Exploring the Antibacterial Potential of Lamiaceae Plant Extracts: Inhibition of Bacterial Growth, Adhesion, Invasion, and Biofilm Formation and Degradation in Pseudomonas aeruginosa PAO1.

In response to the global rise in antibiotic resistance and the prevalence of bacterial biofilm-related infections, the antibacterial efficacy of methanolic, ethanolic, and aqueous extracts of 18 Lamiaceae plants from Serbia was evaluated. The total coumarins and triterpenes were detected spectrophotometrically, while a microdilution assay measured their effects on bacterial growth. Additionally, the impact of these extracts was assessed on Pseudomonas aeruginosa PAO1 adhesion and invasion in human fibroblasts and biofilm formation and degradation. The alcoholic extracts had the highest phytochemical content, with Teucrium montanum and Lavandula angustifolia being the richest in coumarins and triterpenes, respectively. Gram-positive bacteria, particularly Bacillus subtilis, were more susceptible to the extracts. Hyssopus officinalis ethanolic and Sideritis scardica methanolic extracts inhibited bacterial growth the most efficiently. Although the extracts did not inhibit bacterial adhesion, most ethanolic extracts significantly reduced bacterial invasion. Origanum vulgare and H. officinalis ethanolic extracts significantly inhibited biofilm formation, while Teucrium chamaedrys extract was the most active in biofilm degradation. This study significantly contributes to the literature by examining the antibacterial activity of Lamiaceae extracts, addressing major literature gaps, and underscoring their antibacterial potential, particularly Satureja montana and O. vulgare ethanolic extracts, linking their efficacy to coumarins and triterpenes.

Influence of alkylthio and arylthio derivatives of tert-butylquinone on the induction of DNA damage in a human hepatocellular carcinoma cell line (HepG2).

The aim of this study was to investigate the effects of tert-butylquinone (TBQ) and its alkylthio and arylthio derivatives on DNA in vitro, using acellular and cellular test systems. Direct interaction with DNA was studied using the plasmid pUC19. Cytotoxic (MTS assay) and genotoxic (comet assay and γH2AX focus assays) effects, and their influence on the cell cycle were studied in the HepG2 cell line. Our results show that TBQ and its derivatives did not directly interact with DNA. The strongest cytotoxic effect on the HepG2 cells was observed for the derivative 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone (IC50 64.68 and 55.64 µM at 24-h and 48-h treatment, respectively). The tested derivatives did not significantly influence the cell cycle distribution in the exposed cellular populations. However, all derivatives showed a genotoxic activity stronger than that of TBQ in the comet assay, with 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone producing the strongest effect. The same derivative also induced DNA double-strand breaks in the γH2AX focus assay.

Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

Combined use of biomarkers to assess the impact of untreated wastewater from the Danube River, Serbia.

In this study a battery of bioassays, both in vivo (metals and metalloids concentrations, erythrocyte morphometry, comet assay, micronucleus assay, and histopathological analyses) on vimba bream Vimba vimba (L., 1758) and white bream Blicca bjoerkna (L., 1758), and in vitro (treatment of HepG2 cells with native water samples) was applied to assess the harmful potential of untreated wastewater. Faecal indicator bacteria were quantified to assess the microbiological water quality. Vimba bream had significantly higher Fe concentrations in both liver and muscle, while white bream had higher Ca and Cu concentrations in liver. Vimba bream had a significantly higher level of DNA damage in both liver and blood cells, in comparison to white bream. Low levels of micronucleus and nuclear abnormalities were observed in both species. Erythrocytes morphometry did not show significant interspecific differences. Histopathological analyses revealed a similar response of the studied species, with a significantly higher presence of ceroid pigments in the liver of vimba bream. Treatment of HepG2 cells revealed the high genotoxic potential of water downstream of the discharge point. The results of this study clearly demonstrate the importance of effect-based monitoring, in order to enforce more efficient management of natural resources and implementation of wastewater treatment systems.

First report on the occurrence of Vibrio cholerae nonO1/nonO139 in natural and artificial lakes and ponds in Serbia: Evidence for a long-distance transfer of strains and the presence of Vibrio paracholerae.

