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Indoor air pollution is becoming a rising public health problem and is largely resulting from the burning of solid fuels and heating in households. Burning these fuels produces harmful compounds, such as particulate matter regarded as a major health risk, particularly affecting the onset and exacerbation of respiratory diseases. As exposure to polluted indoor air can cause DNA damage including DNA sd breaks as well as chromosomal damage, in this paper, we aim to provide an overview of the impact of indoor air pollution on DNA damage and genome stability by reviewing the scientific papers that have used the comet, micronucleus, and γ-H2AX assays. These methods are valuable tools in human biomonitoring and for studying the mechanisms of action of various pollutants, and are readily used for the assessment of primary DNA damage and genome instability induced by air pollutants by measuring different aspects of DNA and chromosomal damage. Based on our search, in selected studies (in vitro, animal models, and human biomonitoring), we found generally higher levels of DNA strand breaks and chromosomal damage due to indoor air pollutants compared to matched control or unexposed groups. In summary, our systematic review reveals the importance of the comet, micronucleus, and γ-H2AX assays as sensitive tools for the evaluation of DNA and genome damaging potential of different indoor air pollutants. Additionally, research in this particular direction is warranted since little is still known about the level of indoor air pollution in households or public buildings and its impact on genetic material. Future studies should focus on research investigating the possible impact of indoor air pollutants in complex mixtures on the genome and relate pollutants to possible health outcomes., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at - 80 °C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Biomonitoring of human populations exposed to chemical substances that can act as potential mutagens or carcinogens, may enable the detection of damage and early disease prevention. In recent years, the comet assay has become an important tool for assessing DNA damage, both in environmental and occupational exposure contexts. To evidence the role of the comet assay in human biomonitoring, we have analysed original research studies of environmental or occupational exposure that used the comet assay in their assessments, following the PRISMA-ScR method (preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews). Groups of chemicals were designated according to a broad classification, and the results obtained from over 300 original studies (n = 123 on air pollutants, n = 14 on anaesthetics, n = 18 on antineoplastic drugs, n = 57 on heavy metals, n = 59 on pesticides, and n = 49 on solvents) showed overall higher values of DNA strand breaks in the exposed subjects in comparison with the unexposed. In summary, our systematic scoping review strengthens the relevance of the use of the comet assay in assessing DNA damage in human biomonitoring studies.

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide., Competing Interests: Declaration of Competing Interest On behalf of, and having obtained permission from all the authors, Goran Gajski declares that the material has not been published elsewhere, the paper is not currently being considered for publication elsewhere, all authors have been personally and actively involved in substantive work leading to the report and will hold themselves responsible for its content. Goran Gajski also declares that there are no potential conflicts of interest related neither to individual authors’ commitments, project support, or the commitments of editors, journal staff, or reviewers., (Copyright © 2024 Elsevier B.V. All rights reserved.)

More than eight million premature deaths annually can be attributed to air pollution, with 99% of the world's population residing in areas below recommended air quality standards. Hence, the present study aimed to examine the association between primary DNA damage and air pollution data among 123 participants enrolled between 2011 and 2015 in Zagreb, Croatia. While most measured air pollutants adhered to regulatory limits, benzo[a]pyrene concentrations bound to PM 10 exceeded them. Factorial analysis narrowed down air pollution data to four exposure factors (particulate matter, two metal factors, and other pollutants). Despite the absence of significant positive associations between modeled air pollution exposure factors and comet assay descriptors (tail length, tail intensity, tail moment, and highly damaged nuclei), the critical health implications of air pollution warrant further investigations, particularly with biomarkers of exposure and different biomarkers of effect in populations facing air pollution exposure.

DNA damage is known to be associated with many adverse health outcomes, including cancer and chronic diseases, but also with the process of aging. Empirical evidence has shown that environmental exposures, such as certain lifestyle factors, can affect a variety of health-related biomarkers and also impact the stability of DNA through the upregulation of the antioxidant defense system and alteration of its repair capacity. In addition to exercising, diet is an important lifestyle factor that can affect the development of a variety of chronic diseases and growing evidence suggests that plant-based diets, including vegetarianism, may promote health, longevity, and well-being. Therefore, we aimed to assess the primary DNA damage in 32 young healthy females from Zagreb, Croatia, based on their dietary preferences. The participants were divided into two groups: vegetarians and non-vegetarians, where the non-vegetarian group was further divided into omnivores (traditional mixed diet) and pescatarians (consumption of fish and seafood). According to statistical analysis, the DNA damage measured in whole blood cells expressed as the % tail DNA was significantly ( p < 0.05) higher in vegetarians (3.6 ± 1.1%) compared to non-vegetarians (2.8 ± 1.0%). When further dividing the participants into specific sub-groups, lower DNA damage was observed amongst omnivorous subjects (3.2 ± 0.8%) compared to vegetarians, with the lowest DNA damage found in females practicing a pescatarian diet (2.4 ± 1.1%). Although a vegetarian diet can lead to a higher intake of specific vitamins and micronutrients, it can also lead to a deficiency of iron, calcium, and total proteins, which may affect genome stability and induce oxidative stress. Even though our results have shown that the pescatarian diet would be more beneficial in terms of maintaining DNA integrity, further research should be carried out to assess how specific dietary preferences affect DNA integrity on a larger scale.

The aim of this study was to test the phytotoxicity and mode of action of bisphenol A (BPA) on Allium cepa using a multibiomarker approach. A. cepa roots were exposed to BPA in concentration range 0-50 mg L -1 for 3 days. BPA even in the lowest applied concentration (1 mg L -1 ) reduced root length, root fresh weight, and mitotic index. Additionally, the lowest BPA concentration (1 mg L -1 ) decreased the level of gibberellic acid (GA 3 ) in root cells. BPA at concentration 5 mg L -1 increased production of reactive oxygen species (ROS) that was followed by increase in oxidative damage to cells' lipids and proteins and activity of enzyme superoxide dismutase. BPA in higher concentrations (25 and 50 mg L -1 ) induced genome damage detected as an increase in micronucleus (MNs) and nuclear buds (NBUDs). BPA at >25 mg L -1 induced synthesis of phytochemicals. Results of this study using multibiomarker approach indicate that BPA is phytotoxic to A. cepa roots and has shown genotoxic potential to plants, thus its presence in the environment should be monitored.

