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.

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.

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.

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.

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 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.

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.

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

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

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.

Sterigmatocystin, 5-Methoxysterigmatocistin, and Their Combinations Are Cytotoxic and Genotoxic to A549 and Hepg2 Cells and Provoke Phosphorylation of Chk2, but Not Fancd2 Checkpoint Proteins.

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are structurally related mycotoxins with cytotoxic and genotoxic properties. In the present study, we hypothesized that DNA damage induced by non-cytotoxic concentrations of single and combined mycotoxins could alter the phosphorylation of the checkpoint proteins Chk2 and FANCD2 (ELISA) in HepG2 and A549 cells. The cytotoxic potential (MTT test) of single and combined STC and 5-M-STC, the nature of their interaction (additivity, antagonism, or synergy) and DNA damage level (alkaline comet assay) in HepG2 and A549 cells were also investigated. All experiments were performed after 24 h of mycotoxin treatment. 5-M-STC was 10-folds more cytotoxic than STC to both HepG2 and A549 cells. Both mycotoxins are genotoxic to HepG2 and A549 cells by inducing both double and single DNA strand breaks that activate Chk2 (especially in HepG2 cells) but not the FANCD2 protein. STC exerted higher genotoxic potential than 5-M-STC in HepG2 and A549 cells when both toxins were applied individually at the same concentration. Dual combinations of non-cytotoxic mycotoxin concentrations showed additive to antagonizing cytotoxic and genotoxic effects. The absence and low activation of checkpoint proteins during prolonged exposure to non-cytotoxic concentrations of STC and 5-M-STC could support cell proliferation and carcinogenesis.

Effects of low-level imidacloprid oral exposure on cholinesterase activity, oxidative stress responses, and primary DNA damage in the blood and brain of male Wistar rats.

Imidacloprid is a neonicotinoid insecticide that acts selectively as an agonist on insect nicotinic acetylcholine receptors. It is used for crop protection worldwide, as well as for non-agricultural uses. Imidacloprid systemic accumulation in food is an important source of imidacloprid exposure. Due to the undisputable need for investigations of imidacloprid toxicity in non-target species, we evaluated the effects of a 28-day oral exposure to low doses of imidacloprid (0.06 mg/kg b. w./day, 0.8 mg/kg b. w./day and 2.25 mg/kg b. w./day) on cholinesterase activity, oxidative stress responses and primary DNA damage in the blood and brain tissue of male Wistar rats. Exposure to imidacloprid did not cause significant changes in total cholinesterase, acetylcholinesterase and butyrylcholinesterase activities in plasma and brain tissue. Reactive oxygen species levels and lipid peroxidation increased significantly in the plasma of rats treated with the lowest dose of imidacloprid. Activities of glutathione-peroxidase in plasma and brain and superoxide dismutase in erythrocytes increased significantly at the highest applied dose. High performance liquid chromatography with UV diode array detector revealed the presence of imidacloprid in the plasma of all the treated animals and in the brain of the animals treated with the two higher doses. The alkaline comet assay results showed significant peripheral blood leukocyte damage at the lowest dose of imidacloprid and dose-dependent brain cell DNA damage. Oral 28-day exposure to low doses of imidacloprid in rats resulted in detectable levels of imidacloprid in plasma and brain tissue that directly induced DNA damage, particularly in brain tissue, with slight changes in plasma oxidative stress parameters.

Protective effects of olive oil phenolics oleuropein and hydroxytyrosol against hydrogen peroxide-induced DNA damage in human peripheral lymphocytes.

This study investigates antioxidant capacity and protective effects of phenolic compounds oleuropein (OLP) and hydroxytyrosol (HT), present in olive oil and olive leaves, against H2O2-induced DNA damage in human peripheral lymphocytes. Antioxidant potency was determined using the measurement of radical-scavenging activity (ABTS∙+ assay), ferric reducing power (FRAP assay) and cupric reducing antioxidant capacity (CUPRAC assay). Both substances were found to be potent antioxidant agents due to their free radical-scavenging activities. Antigenotoxic effects of oleuropein and hydroxytyrosol against H2O2-induced damage in human lymphocytes were evaluated in vitro by alkaline comet assay. At tested concentrations (1, 5, 10 µmol L-1), oleuropein and hydroxytyrosol did not induce a significant increase of primary DNA damage in comparison with the negative control. Pretreatment of human lymphocytes with each of the substances for 120 min produced a dose-dependent reduction of primary DNA damage in the tested cell type. Hydroxytyrosol showed a better protective effect against H2O2-induced DNA breaks than oleuropein which could be associated with their free radical-scavenging efficacy.

Single-Dose Toxicity of Individual and Combined Sterigmatocystin and 5-Methoxysterigmatocistin in Rat Lungs.

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are mycotoxins produced by common damp indoor Aspergilli series Versicolores. Since both STC and 5-M-STC were found in the dust of indoor occupational and living areas, their occupants may be exposed to these mycotoxins, primarily by inhalation. Thus, STC and 5-M-STC were intratracheally instilled in male Wistar rats using doses (0.3 mg STC/kg of lung weight (l.w.); 3.6 mg 5-M-STC/kg l.w.; toxin combination 0.3 + 3.6 mg/kg l.w.) that corresponded to concentrations detected in the dust of damp indoor areas in order to explore cytotoxicity, vascular permeability, immunomodulation and genotoxicity. Single mycotoxins and their combinations insignificantly altered lactate-dehydrogenase activity, albumin, interleukin-6, tumor necrosis factor-α and chemokine macrophage inflammatory protein-1α concentrations, as measured by ELISA in bronchioalveolar lavage fluid upon 24 h of treatment. In an alkaline comet assay, both mycotoxins provoked a similar intensity of DNA damage in rat lungs, while in a neutral comet assay, only 5-M-STC evoked significant DNA damage. Hence, naturally occurring concentrations of individual STC may induce DNA damage in rat lungs, in which single DNA strand breaks prevail, while 5-M-STC was more responsible for double-strand breaks. In both versions of the comet assay treatment with STC + 5-M-STC, less DNA damage intensity occurred compared to single mycotoxin treatment, suggesting an antagonistic genotoxic action.