Vibrio cholerae are natural inhabitants of specific aquatic environments. Strains not belonging to serogroups O1 and O139 are usually unable to produce cholera toxin and cause cholera. However, non-toxigenic V. cholerae (NTVC) are able to cause a variety of mild-to-severe human infections (via seafood consumption or recreational activities). The number of unreported cases is considered substantial, as NTVC infections are not notifiable and physicians are mostly unaware of this pathogen. In the northern hemisphere, NTVC infections have been reported to increase due to global warming. In Eastern Europe, climatic and geological conditions favour the existence of inland water-bodies harbouring NTVC. We thus investigated the occurrence of NTVC in nine Serbian natural and artificial lakes and ponds, many of them used for fishing and bathing. With the exception of one highly saline lake, all investigated water-bodies harboured NTVC, ranging from 5.4 × 101 to 1.86 × 104  CFU and 4.5 × 102 to 5.6 × 106 genomic units per 100 ml. The maximum values observed were in the range of bathing waters in other countries, where infections have been reported. Interestingly, 7 out of 39 fully sequenced presumptive V. cholerae isolates were assigned as V. paracholerae, a recently described sister species of V. cholerae. Some clones and sublineages of both V. cholerae and V. paracholerae were shared by different environments indicating an exchange of strains over long distances. Important pathogenicity factors such as hlyA, toxR, and ompU were present in both species. Seasonal monitoring of ponds/lakes used for recreation in Serbia is thus recommended to be prepared for potential occurrence of infections promoted by climate change-induced rise in water temperatures.

Single and combined potential of polystyrene microparticles and fluoranthene in the induction of DNA damage in haemocytes of Mediterranean mussel (Mytilus galloprovincialis).

In this study, the possible 'vector effect' within the exposure of Mediterranean mussels (Mytilus galloprovincialis) to polystyrene microplastics with adsorbed fluoranthene was investigated by applying the multibiomarker approach. The major focus was placed on genotoxicological endpoints as to our knowledge there are no literature data on the genotoxicity of polystyrene microparticles alone or with adsorbed fluoranthene in the selected experimental organisms. DNA damage was assessed in haemocytes by comet assay and micronucleus test. For the assessment of neurotoxicity, acetylcholinesterase activity was measured in gills. Glutathione S-transferase was assessed in gills and hepatopancreas since these enzymes are induced for biotransformation and excretion of lipophilic compounds such as hydrocarbons. Finally, differences in physiological response within the exposure to polystyrene particles, fluoranthene, or particles with adsorbed fluoranthene were assessed by the variation of heart rate patterns studied by the noninvasive laser fibre-optic method. The uniform response of individual biomarkers within the exposure groups was not recorded. There was no clear pattern in variation of acetylcholinesterase or glutathione S-transferase activity which could be attributed to the treatment. Exposure to polystyrene increased DNA damage which was detected by the comet assay but was not confirmed by micronucleus formation. Data of genotoxicity assays indicated differential responses among the groups exposed to fluoranthene alone and fluoranthene adsorbed to polystyrene. Change in the heart rate patterns within the studied groups supports the concept of the Trojan horse effect within the exposure to polystyrene particles with adsorbed fluoranthene.

In situ detection of the genotoxic potential as one of the lines of evidence in the weight-of-evidence approach-the Joint Danube Survey 4 Case Study.

Environmental studies which aim to assess the ecological impact of chemical and other types of pollution should employ a complex weight-of-evidence approach with multiple lines of evidence (LoEs). This study focused on in situ genotoxicological methods such as the comet and micronucleus assays and randomly amplified polymorphic DNA analysis as one of the multiple LoEs (LoE3) on the fish species Alburnus alburnus (bleak) as a bioindicator. The study was carried out within the Joint Danube Survey 4 (JDS4) at nine sites in the Danube River Basin in the Republic of Serbia. Out of nine sampling sites, two were situated at the Tisa, Sava, and Velika Morava rivers, and three sites were at the Danube River. The three additionally employed LoEs were: SumTUwater calculated based on the monitoring data in the database of the Serbian Environmental Protection Agency (SEPA) (LoE1); in vitro analyses of JDS4 water extracts employing genotoxicological methods (LoE2); assessment of the ecological status/potential by SEPA and indication of the ecological status for the sites performed within the JDS4 (LoE4). The analyzed biomarker responses in the bleak were integrated into the unique integrated biomarker response index which was used to rank the sites. The highest pollution pressure was recorded at JDS4 39 and JDS4 36, while the lowest was at JDS4 35. The impact of pollution was confirmed at three sites, JDS4 33, 40, and 41, by all four LoEs. At other sampling sites, a difference was observed regarding the pollution depending on the employed LoEs. This indicates the importance of implementing a comprehensive weight-of-evidence approach to ensure the impact of pollution is not overlooked when using only one LoE as is often the case in environmental studies.

