Single Cell Gel Research Articles

Genotoxic Effects of Rice-Agrochemicals on Channa punctatus (Bloch) and Cyprinus carpio (Linnaeus) Using Micronucleus Assay and Alkaline Single Cell Gel Electrophoresis

Rice-cum-fish culture is a cost-effective practice for marginal farmers but the major constraint is the indiscriminate use of agrochemicals. Present work was designed to assess the genotoxic effects of rice agrochemicals in Channa punctatus (Experiment 1 CP1 to CP3) and Cyprinus carpio (Experiment 2 CC1 to CC3); using micronucleus, chromosome aberration, and single cell gel electrophoresis/Comet assay. Two experiments with three treatments (CP1/CC1: without pesticide; CP2/CC2: recommended doses; CP3/CC3: farmers’ dose) were maintained in triplicates. The presence of tail DNA and micronuclei depicted significant DNA damage (P<0.05) in all the treated fish. The mean percent frequency of MN showed significant (P<0.05) differences with respect to the initial. The chromosomal aberrations and mean frequencies of tail DNA (%) were significantly abundant in CP3 and CC3 indicating high a genotoxic effect. Keeping in view the low genotoxic effects, treatment of CP2 and CC2 with recommended doses of pesticides may be disseminated to farmers.

In vivo and in vitro analysis of cytogenotoxicity in populations living in abnormal conditions from Santos-Sao Vicente estuary.

The aim of the study was to evaluate cyto- and genotoxic effects in populations living in subnormal clusters in Santos São Vicente estuary. For in vivo study, samples of buccal mucosa and peripheral blood cells were collected. Micronucleus assay and single-cell gel (comet) assay were performed. For in vitro study, Chinese ovary hamster (CHO) cells were exposed to contaminated water. The results showed that people living in the contaminated estuary have increased DNA damage in oral mucosa and peripheral blood cells, as detected in the micronucleus and comet assays respectively. In addition, estuarine water was able to promote cytotoxicity at the highest concentrations, as well as decrease the number of cells in the G1 phase. In summary, our results indicate that water from the Santos-São Vicente estuary is capable of inducing cytogenotoxicity in mammalian cells in vivo and in vitro.

Abstract 2562: Functional characterization of Polθ in esophageal squamous cell carcinoma

Abstract Introduction: Wild-type (WT) Polθ, encoded by POLQ gene, is a specialized DNA polymerase which helps to protect cells against genomic instability by allowing DNA double-strand break (DSB) repairs through the alternative end-joining (AltEJ) pathway. In our previous targeted gene sequencing studies, we identified a high frequency of POLQ germline variants associated with esophageal squamous cell carcinoma (ESCC) in an endemic high-risk region of China. Here in this study, we aim to determine the functional impact of POLQ in ESCC, examine the synthetic lethality relationship of POLQ-mediated AltEJ DNA repair pathway and the canonical homologous recombination (HR) pathway, and eventually explore its potential roles as a biomarker or therapeutic target in ESCC. Methods: To determine the functional roles of Polθ, POLQ knock out (KO), FANCD2 KO and POLQ/FANCD2 double KO ESCC cell lines were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique. MTT assay was used to assess cell viabilities with or without additional treatments that induced DNA damage or replication stress. DSB repair efficiency was examined by the H2AX foci recovery assay. The single cell gel electrophoresis/Comet assay was used to evaluate the genome stability. The expression level of POLQ was compared between ESCC patients’ samples and paired non-tumoral tissues using Q-PCR. Results: Among all ESCC cell lines we tested, 8 out of 13 (61.5%) exhibited higher levels of POLQ mRNA/protein than the normal immortalized esophageal epithelial cell line NE1. A 2-fold or more elevation of POLQ was found in 17 out of 28 Hong Kong ESCC patients’ tumor samples when compared with their paired non-tumoral tissues. Depletion of POLQ in high-POLQ expressing ESCC cell lines (KYSE180TS and SLMTorT3) only slightly diminished the cell viability. Double KO POLQ and FANCD2, one of the HR genes, significantly sabotaged cell proliferation, as compared with single KO POLQ or FANCD2, especially after treated with Cisplatin or Hydroxyurea. The percentage of H2AX foci positive cells increased considerably upon POLQ KO at 8 hours post 4Gy ionizing radiation, hinting the compromised DSB repair efficiency in POLQ depleted cells. The POLQ-deficient cells also showed a higher degree of genomic instability as suggested by the Comet assay. Conclusions: The results suggested that Polθ is overexpressed in ESCC tumors and cell lines. The Polθ mediated DSB repair pathway may work as a backup plan of HR to prevent the genomic instability in ESCC. Targeting Polθ might be a potential therapeutic approach for the better management of HR-deficient ESCC. Acknowledgments: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL. We thank Prof. Jean-Sébastien Hoffmann for providing the Polθ antibody. Citation Format: Jian Li, Josephine Ko, Wei Dai, Lvwen Ning, Hoi Yan Ng, Valen Zhuoyou Yu, Maria Lung. Functional characterization of Polθ in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2562.

