Protective Effects of Arbutus unedo L. Honey in the Alleviation of Irinotecan-Induced Cytogenetic Damage in Human Lymphocytes-An In Vitro Study.

Strawberry tree (Arbutus unedo L.) honey (STH) has been used since ancient times as a folk medicine remedy, especially in certain Mediterranean countries. This honey, rich in phenolic content, is well recognized for its antioxidant, anti-inflammatory, and antimicrobial activities, and is used for the treatment of skin lesions as well as gastrointestinal and respiratory disorders. This study investigated whether STH alleviates genome damage in human peripheral blood lymphocytes produced by the cytotoxic drug irinotecan. The phenolic profile of STH was previously estimated by ultra-high-performance liquid chromatography coupled to a linear ion trap-Orbitrap hybrid mass spectrometer. The effects of STH were evaluated at three concentrations (1×, 5×, and 10×), based on the daily consumption of the honey by an adult person. After 2 h of in vitro exposure, standard lymphocyte cultures for the analysis of chromosome aberrations and the cytokinesis-block micronucleus cytome assay were established. Our results demonstrate that STH offered remarkable geno- and cytoprotection when administered with irinotecan. These findings are relevant for drawing preliminary conclusions regarding the in vitro safety of the tested honey. However, further studies are needed with the application of more complex experimental models.

Evaluation of DNA-Damaging Effects Induced by Different Tanning Agents Used in the Processing of Natural Leather-Pilot Study on HepG2 Cell Line.

For a long time, the production and processing of cowhide was based on the use of chrome tanning. However, the growing problem with chromium waste and its negative impact on human health and the environment prompted the search for more environmentally friendly processes such as vegetable tanning or aldehyde tanning. In the present study, we investigated the DNA-damaging effects induced in HepG2 cells after 24 h exposure to leather samples (cut into 1 × 1 cm2 rectangles) processed with different tanning agents. Our main objective was to determine which tanning procedure resulted in the highest DNA instability. The extent of treatment-induced DNA damage was determined using the alkaline comet assay. All tanning processes used in leather processing caused primary DNA damage in HepG2 cells compared to untreated cells. The effects measured in the exposed cells indicate that the leaching of potentially genotoxic chemicals from the same surface is variable and was highest after vegetable tanning, followed by synthetic tanning and chrome tanning. These results could be due to the complex composition of the vegetable and synthetic tanning agents. Despite all limitations, these preliminary results could be useful to gain a general insight into the genotoxic potential of the processes used in the processing of natural leather and to plan future experiments with more specific cell or tissue models.

Liver function and DNA integrity in hepatocytes of rats evaluated after treatments with strawberry tree (Arbutus unedo L.) water leaf extract and arbutin.

Due to their beneficial health effects, strawberry tree (Arbutus unedo L.) leaves have for decades been used as herbal remedy in countries of the Mediterranean region. This pilot study is the first to investigate the liver function and DNA integrity in rat hepatocytes evaluated after 14 and 28 day treatments with strawberry tree water leaf extract and arbutin, administered per os to Lewis rats of both genders at a daily dose 200 mg/kg b.w. We focused on two types of biomarkers: enzyme serum markers of liver function (AST, ALT, and LDH), and primary DNA damage in the liver cells, which was estimated using the alkaline comet assay. At the tested dose, strawberry tree water leaf extract showed acceptable biocompatibility with liver tissue both in male and female rats, especially after shorter exposure. Our results also suggest that oral administration of single arbutin to rats was not associated with significant impairments either in the liver function or DNA integrity in hepatocytes. Considering that prolonged exposure to the tested compounds revealed minor changes in the studied biomarkers, future in vivo studies have to further clarify the biological and physiological relevance of these findings.

DNA Damaging Effects, Oxidative Stress Responses and Cholinesterase Activity in Blood and Brain of Wistar Rats Exposed to Δ9-Tetrahydrocannabinol.