The study aimed to investigate toxicity and the mechanism of toxicity of two Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZEA). DON and ZEA were applied to HepG2 cells as single compounds and in combination at low environmentally relevant concentrations. HepG2 cells were exposed to DON (0.5, 1, and 2 µM), ZEA (5, 10, and 20 µM) or their combinations (1 µM DON + 5 µM ZEA, 1 µM DON + 10 µM ZEA and 1 µM DON + 20 µM ZEA) for 24 h and cell viability, DNA damage, cell cycle and proliferation were assessed. Both mycotoxins reduced cell viability, however, combined treatment with DON and ZEA resulted in higher reduction of cell viability. DON (1 µM) induced primary DNA damage, while DON (1 µM) in combination with higher ZEA concentrations showed antagonistic effects compared to DON alone at 1 µM. DON arrested HepG2 cells in G2 phase and significantly inhibited cell proliferation, while ZEA had no significant effect on cell cycle. The combined treatment with DON and ZEA arrested cells in G2 phase to a higher extend compared to treatment with single mycotoxins. Potentiating effect observed after DON and ZEA co-exposure at environmentally relevant concentrations indicates that in risk assessment and setting governments' regulations, mixtures of mycotoxins should be considered.

One of the ways to impact emerging problems of unhealthy diet such as microbiota dysbiosis, inflammation, and oxidative stress is the application of probiotics and their incorporation into different food matrices. Discovery and selection of appropriate probiotic bacteria is challenging procedure especially for fermented meat products that have also been described as a potential source of resilient probiotic microorganisms. The aim of this study was to investigate probiotic bacteria Lactiplantibacillus plantarum 1K isolated from traditional fermented meat product for its potential beneficial properties. Furthermore, small probiotic metabolites were extracted, and their anti-inflammatory activity was tested in a lipopolysaccharide-stimulated inflammatory model on human peripheral blood mononuclear cells (PBMCs). Safety characteristics of metabolites including cytotoxicity and genotoxicity were also determined. Investigated probiotic strain exerted high antioxidant potential by viable cells but also by metabolite fraction. Viable cells retained the satisfactory antioxidant activity after gastrointestinal transit. Extracted probiotic metabolites significantly inhibited TNF-α production in LPS-stimulated PBMC thus exerting anti-inflammatory activity. Metabolites alone showed no cytotoxic or genotoxic activity toward isolated immune cells. Obtained results indicate the possibility to use fermented meat products as sources for specific probiotics that might provide antioxidant and anti-inflammatory benefits for the consumers., (© The Author(s) 2022. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The health benefit of a vegetarian diet is still under debate as it may result in a higher intake of some beneficial micronutrients, while others may be reduced, thus influencing various metabolic pathways and health-related biomarkers. This scoping review discusses inflammatory, oxidative and DNA damage status in vegetarians and vegans compared to omnivores. Most of the reviewed studies indicated favorable effects of a vegetarian diet on oxidative status compared to omnivores but did not clearly associate particular dietary habits to genome damage. The evidence on the effect of vegetarian diet on the inflammatory and immunological biomarkers is poor, which could at least partly be explained by methodological constraints such as small sample size, short duration of vegetarianism and inconsistent definitions of the omnivorous diet. The only inflammatory biomarker that seems to be associated with the vegetarian diet was inflammatory mediator C-reactive protein, which in several studies showed lower values in vegetarians as compared to omnivores. There were very few studies on immunological markers and the results on the difference between vegetarians and omnivores were inconclusive. Although several biomarkers involved in oxidative stress and inflammation showed a beneficial association with the vegetarian diet, further research in well-defined and sufficiently sized cohorts is needed to provide more evidence.

Diatoms of the genus Pseudo-nitzschia are cosmopolitans spread in seas and oceans worldwide, with more than 50 described species, dozens of which have been confirmed to produce domoic acid (DA). Here, we characterized and investigated the toxicological activity of secondary metabolites excreted into the growth media of different Pseudo-nitzschia species sampled at various locations in the northern Adriatic Sea (Croatia) using human blood cells under in vitro conditions. The results revealed that three investigated species of the genus Pseudo-nitzschia were capable of producing DA indicating their toxic potential. Moreover, toxicological data suggested all three Pseudo-nitzschia species can excrete toxic secondary metabolites into the surrounding media in addition to the intracellular pools of DA, raising concerns regarding their toxicity and environmental impact. In addition, all three Pseudo-nitzchia species triggered oxidative stress, one of the mechanisms of action likely responsible for the DNA damage observed in human blood cells. In line with the above stated, our results are of great interest to environmental toxicologists, the public and policy makers, especially in light of today's climate change, which favours harmful algal blooms and the growth of DA producers with a presumed negative impact on the public health of coastal residents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Non-communicable diseases (NCD) and lifestyle, particularly diet, have a close relationship. Based on the recent statistics, Croatian men and women lead in European overweight lists, which implies pessimistic prognosis in terms of incidence and prevalence of NCDs in the future. One of the possible solutions to overcome weight problems is turn to traditional balanced and sustainable diets, such as the Mediterranean diet. In this study, we assessed adherence towards Mediterranean diet using a validated questionnaire in an online survey and associated adherence scores with several demographic and anthropometric data. Based on the results of a validated Mediterranean Diet Adherence Screener (N = 3326), we assessed the adherence score to be 7.6 ± 2.5. The score tended to depend on sex, residence, age, education, income, and body mass index (BMI); indeed, women, residents of a coastal part of the country, older volunteers, those possessing a higher education degree, those with higher income, and those with lower BMI were associated with higher scores. As income was one of the significant findings related to higher adherence scores, we developed a dietary plan complying with Mediterranean diet principles that, on average, costed less than the average traditional balanced diet menu. Taken together, this study brought new findings regarding target groups who need to be encouraged to make lifestyle changes, and highlighted the first steps on how to make them.