Escherichia Coli Outer Membrane Vesicles Induced DNA Double-Strand Breaks in Intestinal Epithelial Caco-2 Cells.

BackgroundRecent studies have shown that Escherichia coli induced digestive tract diseases may be related to outer membrane vesicles (OMVs) induced intestinal double-strand breaks (DSBs) in intestinal epithelial cells. This study aimed to compare the impact of OMVs forces on DSBs in intestinal epithelial Caco-2 cells, and provide a new treatment for digestive diseases caused by E. coli.Material/MethodsE.coli OMVs were prepared and co-cultured with Caco-2 cells. The uptake of OMVs by Caco-2 cells was observed by confocal microscopy. The γ-H2AX protein was detected by western-blots. The DSBs caused by OMVs was detected by single cell gel electrophoresis.ResultsThe particle size analyzer showed that the average diameters of OMVs centrifuged at 20 000×g and 50 000×g were 217.5±7.29 nm and 186.3±6.59 nm (P<0.05), respectively. Transmission electron microscopy of the OMVs revealed a lipid bilayer structure with a variety of different sizes. Confocal fluorescence microscopy revealed that OMVs almost completely entered Caco-2 cells after 24 hours. The ratio of γ-H2AX protein band gray value normalized data in the OMVs centrifuged at 20 000×g and 50 000×g, and the control group (without OMVs) were 2.23±0.18, 1.58±0.20, 1±0.30 (P<0.05), respectively, while DNA levels of the comet tail (TailDNA%, TDNA%) were 72.21±14.61%, 23.11±4.98%, and 1.02±1.41% (P<0.05), respectively. The corresponding DNA damage was categorized as high (grade 3), moderate (grade 2), and no damage (grade 0).ConclusionsDifferent sizes of OMVs induced different degrees of DNA damage in intestinal epithelial Caco-2 cells.

Abstract 1357: Functional characterization of FANCD2 in esophageal squamous cell carcinoma

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in Northern China, where there is evidence for a significant familial association. We performed targeted next-generation sequencing (NGS) analysis on familial ESCC germline samples compared to non-cancer controls from the same high-risk region and compiled a list of candidate cancer predisposition genes. Interestingly, genes related to the Fanconi Anemia (FA) - BRCA pathway are enriched in the list. Among these FA-BRCA genes, Fanconi anemia complementation group D2 (FANCD2) was one of the top candidates, as it also had a high frequency of somatic mutations in ESCC tumor specimens. Therefore, we aim to characterize the role of FANCD2 in tumor development and explore its translational value. Methods: We knocked out the FANCD2 gene in ESCC cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to evaluate its potential oncogenic function in ESCC. Cell proliferation was measured by a MTT 2D clonogenic assay in vitro. Subcutaneous injection of the FANCD2 knockout ESCC cells into BALB/c-nude mice in vivo was performed to assess its functional impact on tumorigenesis. The single cell gel electrophoresis/comet assay was used to investigate the genome stability. Results: The FANCD2 knockout efficiency was confirmed by western blotting. Surprisingly, in vitro functional analyses showed that ESCC cells with FANCD2 knockout survive, with a greatly reduced growth rate and colony-forming ability. Consistent with the in vitro data, ESCC cells with FANCD2 knockout form significantly smaller subcutaneous tumors in nude mice. By applying the comet assay to examine the genome integrity, ESCC cells with FANCD2 knockout show significantly greater damage to the genome. Conclusion: These results suggest that FANCD2 plays an important role in supporting ESCC tumor growth. We attribute this to its core function in DNA repair ability and genome integrity maintenance. Acknowledgement: We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative Research Fund (C7031.15G to M.L.L.). Citation Format: Lisa Chan Lei, Valen Zhuoyou Yu, Lvwen Ning, Josephine Mun-Yee Ko, Li Dong Wang, Maria Li Lung. Functional characterization of FANCD2 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1357.

