 and 70?kGy at 2% concentration (in cell line), and 1% (in E. coli). Study was also performed with other meat samples such as chicken, fishes (pomfret and rohu) and shrimps by carrying out limited long-term sub-culturing trials in human lymphoblast cell line. Mutation analysis was also carried out using a novel DPAR (Differential loss of Plasmid Antibiotic Resistance) assay followed by sequencing of tc^R (tetracycline resistance) gene of pBR322 plasmid isolated from E. coli cells grown for 1500 generations on HDIMS medium and RAPD (Random Amplified Polymorphic DNA) analysis of the genome.

Results and conclusion: None of the assays exhibited any induced mutation when analyzed at regular time intervals. RAPD analysis also did not indicate any change in its nucleotide sequence, ruling out the occurrence of any silent mutation. Thus, the present findings report absence of mutagenic effect of high dose irradiated meat samples.     

A role for endothelial cells in radiation-induced inflammation

Purpose: To unravel the role of the vasculature in radiation-induced brain tissue damage.

Materials and methods: Postnatal day 14 mice received a single dose of 10?Gy cranial irradiation and were sacrificed 6?h, 24?h or 7 days post-irradiation. Endothelial cells were isolated from the hippocampus and cerebellum using fluorescence-activated cell sorting, followed by cell cycle analysis and gene expression profiling.

Results: Flow cytometric analysis revealed that irradiation increased the percentage of endothelial cells, relative to the whole cell population in both the hippocampus and the cerebellum. This change in cell distribution indicates that other cell types are more susceptible to irradiation-induced cell death, compared to endothelial cells. This was supported by data showing that genes involved in endothelial cell-specific apoptosis (e.g. Smpd1) were not induced at any time point investigated but that genes involved in cell-cycle arrest (e.g. Cdkn1a) were upregulated at all investigated time points, indicating endothelial cell repair. Inflammation-related genes, on the other hand, were strongly induced, such as Ccl2, Ccl11 and Il6.

Conclusions: We conclude that endothelial cells are relatively resistant to ionizing radiation but that they play an active, hitherto unknown, role in the inflammatory response after irradiation. In the current study, this was shown in both the hippocampus, where neurogenesis and extensive cell death after irradiation occurs, and in the cerebellum, where neurogenesis no longer occurs at this developmental age.     

Induction of adaptive response: Pre-exposure of mice to 900 MHz radiofrequency fields reduces hematopoietic damage caused by subsequent exposure to ionising radiation

Purpose:?To investigate whether an adaptive response can be induced in mice which were pre-exposed to 900 MHz radiofrequency fields.

Materials and methods:?Adult male Kunming mice were exposed to 900 MHz radiofrequency fields (RF) at power intensities of 12, 120 and 1200?μW/cm² for 1 h/day for 14 days and then subjected to whole body gamma-irradiation. The results were compared with those in unexposed control animals and those exposed to gamma-irradiation alone (without pre-exposure to RF). The extent of survival and hematopoietic tissue damage (assessed in the form of nucleated colony forming cells in the bone marrow and colony forming cells in the spleen of lethally irradiated ‘recipient’ mice) as well as the expression of cell cycle-related genes were investigated.

Results:?The results indicated a significant increase in survival time, reduction in the hematopoietic tissue damage in RF pre-exposed mice which were gamma-irradiated (as compared with those exposed to gamma-radiation alone). This was accompanied by significantly increased expression of cell cycle-related genes, namely, cyclin-D1, cyclin-E, cyclin-DK4 and cyclin-DK2 in hematopoietic cells.

Conclusions:?Pre-exposure of mice to 900 MHz radiofrequency fields has resulted in a significant reduction in hematopoietic damage caused by subsequent exposure to ionising radiation. This phenomenon appears to be similar to that of the ‘adaptive response’ which is well documented in scientific literature.     

Necrosis is not induced by gadolinium neutron capture in glioblastoma multiforme cells

Abstract

Purpose: A comparative study of the effects of different radiation modalities on cell death was performed.

Materials and methods: Radiation modalities included γ-rays, fast neutrons, a mixed energy neutron beam called the modified enhanced thermal neutron beam and the mixed beam including Auger electron irradiation by gadolinium neutron capture. U87 (human brain tumor cells) cell survival curve data were modeled to predict how cells died. Transmission electron microscopy (TEM) images were assembled into a morphology of cell death (MCD) database and used to determine the fraction of necrotic or autophagic cells.

