FLASH Radiotherapy: Expectations, Challenges, and Current Knowledge.

Major strides have been made in the development of FLASH radiotherapy (FLASH RT) in the last ten years, but there are still many obstacles to overcome for transfer to the clinic to become a reality. Although preclinical and first-in-human clinical evidence suggests that ultra-high dose rates (UHDRs) induce a sparing effect in normal tissue without modifying the therapeutic effect on the tumor, successful clinical translation of FLASH-RT depends on a better understanding of the biological mechanisms underpinning the sparing effect. Suitable in vitro studies are required to fully understand the radiobiological mechanisms associated with UHDRs. From a technical point of view, it is also crucial to develop optimal technologies in terms of beam irradiation parameters for producing FLASH conditions. This review provides an overview of the research progress of FLASH RT and discusses the potential challenges to be faced before its clinical application. We critically summarize the preclinical evidence and in vitro studies on DNA damage following UHDR irradiation. We also highlight the ongoing developments of technologies for delivering FLASH-compliant beams, with a focus on laser-driven plasma accelerators suitable for performing basic radiobiological research on the UHDR effects.

Artemisia vulgaris L., Artemisia alba Turra and their constituents reduce mitomycin C-induced genomic instability in human peripheral blood lymphocytes in vitro.

This study aimed to evaluate the effect of aqueous and acetone extracts from Artemisia vulgaris L. (AV) and Artemisia alba Turra (AA), and two major polyphenols compounds (3,5-dihydroxybenzoic acid and quercetin-3-O-glucopyranoside) presented in both extracts of the plants against mitomycin C (MMC)-induced genomic instability. Genomic instability was measured using cytokinesis block micronucleus (MN) assay in human peripheral blood lymphocytes (PBLs) in vitro by analyzing two biomarkers - MN and nuclear division index (NDI). Extracts were tested in a concentration-dependent manner (10-250 µg/mL), while 3,5-dihydroxybenzoic acid and quercetin-3-O-glucopyranoside were tested in three different concentrations, in combination with 0.5 µg/mL of MMC. Aqueous and acetone extracts obtained from both plants significantly reduced MMC-induced MN frequency in PBLs, compared to positive control cells (p < 0.05). Extracts from AV did not affect NDI, whereas the concentrations of 10-100 µg/mL of aqueous and acetone AA extracts significantly elevated MMC-decreased NDI values in comparison to positive control cells (p < 0.05). Combined treatment of 3,5-dihydroxybenzoic acid and MMC showed a significant reduction of MMC-induced MN frequency, while quercetin-3-O-glucopyranoside increased MN frequency compared to positive control cells (p < 0.05). Both compounds decreased NDI values but only at the highest tested concentration of quercetin-3-O-glucopyranoside it was of greater significance. In conclusion, all extracts from AV and AA and 3,5-dihydroxybenzoic acid showed protective effect, whereby aqueous AA demonstrated the highest protective effect on MMC- induced genomic instability, while quercetin-3-O-glucopyranoside showed co-mutagen effect.

Comparative study of the genotoxic activity of Artemisia vulgaris L. and Artemisia alba Turra extracts in vitro.

