Genotoxicity analysis of two hydroxyfuranones,byproducts of water disinfection,in human cells treated in vitro

In general, water for human consumption is chemically disinfected, usually by adding chlorine. As well as producing safe drinking water however, the chlorine treatment, also results in a number of disinfection byproducts (DBPs). One important class of these DBPs is made up of hydroxyfuranones (HFs). In this article, we report the results of a recent investigation to assess the genotoxicity of two HFs, namely mucobromic acid (MBA) and mucochloric acid (MCA), in cultured human cells. The comet assay is used to measure the induction of primary DNA damage and to determine the DNA repair kinetics and the ability of the tested compounds to cause oxidative damage. In addition, the micronucleus (MN) assay is applied to evaluate chromosome damage. The results of the comet assay reveal that both HFs are clearly genotoxic leading to high levels of DNA breaks, and that MBA is more effective than MCA. According to the comet results, the DNA damage induced by MBA repairs well over time, but not the one induced by MCA. Furthermore, HFs produce high levels of oxidized bases. In contrast, the results from the MN assay, which measures the induction of clastogenic and/or aneugenic effects, are mainly negative for the two HFs tested, although MCA is able to increase significantly the frequency of micronuclei in binucleated TK cells, at the concentration of 10 μM. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Genotoxicity of 11 heavy metals detected as food contaminants in two human cell lines

Heavy metals, such as arsenic (As), antimony (Sb), barium (Ba), cadmium (Cd), cobalt (Co), germanium (Ge), lead (Pb), nickel (Ni), tellurium (Te), and vanadium (V) are widely distributed in the environment and in the food chain. Human exposure to heavy metals through water and food has been reported by different international agencies. Although some of these heavy metals are essential elements for human growth and development, they may also be toxic at low concentrations due to indirect mechanisms. In this study, the genotoxic and cytotoxic properties of 15 different oxidation statuses of 11 different heavy metals were investigated using high‐throughput screening (γH2AX assay) in two human cell lines (HepG2 and LS‐174T) representative of target organs (liver and colon) for food contaminants. Base on their lowest observed adverse effect concentration, the genotoxic potency of each heavy metal in each cell line was ranked in decreasing order, NaAsO₂ > CdCl₂ > PbCl₂ (only in LS‐174T cells) > As₂O₅ > SbCl₃ > K₂TeO₃ > As₂O₃. No significant genotoxicity was observed with the other heavy metals tested. Cell viability data indicate that several heavy metals (As, Cd, Co, Ni, Sb, and Te) induce cytotoxicity at high concentrations, whereas an increase in the number of cells was observed for lead concentrations >100 µM in both cell lines tested, suggesting that lead stimulates cell growth. All these results highlight the possible human health hazards associated with the presence of heavy metals present in food. Environ. Mol. Mutagen. 59:202–210, 2018. © 2017 Wiley Periodicals, Inc.     

Genotoxicity of a freshwater cyanotoxin,cylindrospermopsin, in two human cell lines: Caco‐2 and HepaRG

Cylindrospermopsin (CYN), a cyanotoxin produced by certain freshwater cyanobacteria, causes human intoxications and animal mortalities. CYN is a potent inhibitor of protein‐ and glutathione‐synthesis. Preliminary evidence for in vivo tumor initiation has been found in mice but the mechanism remains unclear. Several in vitro and in vivo studies demonstrate that CYN is genotoxic and requires metabolic activation. In the present study, the genotoxicity of CYN was assessed in human hepatocyte and enterocyte cell lines, which are models for CYN target organs. The cytokinesis‐block micronucleus assay was conducted on liver‐derived HepaRG cells and colon‐derived Caco‐2 cells. Each cell‐type was exposed to CYN in both the differentiated and the undifferentiated states, and both with and without the cytochrome P450 inhibitor, ketoconazole, to determine the involvement of metabolism in CYN genotoxicity. CYN increased the frequency of micronuclei in binucleated cells (MNBNC) in both Caco‐2 and HepaRG cells. Moreover, ketoconazole reduced both the genotoxicity and cytotoxicity caused by CYN. Our results confirm the involvement of metabolic activation of CYN in mediating its toxicity and suggest that CYN is progenotoxic. Environ. Mol. Mutagen. 2010. © 2009 Wiley‐Liss, Inc.     

