Genotoxicity of selected cannabinoids in human lymphoblastoid TK6 cells.

Natural non-psychoactive cannabinoids such as cannabigerol (CBG), cannabidiol (CBD), cannabichromene (CBC), cannabidivarin (CBDV), and cannabinol (CBN) are increasingly consumed as constituents of dietary products because of the health benefits claims. Cannabinoids may reduce certain types of pain, nausea, and anxiety. Anti-inflammatory and even anti-carcinogenic properties have been discussed. However, there are insufficient data available regarding their potential (geno-)toxic effects. Therefore, we tested CBG, CBD, CBC, CBDV, and CBN for their genotoxic potential and effects on mitosis and cell cycle in human lymphoblastoid TK6 cells. The selected cannabinoids (except CBDV) induced increased micronuclei formation, which was reduced with the addition of a metabolic activation system (S9 mix). CBDV induced micronuclei only after metabolic activation. Mitotic disturbances were observed with all tested cannabinoids, while G1 phase accumulation of cells was observed for CBG, CBD and CBDV. The genotoxic effects occurred at about 1000-fold higher concentrations than are reported as blood levels from human consumption. However, the results clearly indicate a need for further research into the genotoxic effects of cannabinoids. The mechanism of the mitotic disturbance, the shape of the dose-response curves and the possible effects of mixtures of cannabinoids are aspects which need clarification.

Presence of micronuclei and nuclear abnormalities in Caracara (Polyborus) plancus living in an airport area in southern Brazil.

The aviation sector is believed to be responsible for considerable environmental damage attributed to emission of a large number and amount of pollutants. Airports are often surrounded by forest fragments and humid areas that attract birds of prey and hence may potentially serve as useful bioindicators. The aim of the present study was to examine genotoxic potential in raptors exposed to airport pollution using the micronucleus (MN) test and morphological changes as evidenced by bilateral symmetry. This investigation was conducted at Salgado Filho International Airport of Porto Alegre - RS as well as in private and zoological breeding grounds. The presence of metals was measured in the blood cells of the collected birds. Seventeen birds (Caracara (Polyborus) plancus) were used in this study 11 from exposed and 6 from non-exposed group. The nuclear alterations clearly indicate that organisms exposed to airport pollution exhibited a significantly higher frequency of genetic damage compared to non-exposed birds. Further, manganese and chromium were detected exclusively in the blood of the exposed group. In contrast, the analysis of bilateral symmetry did not detect any significant morphologic differences between the two groups. Therefore, data indicate that blood genotoxic stress occurs in birds of prey living in civil aviation areas as evidenced by MN frequency increase and presence of manganese and chromium.

Nabumetone induced photogenotoxicity mechanism mediated by ROS generation under environmental UV radiation in human keratinocytes (HaCaT) cell line.

Nabumetone (NB) is a non-steroidal anti-inflammatory drug (NSAID), prescribed for managing pain associated with acute/chronic rheumatoid arthritis, osteoarthritis and other musculoskeletal disorders. Though some incidences of photosensitivity have been reported, there is limited information available on its phototoxicity potential. In this study, NB photodegraded in a time-dependant manner (0-4 h) under UVA (1.5 mW/cm2), UVB (0.6 mW/cm2) and natural sunlight as observed through UV-vis spectrophotometer and the results were further confirmed with Ultra High-Performance Liquid Chromatography (UHPLC). Photosensitized NB generated reactive oxygen species (ROS) as observed by lipid peroxidation, suggesting oxidative degradation of lipids in cell membrane, thereby resulting in cell damage. MTT and NRU (neutral red uptake) assays revealed that NB induced phototoxicity in concentration-dependent manner (0.5, 1, 5, 10 µg/ml) under UVA, UVB and sunlight exposure (30 min) in human keratinocytes cell line (HaCaT), with significant phototoxicity at the concentration of 5 µg/ml. Photosensitized NB generated intracellular ROS, disrupted mitochondrial and lysosomal membrane integrity, resulting in cell death. UV-induced genotoxicity by NB was confirmed through micronuclei generation, γ-H2AX induction and cyclobutane pyrimidine dimer formation. This is the first study which showed the phototoxicity and photogenotoxicity potential of NB in HaCaT cell line. We also observed that photosensitized NB upregulated inflammatory markers, such as COX-2 and TNFα. This study proposes that sunlight exposure should be avoided by patients using nabumetone and proper guidance should be provided by clinicians regarding photosensitivity of drugs for better safety and efficacy.