In this study, the genotoxic activity of acetone and aqueous extracts of two species of genus Artemisia (Artemisia vulgaris L. and Artemisia alba Turra), and possible role of their polyphenolic composition in the observed activities were investigated. Polyphenolic contents were evaluated by high-performance liquid chromatography (HPLC-PDA), while the genotoxic activity was tested using cytokinesis block micronucleus (CBMN) assay on human peripheral blood lymphocytes (PBLs) in vitro. HPLC-PDA showed that both A. alba extracts were richer in polyphenolic contents than A. vulgaris extracts. The acetone A. alba extract was the richest of polyphenolic content where we detected six phenolic acids and two flavonoids. CBMN assay showed that aqueous extract of A. vulgaris significantly increased micronucleus (MN) frequency in the PBLs treated with all tested concentrations (10, 50, 100, and 250 µg/mL), while A. alba did not significantly affect the mean MN frequency. Further, both acetone extracts were genotoxic in all tested concentrations, except the lowest tested (10 µg/mL) of A. alba. All tested extracts affected the nuclear division index (NDI) except the aqueous A. alba extract (p < 0.05). Based on our results, we can conclude that both acetone and aqueous A. vulgaris extracts and A. alba acetone extract were genotoxic in PBLs in vitro. A. alba aqueous extract was not genotoxic and cytotoxic in tested concentrations. We suggest that the aqueous extract of A. alba can be used in treatment, which has been confirmed by traditional medicine, but with a high dose of caution and not in high concentrations.

Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach.

Translocation of mtDNA in the nuclear genome is an ongoing process that contributes to the development of pathological conditions in humans. However, the causal factors of this biological phenomenon in human cells are poorly studied. Here we analyzed mtDNA insertions in the nuclear genome of human lymphocytes after in vitro treatment with doxorubicin (DOX) using a fluorescence in situ hybridization (FISH) technique. The number of mtDNA insertions positively correlated with the number of DOX-induced micronuclei, suggesting that DOX-induced chromosome breaks contribute to insertion events. Analysis of the odds ratios (OR) revealed that DOX at concentrations of 0.025 and 0.035 µg/mL significantly increases the rate of mtDNA insertions (OR: 3.53 (95% CI: 1.42-8.76, p < 0.05) and 3.02 (95% CI: 1.19-7.62, p < 0.05), respectively). Analysis of the distribution of mtDNA insertions in the genome revealed that DOX-induced mtDNA insertions are more frequent in larger chromosomes, which are more prone to the damaging action of DOX. Overall, our data suggest that DOX-induced chromosome damage can be a causal factor for insertions of mtDNA in the nuclear genome of human lymphocytes. It can be assumed that the impact of a large number of external and internal mutagenic factors contributes significantly to the origin and amount of mtDNA in nuclear genomes.

In vitro genotoxicity assessment of an oxidized dextrin-based hydrogel for biomedical applications.

Hydrogels are three-dimensional, crosslinked networks of hydrophilic polymers swollen with a large amount of water or biological fluids, without dissolving. Dextrin, a low-molecular-weight carbohydrate composed by glucose residues, has been used to develop an injectable hydrogel for biomedical applications. Dextrin was first oxidized to introduce aldehyde groups, which then reticulate with adipic acid dihydrazide, forming the dextrin-based hydrogel (HG). The HG and its components were tested for cyto- and genotoxicity according to the International Standard ISO 10993-3 on the biological evaluation of medical devices. To assess genotoxicity, a battery of in vitro genotoxicity tests employing both eukaryotic and prokaryotic models was performed: comet assay, cytokinesis-block micronucleus assay and Ames test. Our data revealed that the HG (IC50  = 2.8 mg/mL) and oxidized dextrin by itself (IC50  = 1.2 mg/mL) caused a concentration-dependent decrease in cellular viability of human lymphoblastoid TK6 cells after 24 hours of exposure to the test agents. However, these concentrations are unlikely to be reached in vivo. In addition, no significant increase in the DNA and chromosomal damage of TK6 cells exposed to non-cytotoxic concentrations of the HG and its isolated components was detected. Furthermore, neither the HG nor its metabolites exerted a mutagenic effect in different of Salmonella typhimurium strains and in an Escherichia coli mix. Our data demonstrated the genocompatibility of the HG (up to 3.5 mg/mL) for biomedical applications. To our best acknowledge, this is the first report with a detailed genotoxicity assessment of an aldehyde-modified polysaccharide/adipic acid dihydrazide hydrogel.

Multi-parameter dose estimations in radiation biodosimetry using the automated cytokinesis-block micronucleus assay with imaging flow cytometry.

