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In terms of exposome, air pollution is a life-long and constant source of exposure affecting human organs, tissues, cells, and molecules. It is considered responsible for more than 3 million premature deaths annually. Currently ongoing HUMNap project investigates possible associations between air pollutants and biomarkers of exposure and early biological effects. The evaluation of results is divided in two parts. In part 1 we examined historical data (2011-15) of cytogenetic biomarkers in Zagreb population (N=130) and corresponding air quality data. Measured air pollution parameters were largely below regulatory limits, except for B[a]P. There were no significant positive associations indicating contribution of air pollutants to increased genome damage for designated period. In part 2, in 2021- 22 we recruited a new cohort (N=60) and included biomarkers of exposure (benzene, toluene, ethylbenzene, o-, m- and p-xylene) and effect (blood comet assay, buccal and blood micronucleus assay). For air quality data, the results were in agreement with results from part 1. Similarly, tested parameters did not impact biomarkers of exposure nor the genotoxicity biomarkers. The focus of the project will now turn to other cities with different air pollution burden, and will expand the number of evaluated biomarkers in order to find possible links between air pollution and the biomarkers of effect, improve prediction models, and to serve in better risk assessment of general public. Supported by the Croatian Science Foundation (HUMNap project 1192 and the work of doctoral student K. Matković)

Air pollution remains a major issue in environmental and public health and has been recognized by leading world authorities as a risk factor associated with adverse health outcomes. In addition to cancer, exposure to air pollutants has been linked to the onset of cardiovascular and respiratory disease, as well as premature mortality. The most important pollutants to note are particulate matter (PM), polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOC), and toxic metals. Therefore, we aimed to investigate the possible cytogenotoxic effects of a complex mixture of PAHs and toxic metals in vitro in a range of concentrations of air pollutants from 0.5 to 50 cm3/mL. The mixture was prepared based on the outdoor air pollution monitoring data in Zagreb (Croatia) collected during our human biomonitoring study in the colder period of the year 2021 representing the translation of real-scenario exposure to in vitro. Cytogenotoxicity was evaluated on human peripheral blood cells employing the cell viability assay and the alkaline comet assay intended for the assessment of primary DNA damage to better understand the mechanism of action of the tested mixture. Based on the obtained results, we did not observe any impact of the complex mixture on cell viability that remained similar compared to the non-treated cells. As for the DNA damaging effect, only the highest tested concertation (50 cm3/mL) was able to increase primary strand breaks as measured by the % tail DNA as the comet assay descriptor. Our results indicate the necessity of testing complex mixtures that may pose a risk to cell and genome integrity in a complex environment, especially at low environmentally relevant concentrations. Since oxidative stress and inflammation are suspected mechanisms of action of air pollution, further research will include various biomarkers of oxidative stress as well as other biomarkers of genomic instability in order to avoid any possible adverse effects on the environment and human health.

Increased road traffic and industrial and energy production contribute to urban air pollution worldwide. Consequently, cities are hotspots for air pollution, representing a global health threat, resulting in millions of premature deaths annually. The HUMNap project aims to provide scientifically based data on how air pollution can affect genomic instability and our health by determining possible associations between air pollutants and biomarkers of exposure and early biological effects. In the first part of the project, we retrospectively evaluated genomic instability using the comet and micronucleus assays on blood cells in a sample (N=130) of the general population living in Zagreb (Croatia) and associated these results with air pollution levels in the period from 2011 to 2015. We did not observe a significant positive association between parameters tested and measured air pollution parameters except for benzo(a)pyrene (B[a]P), which showed a significant negative association. Our results show that the measured air pollution parameters were largely below regulatory limits, except for B[a]P. In the prospective part of the study, we investigated possible effects of air pollution and BTEX (benzene, toluene, ethylbenzene, o-, m- and p-xylene) exposure on genomic instability using the comet and micronucleus assays on blood in addition to buccal cells in a sample (N = 60) of the general population living in Zagreb (Croatia) during colder and warmer periods in 2021-2022. All measured outdoor air pollution parameters agreed with previously reported values and were below the regulatory limit, except for PM10 particles and B[a]P bound to PM10, which exceeded regulatory limit levels. Again, we did not observe the noteworthy impact of air pollution on tested parameters. Since air pollution is identified as a major health threat, further research investigating the effect of air pollution on genome integrity and human health is warranted. It will prioritize other cities where exposure to different air pollutants is expected. Supported by the Croatian Science Foundation (HUMNap)