Results: Linear energy transfer (LET) differences for the different radiation modalities were revealed by modeling. All radiation modalities induced autophagy but only fast neutrons induced significant levels of necrosis. No necrosis, above control levels, was found in cells irradiated with mixed beam irradiation including Auger electrons. The number of autophagosomes increased with increasing time after exposure to all radiation modalities indicating progression of autophagy but only cells irradiated with the mixed beam plus Auger electrons exhibited extreme autophagy.

Conclusions: Mixed neutron beam irradiation plus Auger electron irradiation from gadolinium neutron capture is a moderately high LET modality that kills U87 cells without the induction of necrosis and with progression of autophagy to an extreme state.     

Luminescence characteristics for identifying irradiated black soybeans

Abstract

Purpose: Considering the commercial use of food irradiation and the prevalence of international trade of irradiated food and agricultural commodities, black soybeans originating from China or Korea were irradiated at 0–5 kGy. Photostimulated luminescence (PSL) and thermoluminescence (TL) were investigated for their ability to identify characteristics that would distinguish irradiated from non-irradiated samples.

Materials and methods: Dried black soybeans [Glycine max (L.) Merr.] were irradiated using a Co-60 gamma irradiator or an electron-beam accelerator and then analysed by PSL and TL.

Results: PSL photon counts were higher in irradiated samples than in non-irradiated ones and increased with applied doses, making it possible to distinguish irradiated from non-irradiated samples. The TL analysis revealed glow curves (TL₁) with low intensity for non-irradiated samples but a higher intensity (~200°C) for irradiated samples, showing increased intensities with radiation dose. The minerals were re-irradiated at 1 kGy and the second TL glow curve (TL₂) was measured. Based on the calculated TL ratios (TL₁/TL₂) and the shape of TL₁ glow curves, the irradiated samples could be distinguished from non-irradiated ones.

Conclusions: PSL and TL are effective screening and reference methods for distinguishing gamma ray or electron beam irradiated black soybeans from non-irradiated black soybeans.     

Disruption of Hif-1α enhances cytotoxic effects of metformin in murine squamous cell carcinoma

Purpose: In the present study, we investigated whether the disruption of the Hif-1α gene affects the sensitivity of SCC VII cells to metformin and also if metformin functions as a radiosensitizer using murine squamous cell carcinoma (SCC VII) cells.

Materials and methods: Cultured SCC VII and SCC VII Hif-1α-deficient cells were incubated with metformin under glucose-free and/or hypoxia-mimetic conditions and cell viabilities were measured. Tumor-bearing mice were continuously given 5-bromo-2’-deoxyuridine (BrdU) to label all proliferating cells. Tumor-bearing mice were then subjected to γ-ray irradiation after the metformin treatment. Immediately after irradiation, cells were isolated from some tumors and incubated with a cytokinesis blocker. The responses of quiescent and total (=?proliferating?+?quiescent) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU.

Results: The disruption of Hif-1α increased the sensitivity of SCC VII cells to metformin in glucose-free medium. Metformin-induced decreases in the percentage of dead cells in the presence of CoCl₂ were partially reduced when Hif-1α was disrupted. In vivo, metformin increased the radiosensitivity of SCC VII Hif-1α-deficient cells.

Conclusion: The combination of disruption of Hif-1α and metformin effectively enhanced the radiosensitivity of SCC VII cells.     

Increased susceptibility to delayed genetic effects of low dose X-irradiation in DNA repair deficient cells

Abstract

Purpose: To examine whether the levels of micronuclei induction, as a marker for genomic instability in the progeny of X-irradiated cells, correlates with DNA repair function.

Materials and methods: Two repair deficient cell lines (X-ray repair cross-complementing 1 [XRCC1] deficient cell line [EM9] and X-ray repair cross complementing 5 [XRCC5; Ku80] deficient X-ray sensitive Chinese hamster ovary [CHO] cell line [xrs5]) were used in addition to wild-type CHO cells. These cells were irradiated with low doses of X-rays (up to 1 Gy). Seven days after irradiation, micronuclei formed in binucleated cells were counted. To assess the contribution of the bystander effect micronuclei induction was measured in progeny of non-irradiated cells co-cultured with cells that had been irradiated with 1Gy.

Results: The delayed induction of micronuclei in 1 Gy-irradiated cells was observed in normal CHO and EM9 but not in xrs5. In the clone analysis, progenies of xrs5 under bystander conditions showed significantly higher levels of micronuclei, while CHO and EM9 did not.