The Ameliorative Effects of Pycnogenol® on Liver Ischemia-Reperfusion Injury in Rats.

Pycnogenol® (PYC®), a standardized extract from the bark of Pinus maritima, consists of different phenolic compounds. PYC® has shown to have protective effects on chronic diseases such as diabetes, asthma, cancer, and immune disorders. The aim of this study was to determine the effects of PYC® against the DNA damage and biochemical changes in blood, liver, and lung tissues of ischemia-reperfusion (IR)-induced Wistar albino rats. A sham group, IR injury-induced group, and IR+PYC® group were formed. Ischemia was induced and sustained for 45 min, then the ischemic liver was reperfused, which was sustained for a further 120 min at the end of this period. After anesthesia and before the IR inducement, 100 mg/kg PYC® was given to the IR+PYC® group through intraperitoneal injections. The total oxidant (TOS) and total antioxidant status (TAS), total thiol levels (TTL), advanced oxidation protein products (AOPP), and biochemical parameters [myeloperoxidase (MPO), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH)] in the rats were analyzed using spectrophotometric methods and DNA damage was assessed using single-cell gel electrophoresis. The levels of TOS, TTL, MPO, AOPP, ALT, AST, and LDH were significantly decreased in the IR+PYC® group compared with the IR group (p<0.05). The levels of TAS were significantly increased in the IR+PYC® group compared with the IR group (p<0.05). PYC® reduced the DNA damage when compared with the IR group (p<0.05). The present results suggest that PYC® treatment might have a role in the prevention of IR-induced oxidative damage by decreasing DNA damage and increasing antioxidant status.

The GH/IGF-1 axis in a critical period early in life determines cellular DNA repair capacity by altering transcriptional regulation of DNA repair-related genes: implications for the developmental origins of cancer.

Experimental, clinical, and epidemiological findings support the concept of developmental origins of health and disease (DOHAD), suggesting that early-life hormonal influences during a sensitive period around adolescence have a powerful impact on cancer morbidity later in life. The endocrine changes that occur during puberty are highly conserved across mammalian species and include dramatic increases in circulating GH and IGF-1 levels. Importantly, patients with developmental IGF-1 deficiency due to GH insensitivity (Laron syndrome) do not develop cancer during aging. Rodents with developmental GH/IGF-1 deficiency also exhibit significantly decreased cancer incidence at old age, marked resistance to chemically induced carcinogenesis, and cellular resistance to genotoxic stressors. Early-life treatment of GH/IGF-1-deficient mice and rats with GH reverses the cancer resistance phenotype; however, the underlying molecular mechanisms remain elusive. The present study was designed to test the hypothesis that developmental GH/IGF-1 status impacts cellular DNA repair mechanisms. To achieve that goal, we assessed repair of γ-irradiation-induced DNA damage (single-cell gel electrophoresis/comet assay) and basal and post-irradiation expression of DNA repair-related genes (qPCR) in primary fibroblasts derived from control rats, Lewis dwarf rats (a model of developmental GH/IGF-1 deficiency), and GH-replete dwarf rats (GH administered beginning at 5weeks of age, for 30days). We found that developmental GH/IGF-1 deficiency resulted in persisting increases in cellular DNA repair capacity and upregulation of several DNA repair-related genes (e.g., Gadd45a, Bbc3). Peripubertal GH treatment reversed the radiation resistance phenotype. Fibroblasts of GH/IGF-1-deficient Snell dwarf mice also exhibited improved DNA repair capacity, showing that the persisting influence of peripubertal GH/IGF-1 status is not species-dependent. Collectively, GH/IGF-1 levels during a critical period during early life determine cellular DNA repair capacity in rodents, presumably by transcriptional control of genes involved in DNA repair. Because lifestyle factors (e.g., nutrition and childhood obesity) cause huge variation in peripubertal GH/IGF-1 levels in children, further studies are warranted to determine their persisting influence on cellular cancer resistance pathways.

Acute crack cocaine exposure induces genetic damage in multiple organs of rats.