Cytotoxicity and genotoxicity assays of halobenzoquinones disinfection byproducts using different human cell lines

Recently, halobenzoquinones (HBQs) disinfection byproducts, including 2,6-dichloro-1, 4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1, 4-benzoquinone (DCMBQ), 2,3,6-trichloro-1, 4-benzoquinone (TCBQ), and 2,6-dibromobenzoquinone (DBBQ), have been of increasing concern due to their reported ability to induce oxidative damage, and thus genotoxicity. However, data on the risk of genotoxicity due to chromosomal damage by HBQs are still scarce. Here, the cytotoxicity and genotoxicity of the four HBQs were assessed using human cell lines (bladder cancer 5637 cells, colon carcinoma Caco-2 cells, and gastric MGC-803 cells). The four HBQs exhibited significant concentration-response relationships in all the three cell lines. Cytotoxicity of DCBQ, DCMBQ, TCBQ, and DBBQ, represented by the 50% concentration of inhibition (IC₅₀) values, were 80.8–99.5, 41.0–57.6, 122.1–146.6, and 86.9–93.8 μM, respectively. The lowest effective concentrations for cellular micronuclei induction in the cell lines by DCBQ, DCMBQ, TCBQ, and DBBQ were 50–75, 20–41.5, 87.4–100, and 50 μM, respectively. 5637 and Caco-2 cells were more sensitive to the cytotoxic and genotoxic effects of HBQs than MGC-803 cells. These results show that HBQs can induce chromosomal damage; DCMBQ induced the highest cytotoxicity and genotoxicity in all the cell lines, and TCBQ caused the least toxicity.     

Genotoxic and immunotoxic effects of cellulose nanocrystals in vitro

Nanocellulosics are among the most promising innovations for a wide‐variety of applications in materials science. Although nanocellulose is presently produced only on a small scale, its possible toxic effects should be investigated at this early stage. The aim of the present study was to examine the potential genotoxicity and immunotoxicity of two celluloses in vitro ‐ cellulose nanocrystals (CNC; mean fibril length 135 nm, mean width 7.3 nm) and a commercially available microcrystalline (non‐nanoscale) cellulose (MCC; particle size ～50 µm). Both celluloses showed 55% cytotoxicity at approximately 100 µg/ml after 4‐h, 24‐h, and 48‐h treatment of human bronchial epithelial BEAS 2B cells, as determined by luminometric detection of ATP and cell count (dead cells identified by propidium iodide). Neither of the materials was able to induce micronuclei (MN) in binucleate or mononucleate BEAS 2B cells after a 48‐h treatment (2.5–100 µg/ml). In human monocyte‐derived macrophages, MCC induced a release (measured by enzyme‐linked immunosorbent assay; ELISA) of the pro‐inflammatory cytokines tumor necrosis factor α (TNF‐α) and (after lipopolysaccharide‐priming) interleukin 1β (IL‐1β) after a 6‐h exposure to a dose of 300 µg/ml, but CNC (30–300 µg/ml) did not. In conclusion, our results show that nanosized CNC is neither genotoxic nor immunotoxic under the conditions tested, whereas non‐nanosized MCC is able to induce an inflammatory response. More studies are needed, especially in vivo, to further assess if CNC and other nanocelluloses induce secondary genotoxic effects mediated by inflammation. Environ. Mol. Mutagen. 56:171–182, 2015. © 2014 Wiley Periodicals, Inc.     

