Risk assessment of short-term intake of pyrrolizidine alkaloids in food: derivation of an acute reference dose.

Pyrrolizidine alkaloids (PA) are phytochemicals that are known to act as human hepatotoxins and are also considered to be genotoxic carcinogens. Several plant-derived foods are frequently contaminated with PA, like teas and herbal infusions, spices and herbs or certain food supplements. With respect to the chronic toxicity of PA, the carcinogenic potential of PA is generally regarded as the critical toxicological effect. The risk assessment of the short-term toxicity of PA, however, is internationally less consistent. The characteristic pathological syndrome of acute PA toxicity is hepatic veno-occlusive disease. High PA exposure levels may lead to liver failure and even death as documented by several case reports. In the present report, we suggest a risk assessment approach for the derivation of an acute reference dose (ARfD) for PA of 1 µg/kg body weight per day based on a sub-acute animal toxicity study in rats after oral PA administration. The derived ARfD value is further supported by several case reports describing acute human poisoning following accidental PA intake. The here derived ARfD value may be used for PA risk assessment in cases where the short-term toxicity of PA is of interest in addition to the assessment of the long-term risks.

Interplay of cGAS with micronuclei: Regulation and diseases.

In higher eukaryotes, sophisticate regulation of genome function requires all chromosomes to be packed into a single nucleus. Micronucleus (MN), the dissociative nucleus-like structure frequently observed in aging and multiple disease settings, has critical, yet under-recognized, pathophysiological functions. Micronuclei (MNi) have recently emerged as major sources of cytosolic DNA that can activate the cGAS-STING axis in a cell-intrinsic manner. However, MNi induced from different genotoxic stressors display great heterogeneity in binding or activating cGAS and the signaling responses downstream of the MN-induced cGAS-STING axis have divergent outcomes including autoimmunity, autoinflammation, metastasis, or cell death. Thus, full characterization of molecular network underpinning the interplay of cGAS and MN is important to elucidate the pathophysiological roles of immunogenic MN and design improved drugs that selectively target cancer via boosting the MN-derived cGAS-STING axis. Here, we summarize our current understanding of the mechanisms for self-DNA discrimination by cGAS. We focus on discussing how MN immunogencity is dictated by multiple mechanisms including integrity of micronuclear envelope, state of nucleosome and DNA, competitive factors, damaged mitochondrial DNA and micronucleophagy. We also describe emerging links between immunogenic MN and human diseases including cancer, neurodegenerative diseases and COVID-19. Particularly, we explore the exciting concept of inducing immunogenic MN as a therapeutic approach in treating cancer. We propose a new theoretical framework to describe immunogenic MN as a biological sensor to modulate cellular processes in response to genotoxic stress and provide perspectives on developing novel experimental approaches to unravel the complexity of MN immunogenicity regulation and immunogenic MN pathophysiology.

Cytokinesis-block micronucleus assay of celecoxib and celecoxib derivatives.

Celecoxib is used widely for the acute treatment of pain and for pain relief in various diseases. Furthermore, it shows potential in chemoprevention, although chronic treatment with celecoxib could lead to adverse effects like cardiovascular events. New derivatives of celecoxib were synthesised that may be suitable as chemopreventive agent without inducing adverse effects. Critical endpoint for a safe use of pharmaceuticals is genotoxicity after application. A standard test for the assessment of genotoxicity is the cytokinesis-block micronucleus assay, that evaluates the number micronuclei after treatment of cells with a test compound as biomarker for DNA damage. Various promising derivatives of celecoxib have been assessed with the cytokinesis-block micronucleus assay in HeLa-H2B-GFP cells. It could be demonstrated, that neither celecoxib nor its derivatives were genotoxic in this assay and therefore celecoxib derivatives could be developed further for a safe use as chemopreventive agent.

Micronucleus frequency in buccal mucosa cells of patients with neurodegenerative diseases.

Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer's disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome.

Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells.

Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.

Eugenol Exerts Apoptotic Effect and Modulates the Sensitivity of HeLa Cells to Cisplatin and Radiation.