Air pollution is recognized as one of the most serious public health issues worldwide and was declared to be a leading environmental cause of cancer deaths. At the same time, the cytokinesis-block micronucleus (CBMN) assay serves as a cancer predictive method that is extensively used in human biomonitoring for populations exposed to environmental contamination. The objective of this cross-sectional study is two-fold: to evaluate genomic instability in a sample (N = 130) of healthy, general population residents from Zagreb (Croatia), chronically exposed to different levels of air pollution, and to relate them to air pollution levels in the period from 2011 to 2015. Measured frequencies of CBMN assay parameters were in agreement with the baseline data for the general population of Croatia. Air pollution exposure was based on four factors obtained from a factor analysis of all exposure data obtained for the examined period. Based on the statistical results, we did not observe a significant positive association between any of the CBMN assay parameters tested and measured air pollution parameters for designated time windows, except for benzo(a)pyrene (B[a]P) that showed significant negative association. Our results show that measured air pollution parameters are largely below the regulatory limits, except for B[a]P, and as such, they do not affect CBMN assay parameters' frequency. Nevertheless, as air pollution is identified as a major health threat, it is necessary to conduct prospective studies investigating the effect of air pollution on genome integrity and human health.

Aims: The literature highlights the pathology of inflammation and its role in carcinogenesis, ageing and related diseases. Inflammatory processes induce oxidative stress and reduce antioxidant capacity. This study investigated the antioxidant and anti-inflammatory potential of probiotic bacteria isolated from fermented whey under conditions of induced stress., Methods and Results: Functional antioxidant characterization of potential probiotic bacteria Lactiplantibacillus plantarum S1 was performed under different growth conditions (aerobic, respiratory and anaerobic) and under stress to find the conditions that yield the most effective cells. Since aerobic growth yielded the most potent cells, the free radical scavenging ability of live and heat-killed cells was measured before and after exposure to gastrointestinal conditions. For heat-killed cells and extracted probiotic metabolites, the reduction of DNA damage to immune cells was determined in the hydrogen peroxide exposure comet assay. The combination of inactivated cells and metabolites showed the best reduction in DNA damage. Finally, in the LPS inflammation model, the aforementioned probiotic metabolites significantly reduced Tumour necrosis factor-alpha levels in immune cells., Conclusions: Whey-derived potential probiotic bacteria exert antioxidant and anti-inflammatory effects, and based on this study, we propose a model combining inactivated cells and metabolites to reduce inflammatory and oxidative stress-related adverse effects., Significance and Impact of Study: In this study, a new probiotic model is proposed for continuous use to reduce oxidative and inflammatory stress in the gut., (© 2022 Society for Applied Microbiology.)

Aging-related impaired body structure and functions may be, at least partially, caused by elevated oxidative stress. Melatonin (MEL) and resveratrol (RSV) may act as antioxidant and anti-aging compounds, but these actions in experimental animals and humans are controversial. Herein, a rat model of aging was used to study the long-term sex-related effects of MEL and RSV treatment on body mass and blood/plasma parameters of DNA damage, oxidative status (glutathione and malondialdehyde levels), and concentrations of sex hormones. Starting from the age of 3mo, for the next 9mo or 21mo male and female Wistar rats (n = 4-7 per group) were given water to drink (controls) or 0.1 % ethanol in water (vehicle), or MEL or RSV (each 10 mg/L vehicle). DNA damage in whole blood cells was tested by comet assay, whereas in plasma, glutathione, malondialdehyde, and sex hormones were determined by established methods. Using statistical analysis of data by ANOVA/Scheffe post hoc, we observed a similar sex- and aging-dependent rise of body mass in both sexes and drop of plasma testosterone in control and vehicle-treated male rats, whose pattern remained unaffected by MEL and RSV treatment. Compared with controls, all other parameters remained largely unchanged in aging and differently treated male and female rats. We concluded that the sex- and aging-related pattern of growth and various blood parameters in rats were not affected by the long-term treatment with MEL and RSV at the estimated daily doses (300-400 μg/kg b.m.) that exceed usual moderate consumption in humans., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Domoic acid (DA) is a marine neurotoxin produced as a defence compound by diatom Pseudo-nitzschia. Although its toxicity is well known in marine mammals and fish, data on DA cyto/genotoxicity in human non-target cells is still limited. Hence, we aimed to study the effect of DA (0.001-10 µg/mL) on cell viability and proliferation kinetics of human hepatocellular carcinoma (HepG2) cells as well as DNA damage induction after 4, 24 and 72 h of exposure. The results revealed that DA up to 10 µg/mL did not elicit significant changes in HepG2 cell viability, proliferation and cell cycle at applied conditions. DA did not generate DNA double-strand breaks, while it exhibited significant dose- and time-dependent increase of DNA damage in the form of either DNA single-strand breaks or alkali labile sites. Additionally, increased malondialdehyde level after DA treatment indicated oxidative damage to lipids. Altogether, the results showed that neurotoxin DA induced only minor adverse genotoxic effects in non-target HepG2 cells that most probably occurred resulting from the oxidative stress. However, additional research is needed to further elucidate the mechanisms of DA toxicity, particularly in terms of chronic exposure, as well as to understand its potential influence on human non-target cells., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Polyoxo-noble-metalates (PONMs), a class of molecular noble metal-oxo nanoclusters that combine features of both polyoxometalates and noble metals, are a promising platform for the development of next-generation antitumor metallodrugs. This study aimed to evaluate the antitumor potential against human neuroblastoma cells (SH-SY5Y), as well as toxicity towards healthy human peripheral blood cells (HPBCs), of five polyoxopalladates(II): (Na 8 [Pd 13 As 8 O 34 (OH) 6 ]·42H 2 O (Pd 13 ), Na 4 [SrPd 12 O 6 (OH) 3 (PhAsO 3 ) 6 (OAc) 3 ]·2NaOAc·32H 2 O (SrPd 12 ), Na 6 [Pd 13 (AsPh) 8 O 32 ]·23H 2 O (Pd 13 L), Na 12 [SnO 8 Pd 12 (PO 4 ) 8 ]·43H 2 O (SnPd 12 ), and Na 12 [PbO 8 Pd 12 (PO 4 ) 8 ]·38H 2 O (PbPd 12 )), as the largest subset of PONMs. A pure inorganic, Pd 13 , was found as the most potent and selective antineuroblastoma agent with IC 50 values (µM) of 7.2 ± 2.2 and 4.4 ± 1.2 for 24 and 48 h treatment, respectively, even lower than cisplatin (28.4 ± 7.4 and 11.6 ± 0.8). The obtained IC 50 values (µM) for 24/48 h treatment with SrPd 12 and Pd 13 L were 75.8 ± 6.7/76.7 ± 22.9 and 63.8 ± 3.6/21.4 ± 10.8, respectively, whereas SnPd 12 and PbPd 12 did not remarkably affect the SH-SY5Y viability (IC 50  > > 100 µM). Pd 13 caused depolarisation of inner mitochondrial membrane prior to superoxide ion hyperproduction, followed by caspase activation, DNA fragmentation and cell cycle arrest, all hallmarks of apoptotic cell death, and accompanied by an increase in acidic vesicles content, suggestive of autophagy induction. Importantly, Pd 13 demonstrated the antitumor effect at concentrations not cytogenotoxic for normal HPBCs. On the contrary, SrPd 12 and Pd 13 L at concentrations ≥ 1/3 IC 50  (24 h) decreased HPBC viability and increased % tail DNA up to 42% and 3.05 times, respectively, related to control. SnPd 12 and PbPd 12, previously confirmed promising antileukemic agents, did not exhibit cytogenotoxicity to HPBCs, and thus could be regarded as tumor cell specific and selective drug candidates., (© 2021. Society for Biological Inorganic Chemistry (SBIC).)