The cytokinesis-block micronucleus (CBMN) assay is an established technique in radiation biological dosimetry for estimating the dose to an individual by measuring the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using slide-scoring algorithms, but an automated multiparameter method without the need of the slide-making procedure would be advantageous to further increase throughput for application in mass casualty events. The development of the ImageStreamX (ISX) imaging flow cytometer has made it possible to adapt the CBMN assay to an automated imaging flow cytometry (FCM) method. The protocol and analysis presented in this work tailor and expand the assay to a multiparameter biodosimetry tool. Ex vivo irradiated whole blood samples were cultured, processed, and analyzed on the ISX and BNCs, MN, and mononuclear cells were imaged, identified, and enumerated automatically and simultaneously. Details on development of the method, gating strategy, and dose response curves generated for the rate of MN per BNC, percentage of mononuclear cells as well as the replication index are presented. Results indicate that adapting the CBMN assay for use in imaging FCM has produced a rapid, robust, multiparameter analysis method with higher throughput than is currently available with standard microscopy. We conclude that the ISX-CBMN method may be an advantageous tool following a radiological event where triage biodosimetry must be performed on a large number of casualties.

The cytokinesis-blocked micronucleus assay: dose estimation and inter-individual differences in the response to γ-radiation.

Biological dosimetry plays an important role in case of a radiation accident or incident, either when it is the only way to estimate the dose or when it is used to complement physical dosimetry. A cytogenetic study was conducted in a group of 16 Portuguese individuals by use of the cytokinesis-blocked micronucleus (CBMN) assay. A dose-response curve for micronuclei yield was established with a linear-quadratic model: Y=(0.0122±0.0010)+(0.0241±0.0023)D+(0.0193±0.0007)D(2). Also, baseline values for the micronucleus formation in the 16 donors were analyzed, with results in close agreement with those from other laboratories. A validation experiment was carried out with three individuals. The real and the estimated doses obtained with the dose-response curve were in very good agreement, allowing the use of the micronucleus dose-response calibration curve in biological dosimetry for estimation of radiation dose in case of overexposure. The results obtained for the cytogenetic endpoints, studied in the same group of 16 individuals, were also analyzed as a function of age and gender. A higher inter-variability was observed for the higher dose points and differences in response were identified between genders, above 2Gy, for all endpoints.

Diarylheptanoids from green alder bark and their potential for DNA protection.

Nine diarylheptanoids, 1-9, catechin (11), and a phenolic glucoside, 10, were isolated from the bark of green alder (Alnus viridis). Four of the isolated compounds, i.e., 2, 5, 8, 10, are new. The structures of 1-11 were determined on the basis of spectroscopic data. All isolated compounds were evaluated for their in vitro protective effects on chromosome aberrations in peripheral human lymphocytes using cytokinesis-block micronucleus (CBMN) assay. Almost all of them exerted a pronounced effect of decreasing DNA damage of human lymphocytes, acting stronger than the known synthetic protector amifostine.

Assessment of Biological Damage Induced during Multidetector Computed Tomography (MDCT) Examination.

Computed tomography (CT) is known for its non-invasiveness, fast procedure, and also for providing detailed diagnostic information to physicians. It also utilises low-dose-rate ionising radiation (X-rays) as a source for imaging. Multidetector computed tomography (MDCT) is an advanced system that uses iodinated contrast media for more accurate diagnostic results. Studies suggest using these contrasts will lead to greater radiation adsorption with significant DNA damage. No studies have been taken comparing the physical dose with the biological effect. The present study sheds light on the same by assessing the biological effect of CT with and without contrast intervention. The present study is timebound; thus, 21 participants attending for CT thorax and abdomen with no history of any cancer were included. The same participants underwent both pre-contrast and post-contrast studies. The blood sample was taken before the procedure and used as a control. Physical parameters like DLP and CTDI obtained from the instrument were compared with the MN frequency obtained (CBMN Assay). The study showed a significant increase (p-value < 0.05) in the Physical and MN frequency in the Post-Contrast group compared to the pre-contrast group. Although a positive correlation was observed between pre and post-contrast groups, the results were not found to be statistically significant (p-value < 0.05). The study confirms increased physical dose and MN frequency upon contrast intervention. This study recommends the judicial use of MDCT in disease diagnostics.