Estimation of genome damage after exposure to ionizing radiation and ultrasound used in industry

The application of ionizing radiation in industry for nondestructive testing entails a specific framework of working conditions that include field work, facilities with different radioactive sources, maintenance thereof without halting production, use of nonionizing radiation, and exposure to chemical agents. The present study gives an estimation of recent genome damage in two groups of subjects using chromosome aberration assay and micronucleus assay. The first group was exposed to (192)Ir and the second was simultaneously exposed to (192)Ir and ultrasound. The results show that both groups had higher values of chromosome aberrations and micronucleus frequency than controls. The group of examinees exposed both to (192)Ir and ultrasound had significantly more chromatid breaks, acentric fragments, and dicentric chromosomes, and had a significantly higher frequency of micronuclei than subjects exposed to (192)Ir only. The study suggests that the detected differences in the genome damage may be attributed to the action of ultrasound. This study confirms the dosimetry data for ionizing radiation, which indicate that the methods used in industrial radiography and the usage of nonionizing radiation entail an increased health risk. In the absence of personal dosimeters for nonionizing radiation and chemical agents, biomonitoring provides reliable parameters for estimation of genome damage and may lead to improvements in working conditions and radiation safety programs.     

In vitro evaluation of the genotoxic and cytotoxic effects of artesunate, an antimalarial drug, in human lymphocytes

Artesunate is one of the main antimalarial drugs used in several countries. It is a semisynthetic compound derived from artemisinin, a substance extracted from the Chinese plant, Artemisia annua L. Despite the widespread use of artesunate as an antimalarial drug, there is a lack of data regarding its genotoxic effects in human lymphocytes. Therefore, in this study, we used the comet assay and micronucleus test to evaluate the possible genotoxic effects of artesunate in cultured human lymphocytes. In addition, cell death by necrosis and apoptosis was also assessed. Cells exposed to different concentrations of artesunate showed a significant concentration-dependent increase (P < 0.05) in DNA damage index and micronuclei frequency. A significant increase in the frequency of apoptotic and necrotic cells was also observed. Our results showed that artesunate is a genotoxic and cytotoxic compound in cultured human lymphocytes.     

Genotoxicity testing of the herbicide trifluralin and its commercial formulation Treflan using the piscine micronucleus test

In this study, the genotoxic effects of a widely used herbicide, trifluralin, and its commercial formulation, Treflan, were evaluated using the micronucleus test in a commercially important fish species, Oreochromis niloticus (Nile Tilapia). Fish were exposed to 1, 5, and 10 microg/L doses of trifluralin and Treflan for 3, 6, and 9 days under laboratory conditions. Ethylmethanesulfonate, at a single dose of 10 mg/L, was used as positive control. Micronuclei were evaluated on the peripheral erythrocytes. Both Treflan and trifluralin treatments significantly increased the micronucleus frequencies in peripheral erythrocytes of O. niloticus. Furthermore, the genotoxicity of the active ingredient, trifluralin, was observed to be higher than that of the commercial formulation Treflan. Our results indicate that herbicide trifluralin has genotoxic potential in fish.     

Analysis of repair and PCNA complex formation induced by ionizing radiation in human fibroblast cell lines

Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase delta and epsilon, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy. The results indicate that exposure to X-rays triggers the induction of PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway. X-ray-induced repair synthesis was greatly inhibited by treatment of cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step.     

In vitro studies of the genotoxicity of ionizing radiation in human G(0) T lymphocytes

In an effort to mimic human in vivo exposures to ionizing irradiation, G(0) phase T lymphocytes from human peripheral blood samples were utilized for in vitro studies of the genotoxic effects of (137)Cs low-LET irradiation and (222)Rn high-LET irradiation. Both types of radiation induced mutations in the HPRT gene in a dose-dependent manner, with a mutant frequency (MF) = 4.28 + 1.34x + 7.51x(2) for (137)Cs (R(2) = 0.95) and MF = 4.81 + 0.67x for (222)Rn (R(2) = 0.51). Post (137)Cs irradiation incubation in the presence of cytosine arabinoside, a reversible inhibitor of DNA repair, caused an increase in the MF over irradiation alone, consistent with a misrepair mechanism being involved in the mutagenicity of low-LET irradiation. The spectrum of (137)Cs irradiation-induced mutation displayed an increase in macro-deletions (in particular total gene deletions) and rearrangement events, some of which were further defined by either chromosome painting or direct DNA sequencing. The spectrum of (222)Rn irradiation-induced mutation was characterized by an increase in small alterations, especially multiple single base deletions/substitutions and micro-deletions. These studies define the specific response of human peripheral blood T cells to ionizing irradiation in vitro and form a basis for evaluating the genotoxic effects of human in vivo exposure.     