Cardiotoxicity is a frequent undesirable phenomenon observed during oncological treatment that limits the therapeutic dose of antitumor drugs and thus may decrease the effectiveness of cancer eradication. Almost all antitumor drugs exhibit toxic properties towards cardiac muscle. One of the underlying causes of cardiotoxicity is the stimulation of oxidative stress by chemotherapy. This suggests that an appropriately designed diet or dietary supplements based on edible plants rich in antioxidants could decrease the toxicity of antitumor drugs and diminish the risk of cardiac failure. This comprehensive review compares the cardioprotective efficacy of edible plant extracts and foodborne phytochemicals whose beneficial activity was demonstrated in various models in vivo and in vitro. The studies selected for this review concentrated on a therapy frequently applied in cancer, anthracycline antibiotic-doxorubicin-as the oxidative stress- and cardiotoxicity-inducing agent.

Imatinib mesylate (IM) is an anticancer drug that belongs to tyrosine kinase inhibitors. We report the results of the first investigation of the chronic exposure of zebrafish (Danio rerio) to IM. The exposure to IM (0.01, 1 and 100 μg/L) was initiated in adult fish and continued through hatching and the offspring generation for seven months. In addition to standard toxicological endpoints, induction of genotoxic effects and whole-genome transcriptome of liver samples of offspring generation of zebrafish were analysed. Exposure to IM did not affect the survival and growth of zebrafish, did not cause any histopathological changes, but it induced a marginal increase in the chromosomal damage in blood cells. The whole-genome transcriptome analyses demonstrated dose-dependent increase in the number of differentially expressed genes with a significantly higher number of deregulated genes in female fish compared to male. Differentially expressed genes included genes involved in response to DNA damage, cell cycle control and regulation of circadian rhythm. Based on the low genotoxic activity and the pattern of the changes in DNA damage responsive genes we consider that at current environmental exposure levels, IM represents low risk for genotoxic effects in aquatic organisms. Exposure to IM also induced deregulation of the expression of genes associated with steroidogenesis and hormone metabolism and function, which indicates hormone-disrupting activity of IM that has not been studied so far. The study provide new information on the potential consequences of chronic exposure to the residues of tyrosine kinase inhibitors, which remain to be further explored., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: The beneficial influence of a vegetarian diet in reducing the risk for metabolic syndrome has been demonstrated. However, adiponectin production and secretion are scarcely studied in vegetarians, despite their important role in recovering metabolic homeostasis by reducing visceral obesity, inflammation, and insulin resistance. The aim of this study was to evaluate the effect of a vegetarian diet on serum adiponectin levels and its association with the established biomarkers of insulin sensitivity and inflammation in healthy, non-obese individuals., Methods: Adiponectin, C-reactive protein, uric acid, glucose, insulin, lymphocyte and polymorphonuclear cell counts were determined in the blood of sex- and age-matched healthy vegetarian (n = 40) and omnivore (n = 36) individuals. The homeostatic model assessment (HOMA-2) calculator was used for the β-cell function (HOMA2-%B) and insulin resistance index (HOMA2-IRI) estimation., Results: Adiponectin levels were significantly higher in female vegetarians than the respective omnivore controls (P = 0.03), whereas no dietary-associated difference was observed in men. HOMA2-%B was significantly higher in vegetarians than in omnivore controls (P = 0.04), whereas no diet-dependent differences were found in insulin, HOMA2-IRI, inflammatory, and metabolic biomarkers. Multiple regression analysis showed that adiponectin levels were significantly predicted by the type of diet only in women (P = 0.042), whereas no associations were found in men., Conclusions: A vegetarian diet resulted in improved β-cell function. Favorable adiponectin and insulin sensitivity responses in women reveal a distinct effect of diet-to-metabolic homeostasis, indicating an interesting pattern of sexual dimorphism regarding the beneficial metabolic effect of a vegetarian diet., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Domoic acid (DA) is an excitatory marine neurotoxin produced by diatoms Pseudo-nitzschia spp. as a defence compound that accumulates in the food web and is associated with amnesic shellfish poisoning in humans. Although its toxicity has been well established in marine species, there is limited data on DA cytogenotoxicity in human non-target cells. Therefore, we aimed to investigate the cytogenotoxic potential of DA (0.01-10 μg/mL) in human peripheral blood cells (HPBCs) using a battery of bioassays in vitro. In addition, the influence of DA on oxidative stress parameters as a possible mechanism of action was assessed. Results revealed that DA induced dose- and time-dependent cytotoxic effects. DA significantly affected genomic instability by increasing the frequency of micronuclei and nuclear buds. Furthermore, a slight induction of primary DNA strand breaks was detected after 24 h of exposure accompanied by a significant increase in the number of abnormal size tailed nuclei. No induction of hOGG1 (human 8-oxoguanine DNA glycosylase) sensitive sites was determined upon exposure to DA. Additionally, DA induced oxidative stress by increased production of reactive oxygen species accompanied by changes in glutathione, superoxide dismutase, malondialdehyde and protein carbonyl levels. Overall, the obtained results showed adverse genotoxic effects of DA in non-target HPBCs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study aimed to evaluate occupational exposure to a styrene and xylene mixture through environmental exposure assessment and identify the potential genotoxic effects through biological monitoring. Secondly, we also exposed human peripheral blood cells in vitro to both xylene and styrene either alone or in mixture at concentrations found in occupational settings in order to understand their mechanism of action. The results obtained by air monitoring were below the occupational exposure limits for both substances. All biomarkers of effect, except for nucleoplasmic bridges, had higher mean values in workers (N = 17) compared to the corresponding controls (N = 17). There were statistically significant associations between exposed individuals and the presence of nuclear buds and oxidative damage. As for in vitro results, there was no significant influence on primary DNA damage in blood cells as evaluated by the comet assay. On the contrary, we did observe a significant increase of micronuclei and nuclear buds, but not nucleoplasmic bridges upon in vitro exposure. Taken together, both styrene and xylene have the potential to induce genomic instability either alone or in combination, showing higher effects when combined. The obtained data suggested that thresholds for individual chemicals might be insufficient for ensuring the protection of human health., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Diagnostic X-ray examinations of paranasal sinuses use a low-dose ionizing radiation to achieve medically indicated purposes. The effects of low-dose radiation are still controversial, making it a highly prioritized field of research. As there is a need to evaluate the effects of low-dose ionizing radiation and that children might be a more vulnerable population, we performed simultaneous physical dosimetry and buccal cell micronucleus cytome assay on pediatric patients before and after an X-ray examination of the sinuses., Methods: The study comprised 20 subjects aged 11.9 ± 3.6 years, and BMI < 25 kg/m 2 . Physical dosimetry was performed using radiophotoluminiscent (RPL) glass dosimeters placed on four positions on the head. The buccal cell micronucleus cytome assay was performed before and 14 ± 1 days after the X-ray exam, to monitor DNA damaging, replicative, cytostatic, and cell death effects., Results: The doses in the primary beam ranged 371-1106 μGy and were several fold higher than at the other positions on the head. As for biological changes, we did not observe any DNA damaging effects. However, a significant increase in cells with condensed chromatin was observed, indicating more cells undergoing early stages of apoptosis. We also observed inter-individual differences between the subjects. A correlation between the doses detected and biological effects was not observed., Conclusion: Although we did not observe significant increase in DNA damage, further studies are needed to increase the statistical power of the results and ensure patients' safety and optimal health care., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study proposes the application of the comet assay for the evaluation of DNA damage from frozen human whole blood samples that could be readily used in human biomonitoring and epidemiological studies. It was done on simply frozen whole blood samples collected from male volunteers (N = 60) aliquoted in small volumes and stored at -80 °C without the addition of cryopreservatives for a period of 5 years. To test the applicability of the alkaline comet assay for the evaluation of DNA damage in frozen whole blood, samples were quickly thawed at 37 °C and immediately embedded in an agarose matrix followed by an alkaline comet assay procedure. We concluded that the whole blood freezing and prolonged storage do not severely affect comet assay values, although background values were higher compared to our historical control data from the fresh whole blood. Even the influence of the variables tested, such as age, body mass index, smoking habit and alcohol consumption were in agreement with our previous data using fresh blood. The obtained results suggest that the comet assay could be applied to frozen blood samples, if properly stored, even for decades, which would certainly facilitate large-scale human biomonitoring and long-term epidemiological studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An adequate level of low molecular weight thiols (LMW-SH, especially glutathione (GSH)) protects cellular macromolecules against toxic agents, and is used as a sensitive biomarker of exposure to toxic compounds. During sample collection, storage and preparation, non-enzymatic and enzymatic oxidation of LMW-SH can occur leading to analytical inaccuracy. The aim of this study was to optimize a fast and reliable screening method for the determination of LMW-SH, mainly GSH, in blood and plasma samples as well as to investigate the impact of storage conditions on the LMW-SH stability. Based on our results, the described spectrophotometric method allows fast and reliable determination of LMW-SH in blood and plasma samples. Results on incubation of samples at 37 °C imply that synthesis of LMW-SH (probably GSH) as well as dynamic interexchange among various thiols forms can be induced in blood cells in in vitro conditions. Importantly, the level of LMW-SH in blood and plasma stored at -20 °C was constant, indicating that they can be stored at -20 °C for at least 30 days. Therefore, the method is suitable for assessment of LMW-SH in long-term human biomonitoring as well as environmental field studies, especially those involving a large number of samples such as epidemiological studies.