Fluoride subacute testicular toxicity in Wistar rats: Benchmark dose analysis for the redox parameters, essential elements and DNA damage.

Excessive fluoride (F-) levels in the environment could induce different pathological changes, including comorbidities in reproductive functions. Hence, the aim of the present in vivo study was to explore F- subacute toxicity mechanisms via Benchmark dose (BMD) methodology on rat's testicles. The experiment was conducted on thirty male Wistar rats for 28 days, divided into six groups (n = 5): 1) Control (tap water); 2) 10 mg/L F-; 3) 25 mg/L F-; 4) 50 mg/L F-; 5) 100 mg/L F-; 6) 150 mg/L F-. Testicles were dissected out and processed for the determination of F- tissue concentrations, redox status parameters, essential elements level, and DNA damage. PROASTweb 70.1 software was used for determination of external and internal dose-response relationship. The results confirmed a significant increase in superoxide anion (O2.-), total oxidative status (TOS), copper (Cu), zinc (Zn), iron (Fe), DNA damage levels, and decrease in superoxide dismutase activity (SOD1) and total thiol (SH) groups. The dose-dependent changes were confirmed for SOD1 activity and DNA damage. The most sensitive parameters were SOD1 activity and DNA damage with the lowest BMDLs 0.1 µg F-/kg b. w. Since human and animal populations are daily and frequently unconsciously exposed to F-, this dose-response study is valuable for further research regarding the F- health risk assessment.

Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells.

BACKGROUND: Although still considered a safer alternative to classical cigarettes, growing body of work points to harmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect of e-cigarettes needs to be investigated in more detail considering their widespread use. METHODS: In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, with and without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cellular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performed via high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry. RESULTS: E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however it negatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletion in total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteins were upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of posttranslational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data are available via ProteomeXchange with identifier PXD032071. CONCLUSIONS: Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMs alterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact.

Wastewater-based epidemiology in countries with poor wastewater treatment - Epidemiological indicator function of SARS-CoV-2 RNA in surface waters.

Wastewater-based epidemiology (WBE) surveillance of COVID-19 and other future outbreaks is a challenge for developing countries as most households are not connected to a sewerage system. In December 2019, SARS-CoV-2 RNA was detected in the Danube River at a site severely affected by wastewaters from Belgrade. Rivers are much more complex systems than wastewater systems, and efforts are needed to address all the factors influencing the adoption of WBE as an alternative to targeting raw wastewater. Our objective was to provide a more detailed insight into the potential of SARS-CoV-2 surveillance in Serbian surface waters for epidemiological purposes. Water samples were collected at 12 sites along the Sava and Danube rivers in Belgrade during the fourth COVID-19 wave in Serbia that started in late February 2021. RNA was concentrated using Amicon Ultra-15 centrifugal filters and quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (faecal indicator bacteria and human and animal genetic faecal source tracking markers), epidemiological, physicochemical and hydromorphological parameters were analysed in parallel. From 44 samples, SARS-CoV-2 RNA was detected in 31, but only at 4 concentrations above the level of quantification (ranging from 8.47 × 103 to 2.07 × 104 gc/L). The results indicated that surveillance of SARS-CoV-2 RNA in surface waters as ultimate recipients could be used as an epidemiological early-warning tool in countries lacking wastewater treatment and proper sewerage infrastructure. The performance of the applied approach, including advanced sampling site characterization to trace and identify sites with significant raw sewage influence from human populations, could be further improved by adaptation of the methodology for processing higher volumes of samples and enrichment factors, which should provide the quantitative instead of qualitative data needed for WBE.

A Study of Phytochemistry, Genoprotective Activity, and Antitumor Effects of Extracts of the Selected Lamiaceae Species.