Genetic instability of in vitro cell lines: Implications for genetic toxicity testing

Cell line-based in vitro testing has been widely used as an important component of the genotoxicity testing battery; however, the use of cell lines is constrained by several limitations, including the genetic drift and variability. A study recently reported in the literature comprehensively examined genomic changes in a large number of cell lines and reported extensive genetic variations within the same cell lines across passage numbers and laboratories, even for single-cell derived subclones. The primary objective of this communication is to raise awareness and stimulate discussion within the genotoxicity testing community of the extent of genetic variability of cell lines in general and how these variables could potentially influence the results and reproducibility of genotoxicity testing. Meanwhile, some recommendations for good cell culture practices are highlighted to mitigate, at least to some extent, the concern about genetic variation. Environ. Mol. Mutagen. 60:559–562, 2019. © 2019 Wiley Periodicals, Inc.     

Validation of a high throughput flow cytometric in vitro micronucleus assay including assessment of metabolic activation in TK6 cells

Genotoxicity is an unacceptable property for new drug candidates and we employ three screening assays during the drug discovery process to identify genotoxicity early and optimize chemical series. One of these methods is the flow cytometric in vitro micronucleus assay for which protocol optimizations have been described recently. Here, we report further validation of the assay in TK6 cells including assessment of metabolic activation. We first optimized assay conditions to allow for testing with and without metabolic activation in parallel in a 96‐well plate format. Then, we tested a set of 48 compounds carefully selected to contain known in vivo genotoxins, nongenotoxins and drugs. Avoidance of irrelevant positives, a known issue with mammalian cell‐based genotoxicity assays, is important to prevent early deselection of potentially promising compounds. Therefore, we enriched the validation set with compounds that were previously reported to produce irrelevant positive results in mammalian cell‐based genotoxicity assays. The resulting dataset was used to set the relevant cut‐off values for scoring a compound positive or negative, such that we obtained an optimal balance of high sensitivity (88%) and high specificity (87%). Finally, we tested an additional set of 16 drugs to further probe assay performance and 14 of them were classified correctly. To our knowledge, the present study is the most comprehensive validation of the in vitro flow cytometric micronucleus assay and the first to report parallel assessment with metabolic activation in reasonable throughput. The assay allows for rapidly screening novel compounds for genotoxicity and is therefore well‐suited for use in early drug discovery projects. Environ. Mol. Mutagen. 55:704–718, 2014. © 2014 Wiley Periodicals, Inc.     

Genetic toxicity of methamphetamine in vitro and in human abusers

Methamphetamine (METH) is a widely abused psychomotor stimulant. Although numerous studies have examined METH-induced neurotoxicity, its ability to produce genotoxic effects has not been evaluated. In this article, we report on the genotoxicity of METH in vitro and in human METH abusers. METH induced his(+) revertants in Salmonella typhimurium strains TA98 and TA100, and increased the frequency of hprt mutants, micronuclei, and sister chromatid exchange (SCE) in cultured Chinese hamster ovary K1 (CHO-K1) cells. These METH-induced genotoxic effects were eliminated if METH exposure was conducted in the presence of rat liver S9, indicating that the genotoxicity was caused by METH, and not by metabolites of METH. In addition, reactive oxygen species (ROS) scavengers inhibited the METH-induced micronuclei in CHO-K1 cells. Further investigation with 76 human long-term METH abusers and 98 unexposed controls demonstrated that total METH exposure correlated with micronucleus and SCE frequencies in cultured lymphocytes. The results of this study indicate that METH is a genotoxic agent and that ROS may play a role in METH-induced genotoxicity.     