Imatinib mesylate (IM) is the first developed protein kinase inhibitor and recently it has topped consumption rates among targeted and total anticancer drugs. Although there are indications that IM possesses cyto/genotoxic activities against normal non-target cells as well, there is a lack of information regarding the underlying mechanism involved in those actions. Therefore, we aimed to evaluate the response of human circulating blood cells towards oxidative stress after IM treatment (0.0001-10 µg/mL) in vitro . Based on the results, IM had an influence on all of the oxidative stress parameters tested. Lower concentrations of IM induced an increase of glutathione level, following its decrease at higher IM concentrations indicating impairment in oxidative stress defences. Concomitant to a glutathione decrease, an increase of malondialdehyde and protein carbonyls level was observed indicating oxidative damage of lipids and proteins. The observed effects overlapped with the observed formation of oxidative base damage detected by formamidopyrimidine-DNA glycosylase modified-comet assay indicating that IM managed to induce oxidative DNA damage. Our results provide novelty in their mechanistic approach to IM-induced toxicity in non-target cells and suggest that IM can affect blood cells and induce oxidative stress., Competing Interests: None declared., (© 2019 The Author(s).)

Sodium copper chlorophyllin (CHL) is a food colorant that exhibits many beneficial properties, including potential for use in radiotherapy. Nevertheless, genotoxicity studies investigating radioprotective properties against γ-radiation on human cells are rather scarce. The aim of this study was to assess the cytotoxicity, genotoxicity and induction of malondialdehyde formation on CHL pre-treated whole blood cells after an absorbed dose of 5 Gy γ-radiation. Irradiated whole blood cells pre-treated with 100, 500, and 1000 μg/mL CHL showed less DNA-strand breaks (10.92 ± 0.74%, 10.69 ± 0.68%, and 8.81 ± 0.69%, respectively) than untreated irradiated cells (12.58 ± 0.88%). At the same time, the level of malondialdehyde was lower in CHL pre-treated samples with 100, 500, and 1000 μg/mL CHL (14.11 ± 0.43, 16.35 ± 2.82, and 13.08 ± 1.03 μmol/L, respectively) compared to untreated irradiated samples (24.11 ± 0.25 μmol/L). Regarding cytotoxicity, no changes were observed in the samples tested. Another important finding is that CHL had no cyto/genotoxic properties toward human blood cells. Taken together, since CHL had no cyto/genotoxic effects and showed good radioprotective properties in human blood cells, further studies should be conducted in order to find its possible application in radiotherapy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Interventional radiology unit workers represent one of the occupationally most exposed populations to low-dose ionizing radiation. Since there are many uncertainties in research of doses below 100 mSv, this study attempted to evaluate DNA damage levels in chronically exposed personnel. The study group consisted of 24 subjects matched with a control population by the number of participants, age, gender ratio, active smoking status, the period of blood sampling, and residence. Based on regular dosimetry using thermoluminiscent dosimeters, our study group occupationally received a dose of 1.82 ± 3.60 mSv over the last year. The results of the cytokinesis-block micronucleus assay and the comet assay showed a higher nuclear buds frequency (4.09 ± 1.88) and tail length (15.46 ± 1.47 μm) than in the control group (2.96 ± 1.67, 14.05 ± 1.36 μm, respectively). Differences in other descriptors from both tests did not reach statistical significance. Further investigations are needed to develop algorithms for improving personal dosimetry and those that would engage larger biomonitoring study groups., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Metallothioneins (MTs) exhibit binding affinity for several essential and toxic trace elements. Previous studies in rodents indicated sex differences in the hepatic and renal expression of MTs and concentrations of various elements. The mechanism responsible for these differences has not been resolved. Here, in the liver and kidney tissues of sham-operated and gonadectomized male and female rats we determined the expression of MT1 and MT2 (MT1&2) mRNA by RT-PCR, abundance of MT1&2 proteins by Western blotting and immunocytochemistry, concentrations of essential (Fe, Zn, Cu, Co) and toxic (Cd, Hg, Pb) elements by ICP-MS, and oxidative status parameters (SOD, GPx, MDA, GSH) by biochemical methods. In both organs, the expression of MT1&2 mRNA and MT1&2 proteins was female-dominant, upregulated by castration, and downregulated by ovariectomy. Concentrations of Fe in the liver and Co in the kidneys followed the same pattern. Most other elements (Zn, Cu, Cd, Hg) exhibited female- or male-dominant sex differences, affected by gonadectomy in one or both organs. Pb was sex- and gonadectomy-unaffected. GPx and MDA were elevated and associated with the highest concentrations of Fe only in the female liver. We conclude that the sex-dependent expression of MT1&2 mRNA and proteins in the rat liver and kidneys may include different mechanisms. In the liver, the female-dominant tissue concentrations of Fe may generate oxidative stress which is a potent enhancer of MTs production, whereas in kidneys, the female-dominant expression of MTs may be unrelated to Fe-mediated oxidative stress., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Multielemental analysis of whole blood can provide significant information for the evaluation of nutritional status and diagnosis of certain diseases as well as for the assessment of exposure to potentially toxic metals. However, the quantification of multiple elements in whole blood is not easy partly because of the wide variation in element concentrations (from ng L -1 to g L -1 ) and the complex matrix. The aim of this work was to develop a fast, sustainable, and reliable analytical method, in combination with low-power TXRF, for multielemental analysis of blood samples. Firstly, a set of experiments were carried out to select the best diluent type and dilution factor using the control material SeronormTM Trace Elements Whole Blood L-1. A critical evaluation of the parameters affecting the sample deposition on the reflector was also carried out including a study of the shape and element distribution of the deposited residue on the reflector by micro X-ray fluorescence spectrometry. Using the best analytical conditions, limits of detection estimated were in the low milligrams per kilogram range and similar to those obtained using more complex sample treatments such as digestion. Accuracy and precision of the results were in most cases acceptable (recoveries 89-102%, RSD 6-8%, n = 5). Only underestimated values were obtained for light elements such as potassium. To prove the applicability of the method, several blood samples from control and thyroid disease patients were analyzed. Despite the fact that more samples need to be analyzed, it seems that Zn and Br contents in some of the patients are significantly higher compared to control samples. Graphical abstract.