Manool, a diterpene from Salvia officinalis, exerts preventive effects on chromosomal damage and preneoplastic lesions.

The present study aimed to evaluate the effect of the manool diterpene on genomic integrity. For this purpose, we evaluated the influence of manool on genotoxicity induced by mutagens with different mechanisms of action, as well as on colon carcinogenesis. The results showed that manool (0.5 and 1.0 µg/ml) significantly reduced the frequency of micronuclei induced by doxorubicin (DXR) and hydrogen peroxide in V79 cells but did not influence genotoxicity induced by etoposide. Mice receiving manool (1.25 mg/kg) exhibited a significant reduction (79.5%) in DXR-induced chromosomal damage. The higher doses of manool (5.0 and 20 mg/kg) did not influence the genotoxicity induced by DXR. The anticarcinogenic effect of manool (0.3125, 1.25 and 5.0 mg/kg) was also observed against preneoplastic lesions chemically induced in rat colon. A gradual increase in manool doses did not cause a proportional reduction of preneoplastic lesions, thus demonstrating the absence of a dose-response relationship. The analysis of serum biochemical indicators revealed the absence of hepatotoxicity and nephrotoxicity of treatments. To explore the chemopreventive mechanisms of manool via anti-inflammatory pathways, we evaluated its effect on nitric oxide (NO) production and on the expression of the NF-kB gene. At the highest concentration tested (4 µg/ml), manool significantly increased NO production when compared to the negative control. On the other hand, in the prophylactic treatment model, manool (0.5 and 1.0 µg/ml) was able to significantly reduce NO levels produced by macrophages stimulated with lipopolysaccharide. Analysis of NF-kB in hepatic and renal tissues of mice treated with manool and DXR revealed that the mutagen was unable to stimulate expression of the gene. In conclusion, manool possesses antigenotoxic and anticarcinogenic effects and its anti-inflammatory potential might be related, at least in part, to its chemopreventive activity.

Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001-770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the "misleading" in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

Protective effects of Bidens pilosa on hepatoxicity and nephrotoxicity induced by carbon tetrachloride in rats.

The aim of this study was to assess the protective effects of oral and topical treatment with Bidens pilosa (BP) against carbon tetrachloride (CCl4)- induced toxicity. Fifty-six rats were divided into seven groups: A: CCl4 only; B: CCl4+oral BP; C: CCl4 and topical BP; D: CCl4+oral and topical BP; E: oral BP only; F: negative control; and G: positive control (cyclophosphamide). The animals were treated for 10 weeks. Blood samples were collected for tests of hepatic and renal function, and fragments of the liver, spleen, pancreas, kidney, and intestine were collected for histopathological analyses. Cells from the femoral bone marrow were used for a micronucleus test and 'comet assay'. Statistically significant differences were observed in the levels of gamma-glutamyl transpeptidase (GGT), albumin, urea and creatinine, hepatic inflammation, renal tubular lesion, and inflammation of the intestinal mucosa between the BP-treated groups and untreated group. The median number of micronuclei in group A was 4.00, in group G was 9.00 and in the other groups was 0.00. Group A had the lowest number of cells with a score of 0 and the greatest number with scores of 3 and 4, similar to the results obtained from group G using the 'comet assay'. Thus, BP effectively protected against the toxic effects of CCl4 on the liver, kidney, and intestine and exerted an antimutagenic effect on rats exposed to CCl4.

Oxidative Stress, Mutations and Chromosomal Aberrations Induced by In Vitro and In Vivo Exposure to Furan.

Furan is a volatile compound that is formed in foods during thermal processing. It is classified as a possible human carcinogen by international authorities based on sufficient evidence of carcinogenicity from studies in experimental animals. Although a vast number of studies both in vitro and in vivo have been performed to investigate furan genotoxicity, the results are inconsistent, and its carcinogenic mode of action remains to be clarified. Here, we address the mutagenic and clastogenic activity of furan and its prime reactive metabolite cis-2 butene-1,4-dial (BDA) in mammalian cells in culture and in mouse animal models in a search for DNA lesions responsible of these effects. To this aim, Fanconi anemia-derived human cell lines defective in the repair of DNA inter-strand crosslinks (ICLs) and Ogg1-/- mice defective in the removal of 8-hydroxyguanine from DNA, were used. We show that both furan and BDA present a weak (if any) mutagenic activity but are clear inducers of clastogenic damage. ICLs are strongly indicated as key lesions for chromosomal damage whereas oxidized base lesions are unlikely to play a critical role.