Eugenol is a phytochemical present in different plant products, e.g., clove oil. Traditionally, it is used against a number of different disorders and it was suggested to have anticancer activity. In this study, the activity of eugenol was evaluated in a human cervical cancer (HeLa) cell line and cell proliferation was examined after treatment with various concentrations of eugenol and different treatment durations. Cytotoxicity was tested using lactate dehydrogenase (LDH) enzyme leakage. In order to assess eugenol's potential to act synergistically with chemotherapy and radiotherapy, cell survival was calculated after eugenol treatment in combination with cisplatin and X-rays. To elucidate its mechanism of action, caspase-3 activity was analyzed and the expression of various genes and proteins was checked by RT-PCR and western blot analyses. Eugenol clearly decreased the proliferation rate and increased LDH release in a concentration- and time-dependent manner. It showed synergistic effects with cisplatin and X-rays. Eugenol increased caspase-3 activity and the expression of Bax, cytochrome c (Cyt-c), caspase-3, and caspase-9 and decreased the expression of B-cell lymphoma (Bcl)-2, cyclooxygenase-2 (Cox-2), and interleukin-1 beta (IL-1ß) indicating that eugenol mainly induced cell death by apoptosis. In conclusion, eugenol showed antiproliferative and cytotoxic effects via apoptosis and also synergism with cisplatin and ionizing radiation in the human cervical cancer cell line.

Differentiated and exponentially growing HL60 cells exhibit different sensitivity to some genotoxic agents in the comet assay.

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation. A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status. Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing.

The Biological Activity of the Novel Vinca Alkaloids 4-chlorochablastine and 4-chlorochacristine.

BACKGROUND: Vinca alkaloids are important cancer drugs belonging to the class of antimitotic agents. The most commonly used substances are vinblastine and vincristine, other compounds are vinorelbine and vinflunine. All of them are very effective drugs but their use is limited by severe side-effects including neurotoxicity and bone marrow depression. Therefore, it is very important to develop novel vinca alkaloids with similar efficacy but lower toxicity. METHODS: Here, we analyzed two new compounds, 4-chlorochablastine and 4-chlorochacristine, with regard to their biological activity. These novel compounds were applied to a leukemia cell line at clinically relevant concentrations. For comparison, the established vinca alkaloids vinblastine, vincristine, vinorelbine, and vinflunine were also tested. RESULTS: Both novel substances decreased cellular proliferation. Apoptosis was found to be increased using two different methods reflecting early and late apoptosis. Cell cycle analysis revealed a clear decrease in G1-cells and an increase in G2/M-cells indicating an arrest in mitosis. In general, 4- chlorochablastine and 4-chlorochacristine caused these effects at concentrations higher than those needed for vinblastine, vincristine, and vinorelbine, but the potency was approximately in the range of vinflunine. CONCLUSION: Taken together, the results show first indications that these novel vinca alkaloids might be effective and that they warrant further analysis.

Aflatoxin in foodstuffs: Occurrence and recent advances in decontamination.

Aflatoxins are highly toxic compounds produced as secondary metabolites by some Aspergillus species, whose occurrence have been reported predominantly in several types of foods of low moisture content, while aflatoxin biotransformation products have been reported mainly in milk and milk products. This review deals with the occurrence of aflatoxins in some of the major food products in the last 5 years including regulatory aspects, and recent advances in detoxification strategies for contaminated foods. Aflatoxin contamination in cereals including corn and peanut is still a public health problem for some populations, especially in African countries. Despite that most of physical and chemical methods for aflatoxin detoxification may affect the nutritional properties of food, or are not safe for human consumption, gamma-radiation and ozone applications have demonstrated great potential for detoxification of aflatoxins in some food matrices. Biological methods based on removal or degradation of aflatoxins by bacterial and yeast have good perspectives, although further studies are needed to clarify the detoxification mechanisms by microorganisms and determine practical aspects of the use of these methods in food products, especially their potential effects on sensory characteristics of foods.