The non-proteinogenic amino acid ß-N-methylamino-l-alanine (BMAA) is associated with the development of neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC) and amyotrophic lateral sclerosis. BMAA is known to induce neurotoxic effects leading to neurodegeneration via multiple mechanisms including misfolded protein accumulation, glutamate induced excitotoxicity, calcium dyshomeostasis, endoplasmic reticulum stress and oxidative stress. In the present study, for the first time, genotoxic activity of BMAA (2.5, 5, 10 and 20 μg/mL) was studied in human peripheral blood cells (HPBCs) using the comet and cytokinesis-block micronucleus cytome assays. In addition, the influence of BMAA on the oxidative stress was assessed. At non-cytotoxic concentrations BMAA did not induce formation of DNA strand breaks in HPBCs after 4 and 24 h exposure; however, it significantly increased the number of micronuclei after 24 and 48 h at 20 μg/mL and nucleoplasmic bridges after 48 h at 20 μg/mL. The frequency of nuclear buds was slightly though non-significantly increased after 48 h. Altogether, this indicates that in HPBCs BMAA is clastogenic and induces complex genomic alterations including structural chromosomal rearrangements and gene amplification. No influence on oxidative stress markers was noticed. These findings provide new evidence that environmental neurotoxin BMAA, in addition to targeting common pathways involved in neurodegeneration, can also induce genomic instability in non-target HPBCs suggesting that it might be involved in cancer development. Therefore, these data are important in advancing our current knowledge and opening new questions in the understanding of the mechanisms of BMAA toxicity, particularly in the context of genotoxicity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The cytokinesis-block micronucleus cytome (CBMN Cyt) assay was used to evaluate the baseline frequency of cytogenetic damage in peripheral blood lymphocytes of the general population (average age, 38.28 ± 12.83 years) in relation to age, sex, body mass index, seasonal variations (season of sampling, period of sampling and different meteorological parameters) and lifestyle factors (smoking habit, alcohol consumption, exposure to medications and diagnostic radiation, physical activity, and family history of cancer). The background frequency of micronuclei (MNi) for the 200 subjects assayed was 5.06 ± 3.11 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 1.21 ± 1.46 and of nuclear buds (NBUDs) 3.48 ± 2.14. The background frequency of apoptosis and necrosis was 1.58 ± 1.50 and 1.39 ± 1.56, respectively, while the mean nuclear division index (NDI) was 1.99 ± 0.14. The cut-off value, which corresponds to the 95th percentile of the distribution of 200 individual values, was 11 MNi, 4 NPBs and 7 NBUDs. The study also confirmed an association of the above mentioned parameters with age, sex and several lifestyle factors. Moreover, significant confounders based on our results are also sampling season, sampling period and different meteorological parameters that were dependent on the CBMN Cyt assay parameters. In line with the above mentioned, several factors should be taken into account when it comes to the monitoring of exposed populations using cytogenetic biomarkers. Moreover, the normal and cut-off values obtained in this study present background data for the general population, and can later serve as baseline values for further biomonitoring studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cytostatic drugs are highly cytotoxic agents used in cancer treatment and although their benefit is unquestionable, they have been recognized as hazardous to healthcare professionals in occupational settings. In a working environment, simultaneous exposure to cytostatics may occur creating a higher risk than that of a single substance. Hence, the present study evaluated the combined cyto/genotoxicity of a mixture of selected cytostatics with different mechanisms of action (MoA; 5-fluorouracil, cyclophosphamide and paclitaxel) towards human lymphocytes in vitro at a concentration range relevant for occupational as well as environmental exposure. The results suggest that the selected cytostatic drug mixture is potentially cyto/genotoxic and that it can induce cell and genome damage even at low concentrations. This indicates not only that such mixture may pose a risk to cell and genome integrity, but also that single compound toxicity data are not sufficient for the prediction of toxicity in a complex working environment. The presence of drugs in different amounts and with different MoA suggests the need to study the relationship between the presence of genotoxic components in the mixture and the resulting effects, taking into account the MoA of each component by itself. Therefore, this study provides new data sets necessary for scientifically-based risk assessments of cytostatic drug mixtures in occupational as well as environmental settings., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Circulating platinum (Pt) is detectable in the blood of Pt-treated cancer patients for over a decade after the treatment. Prolonged exposure to Pt, in combination with adverse compounds from nutrition and lifestyle, such as cadmium (Cd), could increase the risk from second cancers. The aim of this study was to investigate the effects of simultaneous exposure to Cd- and Pt-compounds on oxidative and DNA damage and the possible protective effects of zinc (Zn) and selenium (Se). The aqueous solutions of PtCl 4 , CdCl 2  ×   H 2 O, ZnCl 2 and Na 2 SeO 3 were added, alone or in combination, to whole blood and isolated erythrocytes to produce the final concentrations of 2000 μg/L of Pt, 8 μg/L of Cd, 100 μg/L of Se, and 1000 μg/L of Zn. The activity of copper, zinc-superoxide dismutase, glutathione peroxidase and glutathione in whole blood was determined after 1 h exposure in in vitro conditions. The induction of DNA strand-breaks in human peripheral blood leukocytes was determined with the alkaline comet assay after 24 h exposure. Exposure to Pt and/or Cd decreased the activities of antioxidant enzymes and elevated DNA damage compared to control. A statistically significant change in the activity of both enzymes and in the induction of DNA strand-breaks was observed in the cells treated with Pt + Cd combination, while the addition of Se and/or Zn resulted in partial recovery of these effects. The results indicate that combined exposure to Pt and Cd could disrupt antioxidant protection of the organism and increase DNA damage, whereas Se and Zn could partially ameliorate these harmful effects.