Molecular Consequences of Depression Treatment: A Potential In Vitro Mechanism for Antidepressants-Induced Reprotoxic Side Effects.

The incidence of depression among humans is growing worldwide, and so is the use of antidepressants. However, our fundamental understanding regarding the mechanisms by which these drugs function and their off-target effects against human sexuality remains poorly defined. The present study aimed to determine their differential toxicity on mouse spermatogenic cells and provide mechanistic data of cell-specific response to antidepressant and neuroleptic drug treatment. To directly test reprotoxicity, the spermatogenic cells (GC-1 spg and GC-2 spd cells) were incubated for 48 and 96 h with amitriptyline (hydrochloride) (AMI), escitalopram (ESC), fluoxetine (hydrochloride) (FLU), imipramine (hydrochloride) (IMI), mirtazapine (MIR), olanzapine (OLZ), reboxetine (mesylate) (REB), and venlafaxine (hydrochloride) (VEN), and several cellular and biochemical features were assessed. Obtained results reveal that all investigated substances showed considerable reprotoxic potency leading to micronuclei formation, which, in turn, resulted in upregulation of telomeric binding factor (TRF1/TRF2) protein expression. The TRF-based response was strictly dependent on p53/p21 signaling and was followed by irreversible G2/M cell cycle arrest and finally initiation of apoptotic cell death. In conclusion, our findings suggest that antidepressants promote a telomere-focused DNA damage response in germ cell lines, which broadens the established view of antidepressants' and neuroleptic drugs' toxicity and points to the need for further research in this topic with the use of in vivo models and human samples.

Genomic instability in Buccal mucosal cells of children living in abnormal conditions from Santos-Sao Vicente Estuary.

The aim of this study was to evaluate genomic instability and cytotoxicity in buccal mucosa cells of children living in abnormal conditions from Santos Sao Vicente estuary. The study area is located between coordinates 23°58'11.8"S and 46°24'26.3"W, in the southwestern zone of the Sao Paulo State, Brazil. A total of 40 children was distributed into two groups: exposed and non-exposed groups. The frequency of micronuclei increased to buccal mucosa cells of children living in Santos Sao Vicente estuary when compared to the non-exposed group (p < 0.05). No remarkable differences on buccal cells were found inpyknosis, karyorrhexis and karyolysi between groups. Taken together, our results suggest that children living in contaminated areas comprise a high group for genomic instability on buccal mucosa cells. Given that the current investigation is a preliminary study, further analysis with a larger sample of children is interesting as a future perspective.

Assesment of hematotoxic, oxidative and genotoxic damage potentials of fipronil in rainbow trout Oncorhynchus mykiss, Walbaum.

In this study, changes in the blood tissue of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) caused by Fipronil (FP) insecticide were investigated using different biomarkers (Hematology parameters, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), paraoxonase (PON), arylesterase (ARE), myeleperoxidase (MPO), micronucleus (MN), 8-hydroxy-2-deoxyguanosine (8-OHdG)) level and caspase-3 activity. Statistically significant alterations in hematology parameters occurred with FP effect. In blood tissue, dose-dependent inhibition was determined in SOD-CAT-GPX-PON and ARE enzyme activities, but MDA and MPO were induced statistically significant. The results of MN assay were compared with the control group and it was obtained that genotoxicity of different dose groups was similar. The level of 8-OHdG and the activity and caspase-3 examined in blood tissue was increased depending on the dose. It was determined with different biomarkers that this insecticide caused physiological stress changes in the tissues examined.

Can a digital slide scanner and viewing technique assist the visual scoring for the cytokinesis-block micronucleus cytome assay?

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring.

Melatonin supplementation over different time periods until ageing modulates genotoxic parameters in mice.