Author Correction: Induction of micronuclei by four cytostatic compounds in human hematopoietic stem cells and human lymphoblastoid TK6 cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Induction of micronuclei by four cytostatic compounds in human hematopoietic stem cells and human lymphoblastoid TK6 cells.

For mutagenicity testing, primary lymphocytes or mammalian cell lines are employed. However, the true target for carcinogenic action of mutagenic chemicals may be stem cells. Since hematopoietic cancers induced by chemical agents originate at the hematopoietic stem cell (HSC) stage and since one of the side effects of chemotherapeutic cancer treatment is the induction of secondary tumors, often leukemias, HSC may be a suitable cell system. We compared the sensitivity of HSC with the genotoxicity testing cell line TK6 for chromosomal mutations. HSC were less sensitive than TK6 cells for the genotoxic effects of the model genotoxins and chemotherapeutic agents doxorubicin, vinblastine, methyl methanesulfonate (MMS) and equally sensitive for mitomycin C (MMC). However, loss of viability after mitomycin C treatment was higher in HSC than in TK6 cells. Among the factors that may influence sensitivity for genomic damage, the generation or response to reactive oxygen species (ROS) and the effectiveness of DNA damage response can be discussed. Here we show that HSC can be used in a standard micronucleus test protocol for chromosomal mutations and that their sensitivity was not higher than that of a classical testing cell line.

Fate of micronuclei and micronucleated cells.

The present review describes available evidence about the fate of micronuclei and micronucleated cells. Micronuclei are small, extranuclear chromatin bodies surrounded by a nuclear envelope. The mechanisms underlying the formation of micronuclei are well understood but not much is known about the potential fate of micronuclei and micronucleated cells. Many studies with different experimental approaches addressed the various aspects of the post-mitotic fate of micronuclei and micronucleated cells. These studies are reviewed here considering four basic possibilities for potential fates of micronuclei: degradation of the micronucleus or the micronucleated cell, reincorporation into the main nucleus, extrusion from the cell, and persistence in the cytoplasm. Two additional fates need to be considered: premature chromosome condensation/chromothripsis and the elimination of micronucleated cells by apoptosis, yielding six potential fates for micronuclei and/or micronucleated cells. The available data is still limited, but it can be concluded that degradation and extrusion of micronuclei might occur in rare cases under specific conditions, reincorporation during the next mitosis occurs more frequently, and the majority of the micronuclei persist without alteration at least until the next mitosis, possibly much longer. Overall, the consequences of micronucleus formation on the cellular level are still far from clear, but they should be investigated further because micronucleus formation may contribute to the initial and later steps of malignant cell transformation, by causing gain or loss of genetic material in the daughter cells and by the possibility of massive chromosome rearrangement in chromosomes entrapped within a micronucleus by the mechanisms of chromothripsis and chromoanagenesis.

IR and IGF-1R expression affects insulin induced proliferation and DNA damage.

Diabetes mellitus type 2 is in its prediagnostic and early phase characterized by hyperinsulinemia. Previously, we pointed out hyperinsulinemia as a potential link between diabetes mellitus and the increased cancer risk that is associated with this disease through its induction of oxidative stress and DNA damage. In the present study, we address the relationship between the induction of proliferation and genomic damage in vitro in cell lines with different expression of the insulin and the IGF-1 receptors after treating the cells with insulin and the insulin analog glargine. Contribution of the IGF-1 receptor was further examined by application of the IGF-1R inhibitor ((5R,5aS,8aR,9R)-9-hydroxy-5,8,8a,9-tetrahydro-5-(3,4,5-trimethoxyphenyl)-furo[3_,4_:6,7]-naphtho[2,3-d]-1,3-dioxol-6(5aH)-one) (PPP). Insulin as well as insulin glargine stimulated cell proliferation in IGF-receptor-dominated MCF-7 cells and not in insulin receptor-dominated BT-474 cells and PPP attenuated this effect. Both insulins induced DNA damage which was reduced by PPP in MCF-7 cells only. Overall, we showed in this study that high levels of insulin and insulin glargine can enhance cell proliferation in cells which highly express IGF-1 receptor and induce DNA damage in cells with high and also in those with low IGF-1 receptor levels.