Phytochemical Profile and Evaluation of the Antioxidant, Cyto-Genotoxic, and Antigenotoxic Potential of Salvia verticillata Hydromethanolic Extract.

This study comprises the phytochemical characterization, the evaluation of the total phenolic content (TPC) and antioxidant activity (AA), and the investigation of the cyto-genotoxic and antigenotoxic potential of hydromethanolic extract derived from Salvia verticillata L. leaves. HPLC-DAD-ESI-MS and HPLC-DAD were used for the characterization of the extract and determination of the major ingredients. Afterwards, the TPC and AA were determined. The cytotoxic and genotoxic effect of the extract on cultured human lymphocytes at concentrations of 10, 25, and 50 µg mL-1 was investigated via the Cytokinesis Block MicroNucleus (CBMN) assay. Moreover, its antigenotoxic potential against the mutagenic agent mitomycin C (MMC) was assessed using the same assay. The hydromethanolic extract comprises numerous metabolites, with rosmarinic acid being the major compound. It had a high value of TPC and exerted significant AA as shown by the results of the Ferric Reducing Antioxidant Power (FRAP) and Radical Scavenging Activity by DPPH• assays. A dose-dependent cytotoxic potential was recorded, with the highest dose (50 µg mL-1) exhibiting statistically significant cytotoxicity. None of the tested concentrations induced significant micronuclei (MN) frequencies, indicating a lack of genotoxicity. All tested concentrations reduced the MMC-mediated genotoxic effects, with the two lowest showing statistically significant antigenotoxic potential.

Molecular studies on coronary artery disease-a review.

Coronary artery disease (CAD) remains the major cause of mortality and morbidity in the entire world population. The conventional risk factors of CAD include hypertension, hyperlipidemia, diabetes mellitus, family history, smoking etc. These factors contribute only 50 % of the total risk of CAD. For providing a complete risk assessment in CAD, it is mandatory to have well-planned clinical, biochemical and genetic studies in patients with CAD and subjects who are at risk of developing CAD. In this review an attempt is made to critically evaluate the conventional and emerging risk factors which predispose the individual to CAD. Specifically, the molecular basis of CAD including high oxidative stress, low antioxidant status and increased DNA damage are covered. A comprehensive and multifactorial approach to the problem is the better way to reduce the morbidity and mortality of the disease.

Cytokinesis Blocked Micronuclei Aberration Analysis.

Cytokinesis blocked micronuclei (CBMN) assay is a rapid and sensitive analysis of chromosome aberrations and miss assortments during cell division. Genotoxic agent exposure produces DNA damage and chromosome fragments. Fragmented chromosomes without centromere failed to attach kinetochore which segregates a pair of homologous chromosomes to each daughter cells at cytokinesis, hence leading to form micronuclei. Chromosome or fragments of chromosome can also form micronuclei when they are not accurately sorted to daughter cells. Using cytochalasin B, an actin inhibitor, blocks cytokinesis of which completion leads serration appearance formed with two daughter cells while nuclei segregation is undergoing. As a result, one cell having two daughter nuclei, i.e., binucleated cell, is produced. By analyzing these binucleated cells, chromosome aberrations can be estimated as well as popular chromosome aberration analysis. Frequency of micronuclei formation predicts the testing agents' genotoxicity. By combining use with centromere-specific probes or DNA damage signal probes, the nature of genotoxicity of tested agents can be estimated.