Currently we are faced with an ever-growing use of Δ9-tetrahydrocannabinol (THC) preparations, often used as supportive therapies for various malignancies and neurological disorders. As some of illegally distributed forms of such preparations, like cannabis oils and butane hash oil, might contain over 80% of THC, their consumers can become intoxicated or experience various detrimental effects. This fact motivated us for the assessments of THC toxicity in vivo on a Wistar rat model, at a daily oral dose of 7 mg/kg which is comparable to those found in illicit preparations. The main objective of the present study was to establish the magnitude and dynamics of DNA breakage associated with THC exposure in white blood and brain cells of treated rats using the alkaline comet assay. The extent of oxidative stress after acute 24 h exposure to THC was also determined as well as changes in activities of plasma and brain cholinesterases (ChE) in THC-treated and control rats. The DNA of brain cells was more prone to breakage after THC treatment compared to DNA in white blood cells. Even though DNA damage quantified by the alkaline comet assay is subject to repair, its elevated level detected in the brain cells of THC-treated rats was reason for concern. Since neurons do not proliferate, increased levels of DNA damage present threats to these cells in terms of both viability and genome stability, while inefficient DNA repair might lead to their progressive loss. The present study contributes to existing knowledge with evidence that acute exposure to a high THC dose led to low-level DNA damage in white blood cells and brain cells of rats and induced oxidative stress in brain, but did not disturb ChE activities.

Irinotecan and Δ8-Tetrahydrocannabinol Interactions in Rat Liver: A Preliminary Evaluation Using Biochemical and Genotoxicity Markers.

There is growing interest regarding the use of herbal preparations based on Cannabis sativa for medicinal purposes, despite the poorly understood interactions of their main constituent Δ8-tetrahydrocannabinol (THC) with conventional drugs, especially cytostatics. The objective of this pilot study was to prove whether the concomitant intake of THC impaired liver function in male Wistar rats treated with the anticancer drug irinotecan (IRI), and evaluate the toxic effects associated with this exposure. IRI was administered once intraperitoneally (at 100 mg/kg of the body weight (b.w.)), while THC was administered per os repeatedly for 1, 3, and 7 days (at 7 mg/kg b.w.). Functional liver impairments were studied using biochemical markers of liver function (aspartate aminotransferase-AST, alanine aminotransferase-ALP, alkaline phosphatase-AP, and bilirubin) in rats given a combined treatment, single IRI, single THC, and control groups. Using common oxidative stress biomarkers, along with measurement of primary DNA damage in hepatocytes, the degree of impairments caused at the cellular level was also evaluated. THC caused a time-dependent enhancement of acute toxicity in IRI-treated rats, which was confirmed by body and liver weight reduction. Although single THC affected ALP and AP levels more than single IRI, the levels of liver function markers measured after the administration of a combined treatment mostly did not significantly differ from control. Combined exposure led to increased oxidative stress responses in 3- and 7-day treatments, compared to single IRI. Single IRI caused the highest DNA damage at all timepoints. Continuous 7-day oral exposure to single THC caused an increased mean value of comet tail length compared to its shorter treatments. Concomitant intake of THC slightly affected the levels of IRI genotoxicity at all timepoints, but not in a consistent manner. Further studies are needed to prove our preliminary observations, clarify the underlying mechanisms behind IRI and THC interactions, and unambiguously confirm or reject the assumptions made herein.

Assessment of multiple anthropogenic contaminants and their potential genotoxicity in the aquatic environment of Plitvice Lakes National Park, Croatia.

In this study, the influence of anthropogenic pollution on the aquatic environment of Plitvice Lakes National Park (PLNP) was investigated during 2011-2012 using a combination of chemical and cytogenetic analyses. Four groups of major contaminants [(volatile organic compounds: benzene, toluene, ethylbenzene, and xylenes (BTEX); persistent organochlorine pollutants: organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs); major and trace elements; anthropogenic radionuclides (90Sr, 134Cs, and 137Cs)] were determined in three aquatic compartments (water, sediment, fish). Mass fractions of inorganic constituents in different compartments reflected the geological background of the area, indicating their origin from predominantly natural sources. Levels of volatile and persistent organic compounds in water and fish, respectively, were very low, at levels typical for remote pristine areas. Analysis of anthropogenic radionuclides in water and sediment revealed elevated activity concentrations of 137Cs in water, and measurable 134Cs in the upper sediment layers from April 2011, possibly as a consequence of the Fukushima nuclear accident in March 2011. The potential genotoxicity of river and lake water and lake sediment was assessed under laboratory conditions using the alkaline comet assay on human peripheral blood lymphocytes, and measured levels of primary DNA damage were within acceptable boundaries. The results showed that despite the protected status of the park, anthropogenic impact exists in both its terrestrial and aquatic components. Although contaminant levels were low, further monitoring is recommended to make sure that they will not rise and cause potentially hazardous anthropogenic impacts.