Cilj je odrediti vrijednost biomarkera izloženosti i ranih bioloških učinika u populaciji stanovnika Slavonskog Broda. U studiju su uključena 22 ispitanika koji žive u blizini industrijske zone u Slavonskom Brodu. U krvi i urinu ispitanika određene su koncentracije benzena, toluena, etilbenzena, i izomera ksilena. Također je korišten mikronukleus test i komet test. Pulsnim oksimetrom je određena zasićenje kisikom . Određena je i izdahnuta frakcija dušikovog oksida. Utvrđene vrijednosti su u skladu s dostupnim vrijednostima očekivanim za hrvastku populaciju i/ili referentnih literaturnih vrijednosti.

Polycyclic aromatic hydrocarbons (PAHs) are recognized as one of the most serious contaminants due to their mutagenic and carcinogenic health effects. They originate from a variety of sources such as forest and peat fires, volcano eruptions, coal combustion and vehicle exhaust emissions. There are several routes of PAH exposure, including water, food and tobacco smoke, but ambient air represents one of the major sources of PAH intake. In this study, PM10 particle samples (particulate matter with a diameter

Air pollution represents a major environmental risk to health, facilitating development of many diseases. The effects of air pollution exposure are difficult to determine because the different chemical species in it are not easily definable or measurable, and they can induce additive, synergistic or antagonistic effects. The comet assay is a method successfully used in monitoring air pollution genotoxicity in exposed populations. Hence, we aimed to investigate possible effects of air pollution on DNA integrity using the alkaline comet assay in human peripheral blood cells. The study was conducted during the colder period of the year 2021 and involved 60 healthy subjects (34 females and 26 males), aged 36.4±9.6 years and BMI

Exposure to high levels of air pollution can cause a variety of adverse health outcomes, and is associated with millions of premature deaths annually. The main sources of air pollution in urban areas are traffic, domestic heating and emissions from manufacturing plants. The air in urban areas is a very complex and variable mixture of numerous contaminants, containing many mutagenic pollutants whose mechanism of action is not fully known. Additionally, the emission of compounds such as BTEX (benzene, toluene, ethylbenzene and isomeric xylenes) is increasing, mainly due to intensive industrialization and urbanization. Therefore, we aimed to investigate the association between air pollutants and biomarkers of exposure and early biological effects, using the alkaline comet and micronucleus assays on peripheral blood cells. The research was conducted during the cold season of 2021, as it was found that the concentration of air pollutants is higher in this period due to the heating season. Our research included 60 healthy volunteers aged 18–55 with a BMI

Vodeće svjetske organizacije poput Europske agencije za okoliš, Svjetske zdravstvene organizacije, i Međunarodne agencije za istraživanje raka ističu onečišćenje zraka kao vodeću globalnu zdravstvenu prijetnju odgovornu za milijune smrtnih slučajeva godišnje. Zrak u urbanim sredinama kompleksna je i promjenjiva mješavina raznih čestica i kemijskih spojeva čiji mehanizam djelovanja nije u potpunosti poznat, iako se sumnja na poticanje oksidacijskog stresa i upalnih procesa. Izloženost vanjskom onečišćenju zraka povezana je sa zdravstvenim učincima, osobito u urbanim područjima, zbog viših koncentracija onečišćujućih tvari uzrokovanih urbanom aglomeracijom, prometom i industrijskim aktivnostima. Dodatno, emisija spojeva poput BTEX- a (benzen, toluen, etilbenzen i izomeri ksilena) postaje veća, uglavnom zbog intenzivne industrijalizacije i urbanizacije. Biomonitoring ljudi ključan je u određivanju mogu li i u kojoj mjeri spojevi iz okoliša utjecati na zdravlje ljudi. Stoga smo željeli istražiti povezanost između razina onečišćujućih tvari iz zraka te biomarkera izloženosti i ranih bioloških učinaka, koristeći alkalni komet i mikronukleus test za procjenu genomskih oštećenja na ljudskim bukalnim stanicama i stanicama periferne krvi. Prvi dio istraživanja proveden je tijekom hladnijeg razdoblja 2021. godine, a drugi dio tijekom toplijeg razdoblja 2022. godine. U istraživanju je sudjelovalo 60 zdravih dobrovoljaca u dobi 18 – 55 godina s BMI