This study was designed to evaluate the genoprotective, antigenotoxic, as well as antitumor potential of methanolic, ethanolic, and aqueous extracts of Melissa officinalis, Mentha × piperita, Ocimum basilicum, Rosmarinus officinalis, Salvia officinalis, and Satureja montana (Lamiaceae), in different model systems. The polyphenols in these extracts were quantified both spectrophotometrically and using HPLC-DAD technique, while DPPH assay was used to assess the antioxidant activity. The genoprotective potential was tested on pUC19 Escherichia coli XL1-blue, and the antigenotoxicity on Salmonella typhimurium TA1535/pSK1002 and human lung fibroblasts, while the antitumor activity was assessed on colorectal cancer cells. Rosmarinic acid, quercetin, rutin, and luteolin-7-O-glucoside were among the identified compounds. Methanolic extracts had the best DPPH-scavenging and SOS-inducing activities, while ethanolic extracts exhibited the highest antigenotoxicity. Additionally, all extracts exhibited genoprotective potential on plasmid DNA. The antitumor effect was mediated by modulation of reactive oxygen species (ROS), nitric oxide (NO) production, and exhibition of genotoxic effects on tumor cells, especially with O. basilicum ethanolic extract. Generally, the investigated extracts were able to provide antioxidant protection for the acellular, prokaryotic, and normal human DNA, while also modulating the production of ROS and NO in tumor cells, leading to genotoxicity toward these cells and their decrease in proliferation.

Temporal variation of biomarkers in common bream Abramis brama (L., 1758) exposed to untreated municipal wastewater in the Danube River in Belgrade, Serbia.

This study was conducted on the Danube River locality Visnjica, exposed to the discharge of the largest wastewater collector in Serbia's capital, Belgrade. Concentrations of metals and metalloids (Al, Fe, Cr, As, Sr, Mn, Cd, Zn, Mo, Cu, Li, Ni, B, Co, Pb, and Ba) and histopathological alterations were investigated in different tissues of common bream during one representative month of each season in 2014. This is the first study in which these two biomarkers were assessed parallelly in common bream. The highest concentrations of examined elements were noticed in gills and the lowest in muscle. Statistically significant differences in element concentrations between different seasons were noticed only in gills for Al, Cu, and Fe. Concentrations of As and Pb in fish muscle were below the maximum acceptable concentrations (MAC). The histopathological index (HI) of the liver showed higher values in comparison to the HI of the gills. Histopathological index of the gills had a significantly higher score in November in comparison to August. The liver HI had the highest score in April, and the lowest in August, while the total histopathological index had the highest score in November, and the lowest in August, both without significant differences between the months. This study endorses gills and liver as reliable organs for studying accumulation and histopathology as biomarkers of environmental changes. A faster reaction of the gills was confirmed since seasonal variations of both biomarkers were observed in this organ. Common bream proved as a good indicator of the state of organisms in polluted environments.

Making Waves: Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.

Detection of SARS-CoV-2 RNA in the Danube River in Serbia associated with the discharge of untreated wastewaters.

In Serbia less than 13% of collected municipal wastewaters is being treated before their release in the environment. This includes all municipal wastewater discharges from Belgrade (capital city of Serbia; population 1,700,000). Previous research has identified the impacts of raw wastewater discharges from Belgrade on the Danube River, and this study investigated if such discharges also provided a pathway for SARS-CoV-2 RNA material. Samples were collected during the most critical circumstances that occurred so far within the COVID-19 pandemics in Serbia. Grab and composite samples were collected in December 2020, during the peak of the third wave (in terms of reported cases) at the site which receives the wastewater loads in Belgrade. Grab samples collected upstream and downstream of Belgrade were also analyzed. RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. SARS-CoV-2 RNA (5.97 × 103 to 1.32 × 104 copies/L) was detected only in samples collected at the site strongly impacted by the wastewaters where all three applied primer sets gave positive signals. Determined concentrations correspond to those reported in wastewater influents sampled at treatment plants in other countries indicating an epidemiological indicator function of used approach for rivers with high pollution loads in countries with poor wastewater treatment.

A comprehensive assessment of the chemical composition, antioxidant, genoprotective and antigenotoxic activities of Lamiaceae species using different experimental models in vitro.