In Vitro Hemocompatibility and Genotoxicity Evaluation of Dual-Labeled [99mTc]Tc-FITC-Silk Fibroin Nanoparticles for Biomedical Applications.

Nuclear imaging is a highly sensitive and noninvasive imaging technique that has become essential for medical diagnosis. The use of radiolabeled nanomaterials capable of acting as imaging probes has shown rapid development in recent years as a powerful, highly sensitive, and noninvasive tool. In addition, quantitative single-photon emission computed tomography (SPECT) images performed by incorporating radioisotopes into nanoparticles (NPs) might improve the evaluation and the validation of potential clinical treatments. In this work, we present a direct method for [99mTc]Tc-radiolabeling of FITC-tagged silk fibroin nanoparticles (SFN). NPs were characterized by means of dynamic light scattering and scanning electron microscopy. In vitro studies were carried out, including the evaluation of stability in biological media and the evaluation of hemocompatibility and genotoxicity using the cytokinesis block micronucleus (CBMN) assay. The radiolabeling method was reproducible and robust with high radiolabeling efficiency (∼95%) and high stability in biological media. Hydrodynamic properties of the radiolabeled NPs remain stable after dual labeling. The interaction of SFN with blood elicits a mild host response, as expected. Furthermore, CBMN assay did not show genotoxicity induced by [99mTc]Tc-FITC-SFN under the described conditions. In conclusion, a feasible and robust dual-labeling method has been developed whose applicability has been demonstrated in vitro, showing its value for further investigations of silk fibroin NPs biodistribution in vivo.

Beneficial properties of Drimia numidica leaf methanolic extract against the cytogenotoxic effects of mitomycin C on human lymphocytes.

This study investigated the phytochemical profile of Drimia numidica leaf methanolic extract, as well as its cyto-genotoxic and cyto/genoprotective potential against mitomycin C (MMC) mediated effects on healthy human lymphocytes. Photosynthetic pigments, trace elements, and secondary metabolites were estimated and/or identified in methanolic extract of mature leaves, and the latter was further used for assessing its in vitro biological effects on MMC-free and/or MMC-treated human lymphocytes (at low, non-toxic concentrations of 0.001 and 0.01% v/v). The results showed that D. numidica leaf methanolic extract, being rich in carotenoids, phenolics, flavonoids, organic acids and bufadienolides, could be protective against MMC mediated cyto/genotoxic potential in healthy human lymphocytes. Biomolecules possessing antioxidant and antitumor potential, such as beta-carotene and lutein among others, chlorogenic acid, caffeic acid and their derivatives, minerals such as Si, as well as apigenin- and luteolin-derived glycosides, either individual or in a mixture, could be beneficial rather than harmful, at least at the extract concentrations tested. Although further in vitro and in vivo studies are still needed for elucidating the beneficial (individual and/or additive/synergistic) role of those compounds, the results of the present study are quite promising, thus encouraging new challenges for the appropriate utilization of D. numidica leaf extract.

Phytochemical Analysis and Genotoxicological Evaluation of Prickly Pear Peel Extracts.

This study investigated the beneficial properties of prickly pear peel (PPP) extracts from Opuntia ficus-indica (L.) Mill. Extracts were obtained via the Soxhlet extraction method using methanol (P1), ethanol (P2) and ethanol-water (P3) as extraction solvents. Their total phenolic and flavonoid content (TPC and TFC, respectively) and their antioxidant activity (AA) were determined. The PPP extracts were characterized in detail using mass spectrometry techniques. Their cyto-genotoxic effect and antigenotoxic potential against mitomycin C were evaluated via the cytokinesis block micronucleus (CBMN) assay on human lymphocytes. Enhanced TPC, TFC and AA values were recorded for all the extracts. Moreover, P1 and P2 were cytotoxic only at the highest concentrations, whereas P3 was found to be cytotoxic in all cases. No significant micronucleus induction was observed in the tested extracts. The PPP extracts contain bioactive compounds such as flavonoids, carboxylic acids, alkaloids, fatty acids and minerals (mainly K, Si, Mg, Ca, P and Zn). The results showed that all three extracts exerted high antigenotoxic activity. Our findings confirm the beneficial and genoprotective properties of PPP extracts and further studies on the bioactive compounds of Opuntia ficus-indica (L.) Mill. are recommended, as it constitutes a promising plant in pharmaceutical applications.