Evaluation of genome damage in subjects occupationally exposed to possible carcinogens.

In occupational exposures, populations are simultaneously exposed to a mixture of chemicals. We aimed to evaluate DNA damage due to possible carcinogen exposure (phenylhydrazine, ethylene oxide, dichloromethane, and 1,2-dichloroethane) in lymphocytes of pharmaceutical industry workers from the same production line. Population comprised 16 subjects (9 females and 7 males) who were exposed to multiple chemicals for 8 months. Genome damage was assessed using alkaline comet assay, micronucleus assay, and comet assay coupled with fluorescent in situ hybridization (comet-FISH). After 8 months of exposure, the issue of irregular use of all available personal protective equipment (PPE) came into light. To decrease the risk of exposure, strict use of PPE was enforced. After 8 months of strict PPE use, micronuclei frequency and comet assay parameters in lymphocytes of pharmaceutical workers significantly decreased compared with prior period of irregular PPE use. Comet-FISH results indicated a significant shift in distribution of signals for the TP 53 gene toward a more frequent occurrence in the comet tail. Prolonged exposure to possible carcinogens may hinder DNA repair mechanisms and affect structural integrity of TP 53 Two indicators of loss of TP 53 gene integrity have risen, namely, TP 53 fragmentation rate in lymphocytes with persistently elevated primary damage and incidence of TP 53 deletions in undamaged lymphocytes.

Antifungal Activity of Oleuropein against Candida albicans-The In Vitro Study.

In the present study we investigated activity of oleuropein, a complex phenol present in large quantities in olive tree products, against opportunistic fungal pathogen Candida albicans. Oleuropein was found to have in vitro antifungal activity with a minimal inhibitory concentration (MIC) value of 12.5 mg·mL-1. Morphological changes in the nuclei after staining with fluorescent DNA-binding dyes revealed that apoptosis was a primary mode of cell death in the analyzed samples treated with subinhibitory concentrations of oleuropein. Our results suggest that this antifungal agent targets virulence factors essential for establishment of the fungal infection. We noticed that oleuropein modulates morphogenetic conversion and inhibits filamentation of C. albicans. The hydrophobicity assay showed that oleuropein in sub-MIC values has significantly decreased, in both aerobic and anaerobic conditions, the cellular surface hydrophobicity (CSH) of C. albicans, a factor associated with adhesion to epithelial cells. It was also demonstrated that the tested compound inhibits the activity of SAPs, cellular enzymes secreted by C. albicans, which are reported to be related to the pathogenicity of the fungi. Additionally, we detected that oleuropein causes a reduction in total sterol content in the membrane of C. albicans cells, which might be involved in the mechanism of its antifungal activity.

Cytotoxic and genotoxic potencies of single and combined spore extracts of airborne OTA-producing and OTA-non-producing Aspergilli in Human lung A549 cells.

Aspergillus sclerotiorum (AS) is a well-known producer of ochratoxin A (OTA) while Aspergillus pseudoglaucus (AP) produces a wide range of extrolites with poorly investigated toxicity. These species are frequently co-occur in grain mill aeromycota. The aim of this study was to determine OTA levels in spore extracts using HPLC and immunoaffinity columns, and to examine the cytotoxicity of pure OTA, OTA-positive (AS-OTA(+)) and OTA-negative (AS-OTA(-)) spore extracts, as well as of AP spore extract, on human lung adenocarcinoma cells A549, individually and in combination, using a colorimetric MTT test (540nm). To establish which type of cell death predominated after treatments, a quantitative fluorescent assay with ethidium bromide and acridine orange was used, and the level of primary DNA damage in A549 cells was evaluated using the alkaline comet assay. OTA was detected in spore extracts (0.3-28µg/mL) of 3/6 of the AS strains, while none of the tested AP strains were able to produce OTA. Taking into account the maximum detected concentration of OTA in the spores, the daily intake of OTA by inhalation was calculated to be 1ng/kg body weight (b.w.), which is below the tolerable daily intake for OTA (17ng/kg b.w.). Using the MTT test, the following IC50 values were obtained: single OTA (53µg/mL); AS-OTA(+) (mass concentration 934µg/mL corresponds to 10.5µg/mL of OTA in spore extract); and 2126µg/mL for AP. The highest applied concentration of AS-OTA(-) spore extract (4940µg/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Single OTA and AS-OTA(+) and combinations (AP+AS-OTA(+) and AP+AS-OTA(-)) in subtoxic concentrations provoked significant primary DNA damage, apoptosis, and to a lesser extent, necrosis in A549 cells. Mixture of AP+AS-OTA(+) and AP+AS-OTA(-) in subtoxic concentrations showed dominant additive interactions. Despite the low calculated daily intake of OTA by inhalation, our results suggest that chronic exposure to high levels of OTA-producing airborne fungi in combination with other more or less toxic moulds pose a significant threat to human health due to their possible additive and/or synergistic interactions.