High Doses of Δ9-Tetrahydrocannabinol Might Impair Irinotecan Chemotherapy: A Review of Potentially Harmful Interactions.

This review proposes the hypothesis that the effectiveness of irinotecan chemotherapy might be impaired by high doses of concomitantly administered Δ9-tetrahydrocannabinol (THC). The most important features shared by irinotecan and THC, which might represent sources of potentially harmful interactions are: first-pass hepatic metabolism mediated by cytochrome P450 (CYP) enzyme CYP3A4; glucuronidation mediated by uridine diphosphate glycosyltransferase (UGT) enzymes, isoforms 1A1 and 1A9; transport of parent compounds and their metabolites via canalicular ATP-binding cassette (ABC) transporters ABCB1 and ABCG2; enterohepatic recirculation of both parent compounds, which leads to an extended duration of their pharmacological effects; possible competition for binding to albumin; butyrylcholinesterase (BChE) inhibition by THC, which might impair the conversion of parent irinotecan into the SN-38 metabolite; mutual effects on mitochondrial dysfunction and induction of oxidative stress; potentiation of hepatotoxicity; potentiation of genotoxicity and cytogenetic effects leading to genome instability; possible neurotoxicity; and effects on bilirubin. The controversies associated with the use of highly concentrated THC preparations with irinotecan chemotherapy are also discussed. Despite all of the limitations, the body of evidence provided here could be considered relevant for human-risk assessments and calls for concern in cases when irinotecan chemotherapy is accompanied by preparations rich in THC.

Evaluation of oxidative stress responses and primary DNA damage in blood and brain of rats exposed to low levels of tembotrione.

Tembotrione is a rather novel pesticide, usually used for post-emergence weed control. Even though its use is rapidly growing, it is not followed by an adequate flow of scientific evidence regarding its toxicity towards non-target organisms. We evaluated the potential of low doses of tembotrione to induce oxidative stress and cytogenetic damage in blood and brain cells of adult male Wistar rats. Parameters of lipid peroxidation, glutathione levels, activities of antioxidant enzymes and primary DNA damage were assessed following 28-day repeated oral exposure to doses comparable with the currently proposed health-based reference values. The results of the alkaline comet assay showed that such low doses of tembotrione have the potency to inflict primary DNA damage in both peripheral blood leukocytes and brain of treated rats, even with only slight changes in the oxidative biomarker levels. The DNA damage in blood and brain cells of Wistar rats significantly increased at all applied doses, suggesting that tembotrione genotoxicity is mainly a result of direct interaction with DNA while the induction of oxidative stress responses contributes to DNA instability in a lesser extent. The findings of the present study call for further research using other sensitive biomarkers of effect and different exposure scenarios.

DNA damage in kidney and parenchymal and non-parenchymal liver cells of adult Wistar rats after subchronic oral treatment with tembotrione.

DNA damage in the liver and kidney cells of adult male Wistar rats was studied using the comet assay after a 28-day oral administration of tembotrione at doses of 0.0007, 0.0013 and 0.7 mg/kg b.w./day [AOEL (acceptable operator exposure level), REL (residual exposure level) and 1000× AOEL]. As a descriptor of DNA damage, tail intensity was used. Antioxidant status was assessed by activity of glutathione peroxidase (GPx). Significant DNA damage was recorded in the kidney cells at all three doses as compared to negative control. In parenchymal liver cells, significant DNA damage was observed in AOEL and 1000× AOEL doses, while in non-parenchymal liver cells, only AOEL-treated group was significantly different compared to negative control. In both types of liver cells, REL and 1000× AOEL doses were significantly different from the AOEL dose. No significant changes in GPx activity compared to control were observed at any exposure level. The results of the present study suggest that repeated in vivo exposure to tembotrione led to low-level DNA instability in kidney and liver cells. Exposure to the highest tembotrione dose showed a relatively weak response with the alkaline comet assay. Further research should focus on the effects of this herbicide in other models along with different exposure scenarios.

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.

The effects of strawberry tree (Arbutus unedo L.) water leaf extract and arbutin upon kidney function and primary DNA damage in renal cells of rats.

Although strawberry tree (Arbutus unedo L.) leaves have long been used as a herbal remedy, insufficient information is available on their nephrotoxicity. We assessed the safety of strawberry tree water leaf extract and its key component arbutin, administered per os to Lewis rats of both genders at 200 mg/kg b.w./day for 14 and 28 days. The effects of the tested compounds on DNA integrity in renal cells was evaluated using alkaline comet assay, while kidney function was studied using serum creatinine and urea levels. Strawberry tree water leaf extract showed high biocompatibility with kidney tissue. It did not impair DNA integrity of renal cells and kidney function, either in male or female rats. However, exposure to single arbutin affected the levels of primary DNA damage in renal cells which could be related to metabolic conversion of arbutin into hydroquinone, whose nephrotoxicity has previously been proven.