Investigation of the Genotoxic, Antigenotoxic and Antioxidant Profile of Different Extracts from Equisetum arvense L.

The present study investigated the cyto-genotoxic and antigenotoxic effects of four different extracts of Equisetum arvense L. (common name: field horsetail) on human lymphocytes. Specifically, Soxhlet's prepared extracts from E. arvense L., using different solvents (S1: methanol (MeOH)-, S2: ethanol (EtOH)-, S3: water-, and S4: ethanol/water (EtOH-W)-) were analyzed for (a) their total phenolic and flavonoid content (TPC and TFC, respectively), (b) their antioxidant activity (AA), via the DPPH, FRAP and ABTS assays, and (c) their cyto-genotoxic and/or protective efficiency against the mutagenic agent mitomycin C, via the Cytokinesis Block MicroNucleus assay. All extracts showed increased TPC, TFC, and AA values in almost all cases. S1, S3 and S4 demonstrated no cytotoxic potential, whereas S2 was cytotoxic only at the highest concentrations. Genotoxicity was not observed in the tested extracts. The highest antigenotoxic activity was observed for EtOH-W (S4) extract, which was found to be rich in flavonoids, flavonoid-O-glycosides, phytosterols, phenolic and fatty acids as well as in minerals and mainly in K, Ca, Mg, Si and P, as assessed by using various mass spectrometry techniques. Those findings confirm that E. arvense L. extracts could be valuable candidates for medicinal applications and pharmaceutical products, thus alleviating the effects of more conventional drugs.

Lymphocyte-based challenge DNA-repair assays for personalized health risk assessment.

Combinations of genetic and environmental factors are responsible for the development of many human diseases, such as cancer, as demonstrated using various biomarkers. Within this scenario, DNA repair holds a gate-keeper position which determines outcomes after appearance of DNA damage and, therefore, adverse cellular consequences, e.g., initiation of carcinogenesis. DNA repair deficiency and some of the subsequent events can be validated from studies using live cells from cancer patients. However, these deficiencies/events are difficult to demonstrate in live cells from normal individuals because individual variations in DNA repair capacities (DRC) are too low to be measured easily. Such lack of information has been hindering progress in developing personalized disease prevention and intervention protocols, especially among exposed populations. However, using a variety of challenge assays as biomarkers, variations in individual's DRC can be amplified in live cells and be determined. Furthermore, evidence indicates that DRC are not only inherited but can also be modified by environmental factors (e.g., nutritional status and exposure to genotoxic substances). Using these challenge assays, e.g., in live lymphocytes, individual's DRC can be holistically and functionally determined as well as quantitated. With the more precise information, assessment of health risk can be better determined on an individual rather than on a population basis. This review provides a succinct summary on the development and application of recent challenge assays in lymphocytes which can provide measurements of individuals' DRC, and on the latest data for more precise disease prevention and intervention.

Potential radioprotective properties of arbutin against ionising radiation on human leukocytes in vitro.