Effects of Strawberry Tree (Arbutus unedo L.) Aqueous Leaf Extract and Arbutin on PK-15 and HepG2 Cells.

The antioxidant properties of the leaves of the Mediterranean strawberry tree (Arbutus unedo L.) are mainly attributed to the main bioactive compound, the phenolic glycoside arbutin. In this study, the safety profile of strawberry tree aqueous leaf extract (STE) and arbutin at the DNA level was assessed in vitro using porcine PK-15 kidney cells and HepG2 cells derived from human hepatomas. To examine the effects on cell viability and DNA damage, cells were treated for 24 h with STE or arbutin at three concentrations presumed to be non-toxic (400, 200, and 11.4 µg/mL). Assessments were performed using the MTS viability assay, dual acridine orange/ethidium bromide fluorescent staining, and alkaline comet assay. Results showed that the highest concentration (400 µg/mL) of both tested compounds had no significant cytotoxic effects on either PK-15 or HepG2 cells. Apoptosis was the predominant type of cell death and the total amount of DNA damage in treated cells was within acceptable limits. These results on the in vitro cytocompatibility of arbutin and STE with PK-15 and HepG2 cells could serve to make more reliable judgements about safe levels of arbutin in cosmetic products and functional foods, given the increased popularity of the compound in recent years.

Citrinin Provoke DNA Damage and Cell-Cycle Arrest Related to Chk2 and FANCD2 Checkpoint Proteins in Hepatocellular and Adenocarcinoma Cell Lines.

Citrinin (CIT), a polyketide mycotoxin produced by Penicillium, Aspergillus, and Monascus species, is a contaminant that has been found in various food commodities and was also detected in house dust. Several studies showed that CIT can impair the kidney, liver, heart, immune, and reproductive systems in animals by mechanisms so far not completely elucidated. In this study, we investigated the CIT mode of action on two human tumor cell lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma). Cytotoxic concentrations were determined using an MTT proliferation assay. The genotoxic effect of sub-IC50 concentrations was investigated using the alkaline comet assay and the impact on the cell cycle using flow cytometry. Additionally, the CIT effect on the total amount and phosphorylation of two cell-cycle-checkpoint proteins, the serine/threonine kinase Chk2 and Fanconi anemia (FA) group D2 (FANCD2), was determined by the cell-based ELISA. The data were analyzed using GraphPad Prism statistical software. The CIT IC50 for HepG2 was 107.3 µM, and for A549, it was >250 µM. The results showed that sensitivity to CIT is cell-type dependent and that CIT in sub-IC50 and near IC50 induces significant DNA damage and cell-cycle arrest in the G2/M phase, which is related to the increase in total and phosphorylated Chk2 and FANCD2 checkpoint proteins in HepG2 and A549 cells.

Effects of Strawberry Tree Water Leaf Extract and Arbutin on Biochemical Markers and DNA Integrity in Brain Cells of Lewis Rats.

There is growing evidence that arbutin and plant extracts rich in arbutin, such as extracts of the strawberry tree (Arbutus unedo L.), exert a range of beneficial effects, including cyto- and genoprotective properties. This study evaluated the effects of strawberry tree water leaf extract (STE) and arbutin in the brain tissue of Lewis rats. STE or arbutin were administered per os to male and female rats at a dose of 200 mg/kg body weight/day for 14 or 28 days. Treatment outcomes were evaluated using biochemical markers (lipid peroxidation and the activities of the antioxidative enzymes catalase and superoxide dismutase). The effects of the tested substances on DNA integrity in brain cells were evaluated using the alkaline comet assay. The results suggest a high biocompatibility of both tested substances with rat brain tissue. No significant harmful disturbances were observed in the oxidative/antioxidative status or impairments of DNA integrity in the rat brain cells. Nearly all post-treatment values were within tolerable limits as compared to the matched control rats. Such encouraging findings support further research using other subtle biomarkers to clarify the safety aspects of arbutin and STE prior to the development of specific nutraceutical products.

Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population.

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.

Cytotoxic and genotoxic effects of water and sediment samples from gypsum mining area in channel catfish ovary (CCO) cells.

Man-made activities such as mining generate certain amounts of metal contaminated wastes which can reach aquatic environment and cause the serious effects on different organisms and ecosystem. Chemical analysis of the environmental samples is the most direct approach to reveal their pollution status but it cannot always provide information on biological effects to different organisms, including fish. This study was aimed to investigate the in vitro cytotoxicity and genotoxicity of water and sediment samples from gypsum mining area using the channel catfish ovary (CCO) cell line. Results obtained by the WST-1 assay and alkaline comet assay revealed that exposure of CCO cells to the same concentrations of contaminated water and sediment samples caused significant decrease in cell viability and increased DNA damages. Chemical analysis of water and sediment samples showed that increased concentrations of strontium, aluminum and iron were mainly responsible for the observed cytotoxic and genotoxic effects in CCO cells. The study suggested that fish CCO cells could be useful biological test-system for water and sediment cytotoxicity and genotoxicity assessments.

In vitro cytotoxicity assessment of imidazolium ionic liquids: biological effects in fish Channel Catfish Ovary (CCO) cell line.

Increasing interest in the application of ionic liquids as green replacement for volatile organic solvents emphasized the need for the evaluation of their toxic effects at different biological systems in order to reduce the risk for human health and environment. To our knowledge, effects of imidazolium ionic liquids on cellular level of fish cell lines have not been studied yet. The cytotoxicity of imidazolium ionic liquids containing different anions and alkyl chain lengths as the substituent at the cation ring towards the fish CCO cell line was determined by WST-1 proliferation assay. Morphological alterations were examined by fluorescent microscopy using acridine orange/ethidium bromide staining and flow cytometry analysis was also performed. The results showed concentration-dependent cytotoxicity of ionic liquids in CCO cells, related to the type of anion and alkyl chain length, while EC50 values showed moderate to high cytotoxicity of tested imidazolium ionic liquids. Distinct morphological changes observed under fluorescence microscope and data obtained by flow cytometry suggest that the toxicity of imidazolium ionic liquids with longer alkyl chains could be related to necrosis. Results presented in here may be helpful for filling existing gaps of knowledge about ionic liquids toxicity and their impact on aquatic environment.

Combined Toxicity of the Most Common Indoor Aspergilli.

The most common Aspergilli isolated from indoor air samples from occupied buildings and a grain mill were extracted and analyzed for their combined (Flavi + Nigri, Versicolores + Nigri) cytotoxic, genotoxic and pro-inflammatory properties on human adenocarcinoma cells (A549) and monocytic leukemia cells induced in macrophages (THP-1 macrophages). Metabolite mixtures from the Aspergilli series Nigri increase the cytotoxic and genotoxic potency of Flavi extracts in A549 cells suggesting additive and/or synergistic effects, while antagonizing the cytotoxic potency of Versicolores extracts in THP-1 macrophages and genotoxicity in A549 cells. All tested combinations significantly decreased IL-5 and IL-17, while IL-1ß, TNF-α and IL-6 relative concentrations were increased. Exploring the toxicity of extracted Aspergilli deepens the understanding of intersections and interspecies differences in events of chronic exposure to their inhalable mycoparticles.

Cytotoxicity and genotoxicity of versicolorins and 5-methoxysterigmatocystin in A549 cells.