The selective tyrosine kinase inhibitor imatinib mesylate (IM) is a widely used anticancer drug. Recent studies showing that IM can induce DNA and chromosomal damage in crustaceans and higher plants prompted us to re-examine its potential genotoxicity. IM was not mutagenic in the Ames assay (Salmonella typhimurium). Cytotoxicity and genotoxicity were evaluated in vitro in zebrafish (Danio rerio) liver (ZFL), human hepatoma (HepG2), and human peripheral blood lymphocyte (HPBL) cells. Genotoxicity was determined with the comet assay and with the cytokinesis-block micronucleus cytome assay. ZFL and HPBL cells showed comparable sensitivity to IM cytotoxicity, while HepG2 cells were less sensitive. At non-cytotoxic concentrations, IM induced DNA strand breaks in ZFL and HepG2 cells. An increase in the number of micronuclei was observed in ZFL and HPBL cells. In HPBLs, IM also induced an increase in the number of nucleoplasmic bridges and nuclear buds. Based on the data of the consumption of IM in European countries the predicted environmental concentrations (PEC) were calculated to be in the range between 3.3 and 5.0ng/L, which are several orders of magnitude lower from those that caused adverse effects in fish and human derived cells. However, based on the in vitro studies it is not possible to quantitatively predict the hazard for wildlife and humans, therefore further studies are warranted to explore the underlying molecular mechanisms of induced IM genotoxic effects as well as the studies of the occurrence of IM in the aquatic and occupational environment to establish the relevance of these observations for aquatic organisms and occupationally exposed personnel., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Since the production of printed circuit boards (PCBs) generates wastewater contaminated with heavy metals and organic matter, PCB factories represent potential pollution sites. The wastewater toxicologically tested in this study contained several metals and the most abundant were copper and iron. At two exposure times tested (4 and 24 h) PCB wastewater (PCBW) proved to be cytotoxic (decreased cell viability) and genotoxic (increased comet assay tail intensity and tail moment) to human blood peripheral lymphocytes in vitro, and the oxidative stress parameter (malondialdehyde concentration) was also found to be higher. After application of combined treatment by waste base, ozone and waste sludge methods, concentrations of metals in purified PCBW were below the upper permitted levels and all tested toxicological parameters did not differ compared to the negative control. Taken together, similar methods could be implemented in PCB factories before discharging potentially toxic wastewater into the environment because purified PCBW does not represent a threat from the aspect of cytotoxicity and genotoxicity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Antineoplastic drugs are highly cytotoxic chemotherapeutic agents that can often interfere directly or indirectly with the cell's genome. In an environmental or medical setting simultaneous exposure may occur. Such multiple exposures may pose a higher risk than it could be assumed from the studies evaluating the effect of a single substance. Therefore, in the present study we tested the combined cyto/genotoxicity of a mixture of selected antineoplastic drugs with different mechanisms of action (5-fluorouracil, etoposide, and imatinib mesylate) towards human lymphocytes in vitro. The results suggest that the selected antineoplastic drug mixture is potentially cyto/genotoxic and that it can induce cell and genome damage even at low concentrations. Moreover, the changes in the measured oxidative stress parameters suggest the participation of reactive oxygen species in the cyto/genotoxicity of the selected mixture. The obtained results indicate not only that such mixtures may pose a risk to cell and genome integrity, but also that single compound toxicity data are not sufficient for the predicting toxicity in a complex environment. Altogether, the results emphasise the need for further toxicological screening of antineoplastic drug mixtures, especially at low environmentally relevant concentrations, as to avoid any possible adverse effects on the environment and human health., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Since the incidence of cancer has increased over the years, adequate prevention programmes are needed. Thyroid cancer is one of the fastest growing cancer types in the world. In this study we performed a case-control study of 100 untreated patients with thyroid diseases (papillary thyroid cancer, follicular thyroid adenoma, and other thyroid diseases) and 100 control volunteers. Oxidative status differed among the two investigated groups. The patients' group had 1.60-fold higher concentrations of malondialdehyde and 1.26-fold higher concentrations of protein carbonyls. At the same time, the concentrations of glutathione and catalase activity were by 32% and 35% lower, respectively. A similar effect was observed for the cytogenetic status where higher comet assay tail intensity (1.84-fold) and the total numbers of chromosome aberrations (1.47-fold), micronuclei (2.32-fold), nucleoplasmic bridges (3.98-fold), and nuclear buds (2.34-fold) were detected. As for protein expression in thyroid tissue, 97.89% were positive for either B-Raf or Ret. Interestingly, the papillary thyroid cancer patients more frequently expressed B-Raf proteins compared to the follicular thyroid adenoma patients and patients with other thyroid diseases. Human biomonitoring studies enable a risk assessment of general population, such data could be used to identify risk subgroups., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell's genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC50) 5.3 to 10.4 μg/mL). ZFL cells were more sensitive towards ET- (IC50 0.4 μg/mL) and HepG2 towards CDDP- (IC50 1.4 μg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 μg/mL for ET, 0.01 μg/mL for 5-FU and 0.1 μg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 μg/mL for ET, 0.1 μg/mL for 5-FU and 1 μg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.