This research was aimed to assess the potential of Glechoma hederacea, Hyssopus officinalis, Lavandula angustifolia, Leonurus cardiaca, Marrubium vulgare and Sideritis scardica (Lamiaceae) methanolic, ethanolic and aqueous extracts against the damaging effects of oxidative stress using different experimental models. The chemical characterization was done spectrophotometrically by quantifying total phenolics, phenolic acids, flavonoids and flavonols in the extracts, as well as by employing HPLC-DAD technique. Moreover, DPPH assay was used to assess the extracts' radical scavenging potential. Genoprotective properties of the extracts were evaluated using plasmid pUC19 Escherichia coli XL1-Blue, whereas their antigenotoxic potential was determined using Salmonella typhimurium TA1535/pSK1002 and normal human lung fibroblasts. All of the extracts showed antioxidant activity in DPPH assay. Furthermore, the results have shown that aqueous extracts provided the best protection for plasmid DNA, while alcoholic extracts most effectively contributed to the preservation of prokaryotic DNA. Additionally, each of the tested samples significantly protected the eukaryotic cells against genomic damages. Finally, despite not showing exceptional results in DPPH assay, S. scardica extracts are regarded as the most favorable in maintaining the integrity of DNA, which might be due to high quantities of phenolics such as quercetin (up to 17.95 mg g-1), naringin (up to 5.07 mg g-1) and luteolin-7-O-glucoside (up to 3.54 mg g-1). Overall, this comprehensive concept highlights the ability of these Lamiaceae species to safeguard the DNA from reactive oxygen species, to curtail the inflicted damage and also improve the efficiency of the DNA repair mechanisms, while emphasizing the importance of polyphenols as their active principles.

Genotoxicity of fluoride subacute exposure in rats and selenium intervention.

The aims of this study were to: (i) examine the toxic effects of sodium fluoride (NaF) in blood, liver, spleen, and brain cells of Wistar rats after the subacute exposure; (ii) explore the potential protective properties of selenium (Se) against fluoride toxicity after the simultaneous administration. Twenty male Wistar rats, eight weeks old, weighing approximately 140-190 g, were divided into four experimental groups (n = 5) as follows: I control-tap water; II NaF 150 ppm; III NaF 150 ppm and Se 1.5 mg/L; IV Se 1.5 mg/L, and had available water with solutions ad libitum for 28 days. DNA damage detected by comet assay was confirmed in the liver, spleen, and brain cells, but not in blood. Selenium supplementation together with NaF decreased DNA damage in liver and spleen cells. According to the histological findings, no changes were observed in spleen and brain tissues after NaF administration. Unlike the observed Se protective effect on the DNA level, no significant reduction of liver tissue injury was observed after the NaF and Se treatment, resulting in mild inflammation. Data of this study suggest that DNA damage after NaF subacute exposure at moderately high concentration was reduced in liver and spleen cells due to Se supplementation, but a similar change was not seen in the brain.

Sensitivity of Bleak (Alburnus alburnus) in Detection of the Wastewater Related Pressure in Large Lowland Rivers.

This study deals with bleak (Alburnus alburnus) sensitivity in detecting of the wastewater related pressure in large lowland rivers. The major objective was to investigate if the response measured in bleak should be linked to a certain stretch of the river and characterised as "stretch specific", or it should be linked to the sampling site and characterised as "site specific". The response was evaluated via condition index, metal pollution index, DNA damage and cell viability using integrated biomarker response approach. The study was conducted at 3 sub-sites characterized by different pollution levels in a relatively short stretch (2 km) of the Sava River (Serbia). Results indicated that the response of the biomarkers in bleak can be interpreted as "site specific". Among the studied biomarkers, DNA damage assessed by comet assay and micronucleus test has shown high sensitivity in differentiation of the sites.

Selection of assay, organism, and approach in biomonitoring significantly affects the evaluation of genotoxic potential in aquatic environments.