Effect of Radiofrequency Radiation on Human Hematopoietic Stem Cells.

Exposure to electromagnetic fields in the radiofrequency range is ubiquitous, mainly due to the worldwide use of mobile communication devices. With improving technologies and affordability, the number of cell phone subscriptions continues to increase. Therefore, the potential effect on biological systems at low-intensity radiation levels is of great interest. While a number of studies have been performed to investigate this issue, there has been no consensus reached based on the results. The goal of this study was to elucidate the extent to which cells of the hematopoietic system, particularly human hematopoietic stem cells (HSC), were affected by mobile phone radiation. We irradiated HSC and HL-60 cells at frequencies used in the major technologies, GSM (900 MHz), UMTS (1,950 MHz) and LTE (2,535 MHz) for a short period (4 h) and a long period (20 h/66 h), and with five different intensities ranging from 0 to 4 W/kg specific absorption rate (SAR). Studied end points included apoptosis, oxidative stress, cell cycle, DNA damage and DNA repair. In all but one of these end points, we detected no clear effect of mobile phone radiation; the only alteration was found when quantifying DNA damage. Exposure of HSC to the GSM modulation for 4 h caused a small but statistically significant decrease in DNA damage compared to sham exposure. To our knowledge, this is the first published study in which putative effects (e.g., genotoxicity or influence on apoptosis rate) of radiofrequency radiation were investigated in HSC. Radiofrequency electromagnetic fields did not affect cells of the hematopoietic system, in particular HSC, under the given experimental conditions.

Antigenotoxic effects of resveratrol: assessment of in vitro and in vivo response.

Experiments were performed to evaluate the in vitro and in vivo dose response for antigenotoxic effects of resveratrol (RES). For the in vitro study, HL-60 cells were co-treated with the test genotoxin and three concentrations of RES. Thereafter, genotoxic effects were assessed in the cytokinesis-block micronucleus test. Results of the in vitro experiments using genotoxins nitroquinoline-1-oxide (NQO) and mitomycin C (MMC) showed maximum inhibition of genotoxicity with the lowest test concentration of RES. The mouse bone marrow micronucleus assay was used for evaluating the in vivo antigenotoxic effects of RES against genotoxins diepoxybutane (DEB), MMC, methyl methanesulfonate and procarbazine (PCB). The experimental animals received RES pre-treatment by gavage 30min, 24 and 48h before injecting the genotoxin intraperitoneally. The in vivo studies demonstrated efficacy of the lowest test dose of RES for exerting maximum protection against chromosomal damage induced by all four genotoxins. The antigenotoxic effect observed with 6.25mg/kg RES was significantly higher than that of 100mg/kg RES against PCB and DEB. In conclusion, the findings from the present study indicate that lower test concentrations/doses of RES are more effective in exerting antigenotoxic effects.

Signaling steps in the induction of genomic damage by insulin in colon and kidney cells.

Diabetes mellitus (DM), a disease with almost 350 million people affected worldwide, will be the seventh leading cause of death by 2030. Diabetic patients develop various types of complications, among them an increased rate of malignancies. Studies reported the strong correlation between DM and several cancer types, of which colon and kidney cancers are the most common. Hyperinsulinemia, the high insulin blood level characteristic of early diabetes type 2, was identified as a risk factor for cancer development. In previous studies, we showed that an elevated insulin level can induce oxidative stress, resulting in DNA damage in colon cells in vitro and in kidney cells in vitro and in vivo. In the present study, we elucidate the signaling pathway of insulin-mediated genotoxicity, which is effective through oxidative stress induction in colon and kidney. The signaling mechanism is starting by phosphorylation of the insulin and insulin-like growth factor-1 receptors, followed by activation of phosphatidylinositide 3-kinase (PI3K), which in turn activates AKT. Subsequently, mitochondria and nicotinamide adenine dinucleotide phosphate oxidase (NADPH) isoforms (Nox1 and Nox4 in colon and kidney, respectively) are activated for reactive oxygen species (ROS) production, and the resulting excess ROS can attack the DNA, causing DNA oxidation. We conclude that hyperinsulinemia represents an important risk factor for cancer initiation or progression as well as a target for cancer prevention in diabetic patients.