Melittin (MEL) is the main constituent and principal toxin of bee venom. It is a small basic peptide, consisting of a known amino acid sequence, with powerful haemolytic activity. Since MEL is a nonspecific cytolytic peptide that attacks lipid membranes thus leading to toxicity, the presumption is that it could have significant therapeutic benefits. The aim was to evaluate the cyto/genotoxic effects of MEL in human peripheral blood lymphocytes (HPBLs) and the molecular mechanisms involved using a multi-biomarker approach. We found that MEL was cytotoxic for HPBLs in a dose- and time-dependent manner. It also induced morphological changes in the cell membrane, granulation and lysis of exposed cells. After treating HPBLs with non-cytotoxic concentrations of MEL, we observed increased DNA damage including oxidative DNA damage as well as increased formation of micronuclei and nuclear buds, and decreased lymphocyte proliferation determined by comet and micronucleus assays. The observed genotoxicity coincided with increased formation of reactive oxygen species, reduction of glutathione level, increased lipid peroxidation and phospholipase C activity, showing the induction of oxidative stress. MEL also modulated the expression of selected genes involved in DNA damage response (TP53, CDKN1A, GADD45α, MDM), oxidative stress (CAT, SOD1, GPX1, GSR and GCLC) and apoptosis (BAX, BCL-2, CAS-3 and CAS-7). Results indicate that MEL is genotoxic to HPBLs and provide evidence that oxidative stress is involved in its DNA damaging effects. MEL toxicity towards normal cells has to be considered if used for potential therapeutic purposes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Owing to its proven toxicity and mutagenicity, arsenic is regarded a principal pollutant in water used for drinking. The objective of this study was the toxicological and chemical evaluation of groundwater samples obtained from arsenic enriched drinking water wells before and after electrochemical and ozone-UV-H2O2-based advanced oxidation processes (EAOP). For this purpose, acute toxicity test with Daphnia magna and chronic toxicity test with Lemna minor L. were employed as well as in vitro bioassays using human peripheral blood lymphocytes (HPBLs). Several oxidative stress parameters were estimated in L.minor. Physicochemical analysis showed that EAOP treatment was highly efficient in arsenic but also in ammonia and organic compound removal from contaminated groundwater. Untreated groundwater caused only slight toxicity to HPBLs and D. magna in acute experiments. However, 7-day exposure of L. minor to raw groundwater elicited genotoxicity, a significant growth inhibition and oxidative stress injury. The observed genotoxicity and toxicity of raw groundwater samples was almost completely eliminated by EAOP treatment. Generally, the results obtained with L. minor were in agreement with those obtained in the chemical analysis suggesting the sensitivity of the model organism in monitoring of arsenic-contaminated groundwater. In parallel to chemical analysis, the implementation of chronic toxicity bioassays in a battery is recommended in the assessment of the toxic and genotoxic potential of such complex mixtures., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In the present study toxicity of Frangula alnus Mill. bark, widely used as laxative, was investigated. Human peripheral blood lymphocytes (HPBLs) were treated with F. alnus bark extract or emodin (emodin is bark component with laxative property), and cytotoxicity, genotoxicity and parameters of oxidative stress were assessed. Also, polyphenol content of bark extract and antioxidant activity of the extract and emodin measured by DPPH, ABTS and FRAP methods were examined. The bark extract (500 μg/ml) produced cell death and DNA damage, while level of ROS changed at 250 μg/ml. Emodin induced cell death and DNA damage at 150 μg/ml and 200 μg/ml, respectively, and the increase of ROS was observed at 25 μg/ml. These results suggest that both, bark extract and emodin, are cyto/genotoxic to HPBLs and that oxidative stress is involved in the mechanism of their toxicity. The results on antioxidant activity showed that, unlike emodin, bark extract possess moderate antioxidant capacity (44.6%, 46.8% and 2.25 mmol Fe(2+)/g measured by DPPH, ABTS and FRAP assay, respectively) that can be related to relatively high phenolic content (116.07 mg/g). However, due to toxicological properties use of F. alnus bark as well as emodin-containing preparations should be taken with caution., (Copyright © 2015 Elsevier Inc. All rights reserved.)