Mechanisms of aging, and cancer as one of main associated pathology are only on verge of understanding. Even in healthy aging gradually loss of function in excretion organs kidneys and liver appears. Previously, we did not find expected elevated tissue concentrations of reactive oxidative species (ROS), that are assumed to be present in old humans and experimental animals and that they can be reversed by antioxidants (AO) treatment. The new hypothesis was that the expression of ROS inducible, potent antioxidant and metal scavenging proteins, metallothioneins Mt1 and Mt2 (MTs), among others, increases in old age to maintain ROS levels and that they can be further modulated with AO. To that aim we studied male and female Wistar rats regularly aged together with these treated with melatonin and resveratrol. Starting from 3 months of age, for the next 9 or 21 months treated rats were drinking AO in water (~1 mg/kg b.w./day), whereas the control animals were drinking water or AO vehicle (0.01% ethanol). Trace elements (TE) were analysed by ICP-MS and the expression of MTs mRNA and proteins in kidneys and liver were determinate by end-point RT-PCR and immunochemical methods, respectively. AO did not change TE accumulation and expression of MTs in both sexes, but known iron accumulation in aged animals was followed by copper and toxic metal cadmium with known female domination. After 1 year, aging increased MTs expression in both sexes that did only slightly further increased after 2 years. We conclude that Fenton reactive metals accumulated in aged animals may influence MTs mRNA and protein expression to help retain physiological ROS level in old rats. (Croatian Science Foundation project IP-2013-11-1481).

Oxidative stress is characterized by the inability of the organism to detoxify reactive oxygen species (ROS) and can be defined as a condition in which the balance between prooxidants and antioxidants in the cell is disturbed, leading to DNA hydroxylation, protein denaturation, lipid peroxidation, and cell apoptosis. The antioxidant effects of probiotic cells and probioticenriched foods have been previously noticed, but the exact mechanisms of antioxidant action are not fully understood. Literature suggests that probiotic therapy improves host oxidative status, which may lead to prevention of some chronic diseases. The concept of paraprobiotics has been proposed to refer to the use of inactivated microbial cells or cell fractions that provide health benefits to the host. Hence, we aimed to exert H2O2-induced oxidative stress on isolated human lymphocytes and investigate the protective effect of heat killed probiotic bacteria Lactiplantibacillus plantarum S1 and extracted bacterial metabolites smaller than 2.000 Daltons on nuclear DNA of isolated lymphocytes, using the comet assay. Both, metabolites and heat killed cells significantly reduced the H2O2-induced DNA damage (from 7.59±1.51% in induced stress to 4.07±1.61% for metabolites and 3.86±1.58% for heat killed cells), displaying a protective role. In combined treatment of heat killed cells and metabolites, the DNA damage was significantly lower (2.62±1.02%) than oxidative stress sample and the levels of primary DNA strand breaks did not differ compared to control sample (1.33±0.14%). Probiotic metabolites and heat-killed cells could potentially be used in a modified model to reduce the adverse effects associated with oxidative stress in the lower parts of the gastrointestinal tract.