Crack cocaine is a very toxic product derived from cocaine. The aim of this study was to evaluate genetic damage in multiple organs of rats following acute exposure to crack cocaine. A total of 20 Wistar rats were distributed into four groups (n = 5), as follows: 0, 4.5, 9, and 18mg/kg body weight (b.w.) of crack cocaine administered by intraperitonealroute (i.p.). All animals were killed 24h after intraperitoneal (i.p.) injection. The results showed that crack cocaine increased the number of micronucleated cells in bone marrow cells exposed to 18mg/kg crack cocaine (p < 0.05). Peripheral blood and liver cells presented genetic damage as depicted by single cell gel (comet) assay at 9 and 18mg/kg doses (p < 0.05). Immunohistochemistry data revealed significant increase in 8-hydroxy-20-deoxyguanosine (8-OHdG) immunoexpression in hepatocytes of animals exposed to crack cocaine at 9 and 18mg/kg (p < 0.05) when compared with negative controls. Taken together, our results demonstrate that crack cocaine is able to induce genomic damage in multiple organs of Wistar rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin: Genotoxicity and Oxidative Damage Potential in Human Peripheral Blood Lymphocytes

Dioxin-like compounds, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzo-furans (PCDFs) and dioxin-like polychlorinated biphenyls (PCBs) are a widespread and diverse group of persistent, lipophilic and hazardous environmental pollutants. Additionally, because they are by-products of chlorine- containing manufacturing process and incineration, they represent a serious environmental problem. In this research study; we investigated the genotoxic and oxidative effects of TCDD using single cell gel electrophoresis/COMET assay and measuring levels of catalase, superoxide dismutase enzymes, and malondialdehyde values for lipid peroxidation in peripheral blood lymphocyte cultures at three different doses. Blood samples were taken from healthy non-smoking male subjects by venipuncture. In this study, the three doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin were used as 62.5 ng/ml, 31.25 ng/ml, 15.625 ng/ml. In comet assay, two different parameters were evaluated. Damaged cell percent (DCP). Genetic damage index (GDI). Both GDI and DCP significantly increased in a dose-dependent manner at statistical level. There is no statistically significant difference the levels of catalase, superoxide dismutase enzymes, malondialdehyde values compared with negative control.

Genotoxicity and mutagenicity induced by acute crack cocaine exposure in mice

Context: Crack cocaine is an illicit drug derived from cocaine, in which use and abuse have increased around the world, especially in developing countries. Objectives: The aim of this study was to evaluate genomic damage in multiple organs of mice following acute exposure to crack cocaine. For this purpose, single cell gel (comet) assay in peripheral blood, liver, kidney, and brain cells was performed and micronucleus test for bone narrow and liver cells was also made in this setting. Material and methods: A total of 20 C57BL/10 male mice were distributed into four groups, as follows: 0, 4.5, 9, and 18 mg/kg b.w. of crack cocaine dissolved to 1% dimethyl sulfoxide by intraperitoneal (i.p.) route. All animals were sacrificed 24 h after i.p. injection. Results: The results showed that crack cocaine induced DNA damage in peripheral blood, and brain cells for higher doses used as depicted by single cell gel (comet) assay data. Analysis of kidney cells showed no genetic damage for all groups tested. The number of micronucleated cells did not increase after crack cocaine exposure in bone narrow or liver cells. Conclusion: In summary, crack cocaine is a genotoxic agent in peripheral blood, liver, and brain cells but not mutagenic in multiple organs of mice.

Curcumin prevents perfluorooctane sulfonate-induced genotoxicity and oxidative DNA damage in rat peripheral blood

Perfluorooctane sulfonate (PFOS) is a man-made fluorosurfactant and global pollutant. PFOS a persistent and bioaccumulative compound, and it is widely distributed in humans and wildlife. Therefore, it was added to Annex B of the Stockholm Convention on Persistent Organic Pollutants in May 2009. Curcumin is a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric. It is commonly used as a dietary spice and coloring agent in cooking and anecdotally as an herb in traditional Asian medicine. In this study, male rats were treated with three different PFOS doses (0.6, 1.25, and 2.5 mg/kg) and one dose of curcumin, from Curcuma longa (80 mg/kg), and combined three doses of PFOS with 80 mg/kg dose of curcumin by gavage for 30 d at 48 h intervals. Here, we investigated the DNA damage via single-cell gel electrophoresis/comet assay and micronucleus test in rat peripheral blood in vivo. It is found that all doses of PFOS increased micronucleus frequency (p < 0.05) and strongly induced DNA damage in peripheral blood in two different parameters; the damaged cell percent and genetically damage index, and curcumin prevented the formation of DNA damage induced by PFOS. Results showed that curcumin inhibited DNA damage including GDI at certain levels at statistical manner, 30.07%, 54.41%, and 36.99% for 0.6 mg/kg, 1.25 mg/kg, and 2.5 mg/kg.