The ageing process is a multifactorial phenomenon, associated with decreased physiological and cellular functions and an increased propensity for various degenerative diseases. Studies on melatonin (N-acetyl-5-methoxytryptamine), a potent antioxidant, are gaining attention since melatonin production declines with advancing age. Hence, the aim of this study was to evaluate the effects of chronic melatonin consumption on genotoxic and mutagenic parameters of old Swiss mice. Herein, 3-month-old Swiss albino male mice (n = 240) were divided into eight groups and subdivided into two experiments: first (three groups): natural ageing experiment; second (five groups): animals that started water or melatonin supplementation at different ages (3, 6, 12 and 18 months) until 21 months. After 21 months, the animals from the second experiment were euthanized to perform the comet assay, micronucleus test and western blot analysis. The results demonstrated that melatonin prolonged the life span of the animals. Relative to genomic instability, melatonin was effective in reducing DNA damage caused by ageing, presenting antigenotoxic and antimutagenic activities, independently of initiation age. The group receiving melatonin for 18 months had high levels of APE1 and OGG1 repair enzymes. Conclusively, melatonin presents an efficient antioxidant mechanism aiding modulating genetic and physiological alterations due to ageing.

Detection of urethane-induced genotoxicity in vitro using metabolically competent human 2D and 3D spheroid culture models.

In vitro genotoxicity studies are a quick and high throughput approach to assess the genotoxic potential of chemicals; however, the reliability of these tests and their relevance to in vivo effects depends on the choice of representative cell line and optimisation of assay conditions. For chemicals like urethane that require specific metabolic activation to cause genotoxicity, it is important that in vitro tests are conducted using cell lines exhibiting the activity and induction of CYP450 enzymes, including CYP2E1 enzyme that is important in the metabolism of urethane, at a concentration representing actual or perceived chemical exposure. We compared 2D MCL-5 cells and HepG2 cells with 3D HepG2 hanging drop spheroids to determine the genotoxicity of urethane using the micronucleus assay. Our 2D studies with MCL-5 did not show any statistically significant genotoxicity [99% relative population doubling (RPD)] compared to controls for concentrations and time point tested in vitro. HepG2 cells grown as 2D indicated that exposure to urethane of up to 30 mM for 23 h did not cause any genotoxic effect (102% RPD) but, at higher concentrations, genotoxicity was produced with only 89-85% RPD. Furthermore, an exposure of 20-50 mM for 23 h using 3D hanging drop spheroid assays revealed a higher MN frequency, thus exhibiting in vitro genotoxicity of urethane in metabolically active cell models. In comparison with previous studies, this study indicated that urethane genotoxicity is dose, sensitivity of cell model (2D vs. 3D) and exposure dependent.

Evaluation of the genotoxic effect of heavy metals in pigeons from urban and rural habitat in Monterrey, Mexico, using the chromatin dispersion assay.

PURPOSE: Evaluate genotoxic effect of heavy metals on Pigeon Erythrocytes (PE) from urban and rural habitat (outside of the city) in Monterrey, Mexico, using the chromatin dispersion assay. MATERIALS AND METHODS: We quantified metals concentrations (Cd, Hg, Cu and Pb) in tail feathers of 22 pigeons from an urban and a rural site in northeastern Mexico. DNA damage in peripheral blood erythrocytes was measured by chromatin dispersion assay in 13 pigeon living in urban habitat and in nine living in rural habitat as the control. MicroNucleus (MN) test was used to confirm levels of DNA damage. RESULTS: Birds in urban habitat had highest concentrations in feathers for all the metallic elements analysed with respect to birds in rural habitat. Concentrations of Cu and Hg showed a significant increase (p < 0.05). Our results showed a significant increase of DNA damage in urban-habitat pigeons compared with that of pigeons in rural area. These results were confirmed by a MN test. CONCLUSIONS: Our preliminary findings demonstrate that PE examination via chromatin dispersion assay is a reliable, precise and inexpensive morphological bioassay for evaluating environmental genotoxicity associated with heavy metals. Further studies for evaluating the individual participation of contaminants in DNA damage are needed.

LiCoO2 particles used in Li-ion batteries induce primary mutagenicity in lung cells via their capacity to generate hydroxyl radicals.