Arbutin is a simple phenolic glucoside biosynthesised in many plant families. Some of the everyday foods that contain arbutin are species of the genus Origanum, peaches, cereal products, coffee and tea and Arctostaphyllos uva ursi L. leaves. Arbutin possesses various beneficial effects in the organism, and was confirmed effective in the treatment of urinary tract infections as well as in preventing skin hyperpigmentation. It shows antioxidant and anti-inflammatory properties, and antitumor activity. The aim of this study was to explore potential radioprotective properties of arbutin in concentrations of 11.4 µg/mL, 57 µg/mL, 200 µg/mL and 400 µg/mL administered as a pre-treatment for one hour before exposing human leukocytes to ionising radiation at a therapeutic dose of 2 Gy. The alkaline comet assay was used to establish the levels of primary DNA damage, and cytokinesis-block micronucleus (CBMN) cytome assay to determine the level of cytogenetic damage. None of the tested concentrations of single arbutin showed genotoxic and cytotoxic effects. Even at the lowest tested concentration, 11.4 µg/mL, arbutin demonstrated remarkable potential for radioprotection in vitro, observed both at the level of primary DNA damage, and using CBMN cytome assay. The best dose reduction compared with amifostine was observed after pre-treatment with the highest concentration of arbutin, corresponding to 400 µg/mL. Promising results obtained on the leukocyte model speak in favour of extending similar experiments on other cell and animal models.

Biopharmaceutical-Type Chinese Hamster Ovary Cell Cultivation Under Static Magnetic Field Exposure: A Study of Genotoxic Effect.

The lack of a sufficient research base is the reason for the ongoing discussion regarding the genotoxic effect of magnetic field (MF) exposure on mammalian cell cultures. Chinese hamster ovary (CHO) suspension-type cells, which are widely used for biopharmaceutical production, are potentially subjected to an increased MF when cultivated in bioreactors equipped with bottom-placed magnetically coupled stirring mechanisms. The main challenge for conducting research in this field remains the availability of a suitable experimental setup that generates an appropriate MF for the raised research question. In the present study, a simple and cost-effective experimental setup was developed that generated a static MF, similar to what has been modeled in large-scale bioreactors and, at the same time, was suitable for experimental cell cultivation in laboratory conditions. The measured maximum magnetic flux density to which the cells were exposed was 0.66 T. To assess the possible genotoxic effect, cells were continuously subcultivated in laboratory petri dishes for a period of 14 days, corresponding to a typical duration of a biopharmaceutical production process in a conventional fed-batch regime. The genotoxic effect was assessed using the cytokinesis-block micronucleus assay with fluorescent staining. Results showed that a 0.66-T static MF exposure had no significant long-term effect on cell viability and chromosomal damage but demonstrated a short-term effect on cell apoptosis. Significant increase in nuclear bud formation was observed. These findings may encourage other researchers in future studies investigating cellular responses to MF exposure and contribute relevant data for comparison.

Assessing the human risk and the environmental fate of pharmaceutical Tramadol.

Tramadol (TRA) is a widely used human pharmaceutical and a well-established emerging pollutant and its potential genotoxic and cytotoxic effects on humans as well as its fate in aqueous systems demand full investigation. The present study is a multidisciplinary approach and provides important insights on the potential risks of Tramadol on humans accompanied by its photolytic transformation under simulated solar irradiation. The present study revealed that Tramadol can induce genotoxic and cytotoxic effects under the specific experimental conditions, significantly depended on the tested concentration. In addition, the photolytic transformation of Tramadol was investigated in detail under simulated solar irradiation in two different water matrices: ultrapure water (UW) and treated wastewater (WW). Differences in the degradation rates were observed between UW and WW, being slower in WW. The results showed that more than 70% of Tramadol was removed after 240 min in UW ([TRA] = 10 mg L-1, I = 500 W m-2). After this period, TOC removal was found to be about 40%. Transformation of N atoms into NO3- and NH4+ followed a similar trend reaching up to 38% release. Τramadol degraded mainly by HO radicals and 1O2 through a self-sensitizing process while direct photolysis was also significant. Hydroxylation, demethylation and N-oxidation of the parent compound were found to be the main degradation pathways confirming the important role of HO and 1O2 in the photolytic process. Toxicity measurements showed a noticeable increase of the inhibition for Vibrio fischeri at the first stages which coincide with the formation of the major TPs.