电离辐射对淋巴细胞影响的研究进展

电离辐射导致的免疫系统功能低下是日常生活和临床治疗中的棘手问题.随着辐射免疫学的飞速发展,电离辐射对免疫系统尤其是对淋巴系统的影响,越来越受到重视.本文综述了近年来有关淋巴细胞及其各功能亚群辐射效应的研究进展.     

Genotoxicity of inorganic lead salts and disturbance of microtubule function

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 microM lead chloride and 0.05 microM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 microM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 microM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 microM and reached half-maximal inhibition of motility at about 50 microM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.     

人Egr-1启动子的克隆及其在肿瘤细胞中的辐射诱导反应

目的:克隆人Egr-1基因的启动子, 插入荧光素酶报告基因载体中, 并检测电离辐射对其活性的影响.方法:采用PCR技术从人乳腺癌细胞系MCF-7基因组中扩增出Egr-1启动子, 将其克隆到pGL3-basic载体中;将重组质粒转染人肿瘤细胞, 测定Egr-1启动子在不同辐射条件下转录活性的改变.结果:成功构建了Egr-1启动子的荧光素酶报告基因;在不同剂量的γ射线照射后, Egr-1的启动子活性均明显高于未照射组;在同一剂量照射后48 h, Egr-1的启动子活性达峰值.结论:本实验构建的Egr-1启动子具有辐射激活的功能, 为进一步研究放射-基因治疗奠定了基础.     

Genotoxicity biomonitoring in regions exposed to vehicle emissions using the comet assay and the micronucleus test in native rodent Ctenomys minutus

Exposure to motor vehicle emissions represents an important concern for possible long-term health effects. The present report describes: 1) the application and verification of the alkaline comet assay in Ctenomys minutus to detect the possible genotoxicity of automobile emissions; 2) a comparison of the comet assay results with peripheral blood micronucleus (MN) assay results performed in the same animals; and 3) the identification of agents involved in the responses and in the seasonal variation of the effects. Ctenomys minutus (Octodontidae-Rodentia) were captured in two different fields from both sides of RS/030, a highway on the coastal plain of the Brazilian state of Rio Grande do Sul. Reference animals were obtained from a nearby field that was about 3 km distant from any road. By the end of this study, 123 rodents (73 females and 50 males) were live-trapped. Our results indicate that there was an increase in cells with DNA damage for C. minutus environmentally exposed to automobile emissions, as demonstrated by the alkaline comet assay, but there was no increase in micronucleated cells. The alkaline comet assay showed age and gender differences in the response. The comet assay results suggest that adult females are the principal population affected by air pollutants from vehicle emissions. Chemical data were also collected from areas exposed to automobile exhaust and these indicated that elevated levels of hydrocarbons, metals, and NO(2) were associated with the elevated levels of damaged cells observed in the wild rodent C. minutus. Our results agree with previous data on engine and fuel components, where weak increases in damage for native rodents exposed to emissions have been observed. Other larger, controlled studies are needed to better understand how the metabolism of C. minutus affects its response to emission exposure.     

1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay,but is in mouse liver micronucleus assay

1,4-Dioxane, an animal carcinogen, was not previously genotoxic in in vitro assays. We reevaluated the compound's genotoxic potential in five in vitro genotoxicity tests in the presence and absence of S9 mix using recommended new protocols. We used the bacterial reverse mutation assay with Salmonella TA and E. coli WP2 strains, including the plate and preincubation methods, the CHO chromosomal aberration assay, including examination of polyploid induction and extended sampling time, the CHO sister-chromatid exchange assay with short and long treatment time, the mouse lymphoma tk assay (microtiter method), including longer treatment time (24 hr), and the CHO micronucleus assay with short and long treatment times. The highest concentration we used was five mg/ml or plate. We also evaluated the genotoxic effect of 1,4-dioxane in vivo by conducting peripheral blood and liver micronucleus assays in the same mice after single oral administration of up to 3,000 mg/kg. All in vitro assays and the peripheral blood micronucleus assay were negative. The mouse liver micronucleus assay, on the other hand, was positive, indicating that 1,4-dioxane might be genotoxic. It is also conceivable that the positive result in mouse liver micronucleus assay was due to a nongenotoxic mechanism, i.e., errors in genetic repair following enhancement of hepatocyte proliferation. Environ. Mol. Mutagen. 32:269–280, 1998 © 1998 Wiley-Liss, Inc.     