The aim of this study was to test cytotoxicity, genotoxicity, and oxidative stress as a mechanism of toxicity of bisphenol A (BPA) using Allium test. Allium test is considered as a good model for detecting environmental mutagens and good correlation in cytogenetic studies of Allium test with other test organisms is confirmed. Onion bulbs (Allium cepa L.) were germinated for 24 h, and afterwards, rooted bulbs (n = 10) were exposed to BPA in concentration range 1-50 mg/L or to distilled water (negative control) for 72 h. Following 72 h exposure, morphometry (the root length and the fresh weight) was assessed. In root meristem cells mitotic index (MI) and frequency of micronuclei (MNi) were determined using a light microscope analysing minimum 500 cells per slide. In root homogenate as oxidative stress parameters the level of glutathione (GSH) and protein carbonyls (PC) was assessed spectrophotometrically. The results were analysed by use of one-way ANOVA followed by Dunnett’s test. Statistical significance level was set at p < 0.05. After 72 h exposure to BPA (1-50 mg/L) a concentration-dependent decrease in the root length, root fresh weight as well as decrease in the percentage of MI of meristem cells was observed. Even the lowest concentration of BPA (1 mg/L) inhibited the root growth while the root fresh weight was reduced after exposure to BPA at 5 mg/L compared to negative control. The lowest BPA concentration (1 mg/L) decreased the percentage of MI as well as the number of cells in each of the cell cycle phase. With the increase of BPA concentration, an increase in MNi formation was observed. A decrease in GSH level was observed already after exposure to the lowest BPA concentration (1 mg/L) while an increase of PC was observed after exposure to BPA in concentration 10 mg/L. Based on the obtained results, it can be concluded that BPA induces cyto/genotoxic effects in root meristem cells and that oxidative stress is involved in its toxicity.

Exposure to outdoor air pollution is related to severe health effects, especially in urban areas, due to higher concentrations of pollutants caused by urban agglomeration, traffic, and industrial activities. Additionally, the emission of BTEX (benzene, toluene, ethylbenzene, and xylene) compounds becomes higher, mainly due to intensive industrialization and urbanization. Hence, we aimed to investigate possible effects of air pollution and BTEX exposure on DNA damage detected by the alkaline comet assay and micronucleus assay on human buccal and peripheral blood cells. The first part of the study was conducted during the colder period of the year 2021 and the follow up part during the warmer period of the year 2022. It involved 60 healthy volunteers aged 18-55 years with BMI

Outdoor air pollution in urban and rural areas causes millions of premature deaths worldwide. Cities are more exposed to higher concentrations of pollutants because of the urban agglomeration, traffic, and industrial activities. Additionally, the emission of BTEX (benzene, toluene, ethylbenzene, and xylene) compounds becomes higher, mainly due to intensive industrialization and urbanization. Hence, we aimed to investigate possible effects of air pollution and BTEX exposure on DNA damage detected by the alkaline comet assay in human peripheral blood cells. The study was conducted during the colder period of the year 2021 and involved 60 healthy subjects (34 females and 26 males), aged 36.4±9.6 years and BMI

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.

In this study cytogenotoxicity of mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) as well as their combinations on HepG2 cells was assessed. HepG2 cells were treated with DON (0.25-16 μM), ZEA (1.25-80 μM), and DON+ZEA (1 μM+5 μM, 1 μM+10 μM and 1 μM+20 μM) for 24 h and cytotoxicity was assessed with MTS assay. For genotoxicity experiments cells were treated with DON (0.5, 1 and 2 μM), ZEA (5, 10 and 20 μM) and DON+ZEA (1 μM+5 μM, 1 μM+10 μM and 1 μM+20 μM) for 24 h and the alkaline comet assay was applied. DON at concentration 1 μM reduced cell viability by 14.6 %, ZEA at concentration 40 μM by 22.6 % while their combination (1 μM DON+5 μM ZEA) reduced HepG2 viability by 16.2 % (P < 0.05). However, treatment with DON, ZEA or their combinations did not increase % of tail DNA in the comet assay. Although genotoxicity was not observed, both mycotoxins as well as their combinations were cytotoxic, thus their toxicity and mechanism of toxicity should be further explored.