Terahertz radiation at 0.380 THz and 2.520 THz does not lead to DNA damage in skin cells in vitro.

The question whether nonionizing electromagnetic radiation of low intensity can cause functional effects in biological systems has been a subject of debate for a long time. Whereas the majority of the studies have not demonstrated these effects, some aspects still remain unclear, e.g., whether high-frequency radiation in the terahertz range affects biological systems. In particular for frequencies higher than 0.150 THz, investigations of the ability of radiation to cause genomic damage have not been performed. In the present study, human skin cells were exposed in vitro to terahertz radiation at two specific frequencies: 0.380 and 2.520 THz. Power intensities ranged from 0.03-0.9 mW/cm(2) and the cells were exposed for 2 and 8 h. Our goal was to investigate whether the irradiation induced genomic damage in the cells. Chromosomal damage was not detected in the different cell types after exposure to radiation of both frequencies. In addition, cell proliferation was quantified and found to be unaffected by the exposure, and there was no increase in DNA damage measured in the comet assay for both frequencies. For all end points, cells treated with chemicals were included as positive controls. These positive control cells clearly showed decreased proliferation and increased genomic damage. The results of the present study are in agreement with findings from other studies investigating DNA damage as a consequence of exposure to the lower frequency range (<0.150 THz) and demonstrate for the first time that at higher frequencies (0.380 and 2.520 THz), nonionizing radiation does not induce genomic damage.

Antiproliferative effects of fluoxetine on colon cancer cells and in a colonic carcinogen mouse model.

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

Terahertz electromagnetic fields (0.106 THz) do not induce manifest genomic damage in vitro.

Terahertz electromagnetic fields are non-ionizing electromagnetic fields in the frequency range from 0.1 to 10 THz. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along human exposure to these fields. Up to now, only a limited number of investigations on biological effects of terahertz electromagnetic fields have been performed. Therefore, research is strongly needed to enable reliable risk assessment.Cells were exposed for 2 h, 8 h, and 24 h with different power intensities ranging from 0.04 mW/cm(2) to 2 mW/cm(2), representing levels below, at, and above current safety limits. Genomic damage on the chromosomal level was measured as micronucleus formation. DNA strand breaks and alkali-labile sites were quantified with the comet assay. No DNA strand breaks or alkali-labile sites were observed as a consequence of exposure to terahertz electromagnetic fields in the comet assay. The fields did not cause chromosomal damage in the form of micronucleus induction.

900 MHz radiation does not induce micronucleus formation in different cell types.

The exposure of the population to non-ionising electromagnetic radiation is still increasing, mainly due to mobile communication. Whether low-intensity electromagnetic fields can cause other effects apart from heating has been a subject of debate. One of the effects, which were proposed to be caused by mobile phone radiation, is the occurrence of mitotic disturbances. The aim of this study was to investigate possible consequences of these mitotic disturbances as manifest genomic damage, i.e. micronucleus induction. Cells were irradiated at a frequency of 900 MHz, which is located in one of the main frequency bands applied for mobile communication. Two cell types were used, HaCaT cells as human cells and A(L) cells (human-hamster hybrid cells), in which mitotic disturbances had been reported to occur. After different post-exposure incubation periods, cells were fixed and micronucleus frequencies were evaluated. Both cell types did not show any genomic damage after exposure. To adapt the protocol for the micronucleus test into the direction of the protocol for mitotic disturbances, the post-exposure incubation period was reduced and exposure time was extended to one cell cycle length. This did not result in any increase of the genomic damage. In conclusion, micronucleus induction was not observed as a consequence of exposure to non-ionising radiation, even though this agent was reported to cause mitotic disturbances under similar experimental conditions.