Aspergillus versicolor and A. flavus are primary colonizers in damp dwellings, and they produce sterigmatocystin (ST) and aflatoxin B1 (AFB(1)), respectively. These hepatotoxic and carcinogenic mycotoxins and their precursors and derivates possess a furofuran ring, which has proven responsible for their toxicity. The aim of this study was to investigate the cytotoxicity and genotoxicity of versicolorin A (VER A) and versicolorin B (VER B), as the furofuran precursors of aflatoxins and ST, and of 5-methoxysterigmatocystin (5-MET-ST), a methoxy derivative of ST, in human adenocarcinoma lung cells A549. The IC(50) values of the tested compounds were obtained by the cell proliferation MTT test as follows: 109 ± 3.5 µM (VER A), 172 ± 4 µM (VER B) and 181 ± 2.6 µM (5-MET-ST). The comet assay and micronucleus test were used to assess their genotoxic potential after 24 h of treatment with concentrations corresponding to ½ and » IC(50) in comparison with AFB(1) and ST, applied in concentrations corresponding to ½ IC(50), as previously determined in A549 cells. DNA damage parameters assessed by the comet assay were tail length, tail intensity and tail moment, while the level of DNA damage in the micronucleus test was evaluated by the number of formed micronuclei (MN), nuclear buds (NB) and nucleoplasmic bridges (NPB) in 1,000 binucleated cells. Considering the three comet parameters, all applied toxins exerted significant DNA damage compared to the control, while ST and VER B produced the highest DNA damage. All toxins provoked a statistically significant increase in MN, and a slightly decreased formation of NB and NPB. AFB(1), ST and 20 µM VER A showed a statistically significant increase in all three micronucleus parameters compared to the control, and the highest increase in the number of MN occurred in cells treated with 50 µM VER A. The differences between results obtained by the micronucleus test and comet assay could be explained by the fact that the micronucleus detects irreversible DNA damage, which is usually correlated with the previously determined cytotoxic potential of the AFB(1) precursors.

Evaluation of Toxic Effects Induced by Sub-Acute Exposure to Low Doses of α-Cypermethrin in Adult Male Rats.

To contribute new information to the pyrethroid pesticide α-cypermethrin toxicity profile, we evaluated its effects after oral administration to Wistar rats at daily doses of 2.186, 0.015, 0.157, and 0.786 mg/kg bw for 28 days. Evaluations were performed using markers of oxidative stress, cholinesterase (ChE) activities, and levels of primary DNA damage in plasma/whole blood and liver, kidney, and brain tissue. Consecutive exposure to α-cypermethrin affected the kidney, liver, and brain weight of rats. A significant increase in concentration of the thiobarbituric acid reactive species was observed in the brain, accompanied by a significant increase in glutathione peroxidase (GPx) activity. An increase in GPx activity was also observed in the liver of all α-cypermethrin-treated groups, while GPx activity in the blood was significantly lower than in controls. A decrease in ChE activities was observed in the kidney and liver. Treatment with α-cypermethrin induced DNA damage in the studied cell types at almost all of the applied doses, indicating the highest susceptibility in the brain. The present study showed that, even at very low doses, exposure to α-cypermethrin exerts genotoxic effects and sets in motion the antioxidative mechanisms of cell defense, indicating the potential hazards posed by this insecticide.

Assessment of genotoxic potency of sulfate-rich surface waters on medicinal leech and human leukocytes using different versions of the Comet assay.

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30 min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630 mg/L SO4) and Kosovcica River (823.3 mg/L SO4) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water. The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment.

Homogentisic acid, a main phenolic constituent of strawberry tree honey, protects human peripheral blood lymphocytes against irinotecan-induced cytogenetic damage in vitro.

Homogentisic acid (HGA) is the most abundant phenolic compound in strawberry tree (Arbutus unedo L.) honey and an intermediate in the metabolism of phenylalanine and tyrosine. Since HGA exerts its dual nature (pro-oxidant and antioxidant), which depends on the concentration and cell type, the aim of study was to determine whether HGA possess cytoprotective effects and could counteract the cyto- and genotoxic effects of the antineoplastic drug irinotecan (IRI). Tested concentrations corresponded to HGA content in average daily dose of strawberry tree honey as well as five- and ten-fold higher concentrations. Cyto- and genoprotective effects were tested on human peripheral blood lymphocytes using chromosomal aberrations assay and cytokinesis-block micronucleus cytome assay. HGA, even at concentrations 10-fold higher than the one present in the daily amount of consumed strawberry tree honey, posed a non-significant cytotoxic threat to lymphocytes, had a negligible potential for causing cytogenetic damage in treated cells, and did not significantly impair their proliferation. Results of the chromosomal aberration assay and CBMN Cyt assay also showed that HGA efficiently counteracted the detrimental cytogenetic effects of IRI in vitro. The finding on cyto- and genoprotective effects of HGA merits further research in order to better explain the safety profile of this compound and to assess its potency for the development of novel nutraceutical products.