Conclusion: Genomic instability induced by X-irradiation is associated with DSB (double-strand break) repair, even at low doses. It is also suggested that bystander signals, which lead to genomic instability, may be enhanced when DSB repair is compromised.     

Combined effect of alpha particles and cigarette smoke on human lung epithelial cells in vitro

Abstract

Purpose: The combined toxicity of alpha particles and cigarette smoke to the critical cells in the lungs was investigated to assess the risk of smoking workers who handle naturally occurring radioactive materials.

Materials and methods: The toxicity of alpha particles and cigarette smoke extract (CSE) was evaluated in terms of DNA double-strand break (DSB) induction and clonogenic cell death of human lung epithelial cells in vitro. The cells were exposed to alpha particles at doses of up to 0.25?Gy for gamma-H2AX assay and from 1.25?Gy to 5?Gy for clonogenic assay. CSE exposure of the cells was facilitated in the culture medium at CSE concentrations ranging from 1% to 12%. Additional experiments were performed using mouse endothelial cells for comparison.

Results: The increases in the levels of DNA DSBs were linearly dependent on radiation dose and CSE concentration. The CSE-treated cells also responded with a linearly increasing number of DNA DSBs to the radiation dose. Both human lung epithelial cells and mouse endothelial cells showed exponential decreases in clonogenic surviving fraction as the dose from alpha particle exposure increased. Both cells responded with the clonogenic surviving fractions decreasing in a linear proportion to the CSE concentration in the culture medium.

Conclusion: In our experimental in vitro setup, CSE treatment and alpha particle exposure affected the cells in an additive manner either for DNA DSB production or for clonogenic cell death induction.     

Effect of Oxygen on DNA Strand Breaks in Irradiated Thymocytes

Summary

The ability of gamma-irradiated adenovirus to produce viral structural antigens (Vag) was examined in several normal and Xeroderma pigmentosum (XP) fibroblast strains. The fibroblast cultures were infected with either irradiated or nonirradiated adenovirus and at 48 hours after infection, cells were examined for the presence of Vag using immunofluorescent staining. Survival of Vag synthesis for gamma-irradiated adenovirus had a D₃₇ value of 47 ± 4 × 10⁴ rad following the infection of seven normal fibroblast strains. The survival of this viral function was found to be significantly less following infection of the XP strains. D₃₇ values for Vag synthesis expressed as a percentage of that obtained on normal strains were obtained for a representative strain from each of the XP complementation groups: group A, 57 per cent; group B, 61 per cent; group C, 61 per cent, group D, 59 per cent; group E, 73 per cent; and variant, 75 per cent. These results indicate that XP cells have a reduced repair capacity for some type of gamma-ray-induced DNA damage.     

Radioprotective effect of green tea and grape seed extracts mixture on gamma irradiation induced immune suppression in male albino rats

Purpose: Green tea extract (GTE) and grape seed extract (GSE) have antioxidant and radioprotective effects. The current study aimed to investigate the radioprotective effect of GTE and GSE mixture on radiation-induced immune suppression in rats.

Method: A total of 35 male albino rats were divided into five groups: group 1 (control rats). The 2nd and 3rd groups rats were exposed to a single dose of gamma radiation (5 and 10?Gy), respectively. The 4th and 5th groups of rats were gamma-irradiated with 5 and 10?Gy, respectively, then administrated by gavage with GTE and GSE mixture (100?mg: 200?mg/kg BW), respectively, for 14 consecutive days.

Results: Gamma irradiation induced hematological, immunological and biochemical effects in rats. Treated rats with GTE and GSE mixture (1:2) showed an increase in concentrations of immune cells including CD4 and CD8. The level of pro-inflammatory cytokines Tumor necrosis factor-α and C-reactive protein elevated after γ-irradiation and significantly decreased by mixture administration. Moreover, groups treated with antioxidant mixture showed a significant increase in all hematological parameters and a significant decrease in cholesterol and triglyceride levels.

Conclusion: GTE and GSE mixture is a good radioprotector and immune modulator compound, indicating its possible use as an adjuvant during radiotherapy.     

The importance of serum serotonin levels in the measurement of radiation-induced bystander cell death in HaCaT cells

Abstract

Purpose: The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project.

Materials and methods: Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay.

Results: A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum.

Conclusions: The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.