In this study, few different evaluation concepts were used for the assessment of genotoxic potential at the stretch of the Danube River identified as a significant hotspot of pollution originated through the untreated wastewaters. Three sites were chosen: one site upstream of the wastewater outlet in Novi Sad (Serbia), one at the outlet of wastewaters, and one site few kilometer downstream. Ex situ approach comprised prokaryotic SOS/umuC test on Salmonella typhimurium TA1535/pSK1005 and comet assay on human hepatoma cell line (HepG2). In situ approach was based on the active monitoring (cage approach) using freshwater mussels Sinanodonta woodiana and fish Cyprinus carpio. The comet and micronucleus assays were selected for evaluation of DNA damage in mussel haemocytes and fish blood cells. Within the ex situ part of the study, our results indicated that the eukaryotic model system is more sensitive compared to the prokaryotic one. In situ bioassays are recommended for obtaining a better insight into ecosystem status and in the case of our study the complete insight of genotoxic pressure. However, the choice of animals as bioindicators also has a significant impact on the quality of the obtained information. Differential response between fish and mussels was observed at the highly polluted site suggesting possible involvement of additional protective mechanism such as valve closure in mussels.

The impact of different extracts of six Lamiaceae species on deleterious effects of oxidative stress assessed in acellular, prokaryotic and eukaryotic models in vitro.

The main objective of this research was to evaluate the impact of methanolic, ethanolic and aqueous extracts of Origanum majorana L., Origanum vulgare L., Teucrium chamaedrys L., Teucrium montanum L., Thymus serpyllum L. and Thymus vulgaris L. (Lamiaceae) on the effects of free radicals using different model systems. The extracts were characterized on the basis of the contents of total phenolics, phenolic acids, flavonoids and flavonols, and also using high-performance liquid chromatography with diode-array detection. Antioxidant activity in vitro was assessed using DPPH assay. The genoprotective properties were tested using plasmid relaxation assay on pUC19 E. coli XL1-Blue, while SOS/umuC assay on Salmonella typhimurium TA1535/pSK1002 and Comet assay on human lung fibroblasts were used to assess the antigenotoxicity of the extracts. Ethanolic extracts had the most phenolics (up to 236.20 mg GAE/g at 0.5 mg/mL), flavonoids (up to 42.47 mg QE/g at 0.5 mg/mL) and flavonols (up to 16.56 mg QE/g at 0.5 mg/mL), and they exhibited the highest DPPH activity (up to 92.16% at 0.25 mg/mL). Interestingly enough, aqueous extracts provided the best protection of plasmid DNA (the lowest IC50 value was 0.17 mg/mL). Methanolic extracts, on the other hand, most efficiently protected the prokaryotic DNA, while all the extracts had a significant impact against genomic damages inflicted on human fibroblasts. O. vulgare extracts are considered to be the most promising in preserving the overall DNA integrity against oxidative genomic damages. Moreover, HPLC-DAD analysis highlighted rosmarinic acid as the most abundant in the investigated samples (551.45 mg/mL in total in all the extracts), followed by luteolin-7-O-glucoside (150.19 mg/mL in total), while their presence correlates with most of the displayed activities. The novelty of this study is reflected in the application of a prokaryotic model for testing the antigenotoxic effects of Lamiaceae species, as no previous reports have yet been published on the genoprotective potential of these species.

Evaluation of genotoxic potential of tert-butylquinone and its derivatives in prokaryotic and eukaryotic test models.

Tert-butylquinone (TBQ) and its alkylamino and aralkylamino derivatives are of high interest as a potential antitumor agent. Therefore, it was necessary to investigate if the compounds exert undesirable activities such as interaction with DNA molecule which could result in negative side effects in the case of their use in the diseases treatment. The major aim of this study was to investigate genotoxic potential of TBQ and selected derivatives in an acellular model by using plasmid DNA, in the prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002 and in eukaryotic models by using comet assay in human fetal lung cell line (MRC-5) and human liver cancer cell line (HepG2). Results indicated that in the acellular model TBQ and its derivatives do not interact with plasmid pUC19. In the prokaryotic model, only TBQ exerted weak genotoxic potential and only at highly cytotoxic concentrations. In eukaryotic models, genotoxic potential was detected mainly at the highest concentrations of the tested substances but the effect was lower in both cell lines in comparison with benzo[a]pyrene and etoposide which were used as positive controls. Weak genotoxic potential of tested compounds recommends them as good candidates for further testing in development of new antitumor agents.