Analysis of DNA damage, plasma lipid peroxidation, and antioxidant status in type 2 diabetes mellitus patients among a south Indian population

Oxidative stress is involved in the pathophysiology of diabetes mellitus, which in turn may also contribute to its complications. This case-control study was designed to find out the association between oxidative damage to lipids and DNA in type 2 diabetes mellitus. Sixty individuals with type 2 diabetes and age- and sex-matched 60 clinically healthy individuals without type 2 diabetes were included for the study. Oxidative stress was evaluated by malondialdehyde assay which is a marker of lipid peroxidation and plasma total antioxidant status was evaluated by the ferric reducing antioxidant power assay. Single-cell alkaline gel electrophoresis (comet assay) was employed to assess the level of DNA damage in peripheral lymphocytes. The extent of DNA damage was observed to be high in the diabetic patients when compared with the controls. The total antioxidant status was found to be significantly lower in the patients with diabetes mellitus when compared with the controls (p < 0.05). Lipid peroxidation status was significantly increased in diabetics (p < 0.05) when compared with the control group. The results of this study confirm that hyperglycemia contributes to increased generation of reactive oxygen species and decreased total antioxidant status which may be responsible for the oxidative DNA damage among south Indian population with diabetes mellitus.

Cytogenetic biomonitoring in mucopolyssacharosis I, II and IV patients treated with enzyme replacement therapy

Background and objectives: The aim of this study was to evaluate genotoxicity and mutagenicity in peripheral blood and buccal mucosal cells in mucopolysaccharidosis (MPS) I, II or VI patients.Methods: A total of 12 patients with MPS type I, II and VI attended at the Institute of Genetics and Inborn Errors of Metabolism treated with enzyme replacement therapy (ERT) and 10 healthy control volunteers were included in this study. Mechanically exfoliated cells from cheek mucosa (left and right side) were used to micronucleus test and single cell gel (comet) assay in peripheral blood cells.Results: The results of this study detected the presence of genetic damage in peripheral blood for all individuals with MPS treated with ERT, regardless of type of MPS as depicted by tail moment results. In addition, an increased number of micronucleated cells were found in buccal cells of MPS type II patients. It was also observed an increase of other nuclear alterations closely related to cytotoxicity as depicted by the frequency of pyknosis, karyolysis and karyorrhexis in buccal mucosa cells of MPS VI patients (p < 0.05).Conclusion: Taken together, such results demonstrate that metabolic alterations induced by the enzymatic deficiency characteristic of MPS associated with ERT therapy can induce genotoxicity and mutagenicity in peripheral blood and buccal mucosa cells, respectively. This effect appears to be more pronounced to MPS II.

Single-cell gel (comet) assay detects primary DNA damage in peripheral blood lymphocytes of smokers

Single-cell gel (comet) assay detects primary DNA damage in peripheral blood lymphocytes of smokers

Antimutagenic and antigenotoxic potential of grape juice concentrate in blood and liver of rats exposed to cadmium.

The aim of this study was to evaluate the antimutagenic and antigenotoxic potential of grape juice concentrate in rodent organs exposed to cadmium chloride intoxication. A total of 15 Wistar rats were distributed into three groups (n = 5), as follows: control group (CTRL; nontreated group), cadmium group (Cd), and cadmium-grape juice group (Cd + GJ). Exposed animals received intraperitoneal injection of cadmium chloride (1.2 mg/kg body weight) diluted in water and, after 15 days, Cd + GJ group received grape juice concentrate for 15 days, by gavage (0.8 mL, 1.18 mg of polyphenols kg(-1) day(-1)). Grape juice concentrate was able to decrease genotoxic effects induced by cadmium in peripheral blood and liver cells as depicted by single cell gel (comet) and micronucleus assays. A decrease for anti-8-hydroxy-20-deoxyguanosine (8OHdG) expression in hepatocytes of animals exposed to cadmium and treated with grape juice concentrate was also detected. Higher CuZn-SOD activity was observed in liver cells of the Cd + GJ group. No remarkable differences were seen regarding Mn-SOD activity among groups. Taken together, our results demonstrate that grape juice concentrate was able to exert antimutagenic and antigenotoxic activities in blood and liver cells of rats exposed to cadmium.