Combined treatment of human colorectal tumor cell lines with chemotherapeutic agents and ionizing irradiation can in vitro induce tumor cell death forms with immunogenic potential

Chemotherapeutic agents (CT) and ionizing radiation (X-ray) induce DNA damage and primarily aim to stop the proliferation of tumor cells. However, multimodal anti-cancer therapies should finally result in tumor cell death and, best, in the induction of systemic anti-tumor immunity. Since distinct therapy-induced tumor cell death forms may create an immune activating tumor microenvironment, this study examined whether sole treatment with CT that are used in the therapy for colorectal cancer or in combination with X-ray result in colorectal tumor cell death with immunogenic potential. 5-Fluorouracil (5-FU), Oxaliplatin (Oxp), or Irinotecan (Irino) in combination with X-ray were all potent inhibitors of colorectal tumor cell colony formation. This study then examined the forms of cell death with AnnexinA5-FITC/Propidium Iodide staining. Necrosis was the prominent form of cell death induced by CT and/or X-ray. While only a combination of Irino with X-ray leads to death induction already 1 day after treatment, also the combinations of Oxp or 5-FU with X-ray and X-ray alone resulted in high necrosis rates at later time points after treatment. Inhibition of apoptosis increased the amount of necrotic tumor cells, suggesting that a programmed form of necrosis can be induced by CT + X-ray. 5-FU and Oxp alone or in combination with X-ray and Irino plus X-ray were most effective in increasing the expression of RIP, IRF-5, and p53, proteins involved in necrotic and apoptotic cell death pathways. All treatments further resulted in the release of the immune activating danger signals high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). The supernatants of the treated tumor cells induced maturation of dendritic cells. It is, therefore, concluded that combination of CT with X-ray is capable of inducing in vitro cell death forms of colorectal tumors with immunogenic potential.     

Combined treatment of human colorectal tumor cell lines with chemotherapeutic agents and ionizing irradiation can in vitro induce tumor cell death forms with immunogenic potential

Chemotherapeutic agents (CT) and ionizing radiation (X-ray) induce DNA damage and primarily aim to stop the proliferation of tumor cells. However, multimodal anti-cancer therapies should finally result in tumor cell death and, best, in the induction of systemic anti-tumor immunity. Since distinct therapy-induced tumor cell death forms may create an immune activating tumor microenvironment, this study examined whether sole treatment with CT that are used in the therapy for colorectal cancer or in combination with X-ray result in colorectal tumor cell death with immunogenic potential. 5-Fluorouracil (5-FU), Oxaliplatin (Oxp), or Irinotecan (Irino) in combination with X-ray were all potent inhibitors of colorectal tumor cell colony formation. This study then examined the forms of cell death with AnnexinA5-FITC/Propidium Iodide staining. Necrosis was the prominent form of cell death induced by CT and/or X-ray. While only a combination of Irino with X-ray leads to death induction already 1 day after treatment, also the combinations of Oxp or 5-FU with X-ray and X-ray alone resulted in high necrosis rates at later time points after treatment. Inhibition of apoptosis increased the amount of necrotic tumor cells, suggesting that a programmed form of necrosis can be induced by CT + X-ray. 5-FU and Oxp alone or in combination with X-ray and Irino plus X-ray were most effective in increasing the expression of RIP, IRF-5, and p53, proteins involved in necrotic and apoptotic cell death pathways. All treatments further resulted in the release of the immune activating danger signals high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). The supernatants of the treated tumor cells induced maturation of dendritic cells. It is, therefore, concluded that combination of CT with X-ray is capable of inducing in vitro cell death forms of colorectal tumors with immunogenic potential.