BACKGROUND: Li-ion batteries (LIB) are used in most portable electronics. Among a wide variety of materials, LiCoO2 (LCO) is one of the most used for the cathode of LIB. LCO particles induce oxidative stress in mouse lungs due to their Co content, and have a strong inflammatory potential. In this study, we assessed the mutagenic potential of LCO particles in lung cells in comparison to another particulate material used in LIB, LTO (Li4Ti5O12), which has a low inflammatory potential compared to LCO particles. RESULTS: We assessed the mutagenic potential of LCO and LTO particles in vitro by performing a cytokinesis-block micronucleus (MN) assay with rat lung epithelial cells (RLE), as well as in vivo in alveolar type II epithelial (AT-II) cells. LCO particles induced MN in vitro at non-cytotoxic concentrations and in vivo at non-inflammatory doses, indicating a primary genotoxic mechanism. LTO particles did not induce MN. Electron paramagnetic resonance and terephthalate assays showed that LCO particles produce hydroxyl radicals (•OH). Catalase inhibits this •OH production. In an alkaline comet assay with the oxidative DNA damage repair enzyme human 8-oxoguanine DNA glycosylase 1, LCO particles induced DNA strand breaks and oxidative lesions. The addition of catalase reduced the frequency of MN induced by LCO particles in vitro. CONCLUSIONS: We report the mutagenic activity of LCO particles used in LIB in vitro and in vivo. Our data support the role of Co(II) ions released from these particles in their primary genotoxic activity which includes the formation of •OH by a Fenton-like reaction, oxidative DNA lesions and strand breaks, thus leading to chromosomal breaks and the formation of MN. Documenting the genotoxic potential of the other LIB particles, especially those containing Co and/or Ni, is therefore needed to guarantee a safe and sustainable development of LIB.

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells.

1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 µM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration-response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration-response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.

Ex vivo/in vitro protective effect of myricetin bulk and nano-forms on PhIP-induced DNA damage in lymphocytes from healthy individuals and pre-cancerous MGUS patients.

2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is a central dietary mutagen, produced when proteinaceous food is heated at very high temperatures potentially causing DNA strand breaks. This study investigates the protective potential of a well-researched flavonoid, myricetin in its bulk and nano-forms against oxidative stress induced ex vivo/in vitro by PhIP in lymphocytes from pre-cancerous monoclonal gammopathy of undetermined significance (MGUS) patients and those from healthy individuals. The results from the Comet assay revealed that in the presence of myricetin bulk (10 µM) and myricetin nano (20 µM), the DNA damage caused by a high dose of PhIP (100 µM) was significantly (P < 0.001) reduced in both groups. However, nano has shown better protection in lymphocytes from pre-cancerous patients. Consistent results were obtained from the micronucleus assay where micronuclei frequency in binucleated cells significantly decreased upon supplementing PhIP with myricetin bulk (P < 0.01) and myricetin nano (P < 0.001), compared to the PhIP treatment alone. To briefly determine the cellular pathways involved in the protective role of myricetin against PhIP, we studied gene expression of P53 and ATR kinase (ATM- and Rad3-related), using the real-time PCR technique.

The genotoxicity of an organic solvent mixture: A human biomonitoring study and translation of a real-scenario exposure to in vitro.

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

Mutagenic, genotoxic and cytotoxic studies of invasive corals Tubastraea coccinea and Tubastraea tagusensis.

The high diversity of species in the marine environment gives rise to compounds with unique structural patterns not found as natural products in other systems and with great potential for pharmacological, cosmetic and nutritional use. The genus Tubastraea (Class Anthozoa, Order Scleractinia, Family Dendrophylliidae) is characterized as a hard coral without the presence of zooxanthellae. In species of this genus alkaloids derived from the compound aplysinopsin with pharmacological activity are known. In Brazil T. coccinea and T. tagusensis are characterized as non-indigenous and invasive and are currently found along the Brazilian coast, from Santa Catarina to Bahia states. This study aims to analyze the mutagenic, cytotoxic and genotoxic potential of methanolic and ethanolic extracts from T. coccinea and T. tagusensis collected in Ilha Grande Bay, Rio de Janeiro state, Brazil. Bacterial reverse mutation assay on the standard strains TA97, TA98, TA100, TA102 and TA104, in vitro micronucleus formation test and colorimetric assays for cytotoxic signals on the cell lines HepG2 and RAW264.7 were used. We also synthesized an oxoaplysinopsin derivate alkaloid (APL01) for comparative purposes. No mutagenic (250; 312.5; 375; 437.5 and 500 µg/plate) or genotoxic (0.05; 0.5; 5.0; 50 and 500 µg/mL) effects were observed in any sample tested for all measured concentrations. Cytotoxic responses were observed for eukaryotic cells in all tested samples at 500 and 5000 µg/mL concentrations. Cytotoxicity found in the WST-1 assay was independent of the metabolism of substances present in samples compositions. The cytotoxicity observed in the LDH release assay depended on metabolism.

Use of a common European approach for nanomaterials' testing to support regulation: a case study on titanium and silicon dioxide representative nanomaterials.

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.