Is Lack of Sleep Capable of Inducing DNA Damage in Aged Skin?

Skin naturally changes with age, becoming more fragile. Various stimuli can alter skin integrity. The aim of this study was to evaluate whether sleep deprivation affects the integrity of DNA in skin and exacerbates the effects of aging. Fifteen-month old female Hairless mice underwent 72 h of paradoxical sleep deprivation or 15 days of chronic sleep restriction. Punch biopsies of the skin were taken to evaluate DNA damage by single cell gel (comet) assay. Neither paradoxical sleep deprivation nor sleep restriction increased genetic damage, measured by tail movement and tail intensity values. Taken together, the findings are consistent with the notion that aging overrides the effect of sleep loss on the genetic damage in elderly mice.

Genotoxicity and cytotoxicity of mineral trioxide aggregate and calcium enriched mixture cements on L929 mouse fibroblast cells

Mineral trioxide aggregate (MTA) has shown good biocompatibility in several studies. In the present study, the genotoxic and cytotoxic effects of calcium enriched mixture (CEM) were evaluated compared with MTA using MTT and single-cell gel (comet) assays with serial ascending concentrations (0 to 1,000 µg/mL) of tested materials. Cytotoxicity data indicated that there is no significant difference between CEM and MTA at all concentrations except for the full concentration (1,000 µg/mL); CEM had lower cytotoxicity. Genotoxic effects were more evident with CEM at concentrations of 15.6 and 250 µg/mL; however, was less than that of MTA at concentrations of 500 and 1,000 µg/mL. The cytotoxicity and genotoxicity effects of the two experimental groups generally increased with consistency. Under the conditions of this study, CEM is biocompatible in terms of cyto- and genotoxicity. It appears to be an alternative to MTA as an endodontic biomaterial offering several advantages.

Studies on fate and toxicity of nanoalumina in male albino rats

The purpose of this study is to follow-up the distribution, lethality percentile doses (LDs) and bioaccumulation of aluminium oxide nanoparticles (Al2O3-NPs, average diameter 9.83 ± 1.61 nm) in some tissues of male albino rats, and to evaluate its genotoxicity to the brain tissues, during acute and sublethal experiments. The LDs of Al2O3-NPs, including median lethal dose (LD50), were estimated after intraperitoneal injection. The computed LD50 at 24 and 48 h were 15.10 and 12.88 g/kg body weight (b.w.), respectively. For acute experiments, the bioaccumulation of aluminium (Al) in the brain, liver, kidneys, intestine and spleen was estimated after 48 h of injection with a single acute dose (3.9, 6.4 and 8.5 g/kg b.w.), while for sublethal experiments it was after 1, 3, 7, 14 and 28 days of injection with 1.3 g/kg b.w. once in 2 days. Multi-way analysis of variance affirmed that Al uptake, in acute experiments, was significantly affected by the injected doses, organs (brain, liver, kidneys, intestine and spleen) and their interactions, while for sublethal experiments an altogether effect based on time (1, 3, 7, 14, 28 days), doses (0 and 1.3 g), organs and their interactions was reported. In addition, Al accumulated in the brain, liver, kidney, intestine and spleen of rats administered with Al2O3-NPs were significantly higher than the corresponding controls, during acute and sublethal experiments. The uptake of Al by the spleen of rats injected with acute doses was greater than that accumulated by kidney>brain>intestine>liver, whereas the brain of rats injected with sublethal dose accumulated lesser amount of Al followed by the kidney<intestine<spleen<liver. Bioaccumulation of Al, in all studied tissues, was positively correlated with the injected doses (in acute term) and the experimental periods (in sublethal term). In the acute and sublethal experiments, comet assay parameters such as the tail intensity (i.e. DNA percentage), tail extent moment and olive tail moment were estimated using a single cell gel electrophoresis/comet assay. The results showed significant increase in DNA percentage damage in the brain cells. The obtained results indicate that bioaccumulation of Al was associated with significantly increased levels of comet parameters that depended on the doses and the experimental periods. In conclusion, Al has a high affinity to get accumulated in tissues to a level that is able to induce genotoxicity. Therefore, bioaccumulation is time, dose and organ dependant.

In vitro genotoxicity and cytotoxicity in murine fibroblasts exposed to EDTA, NaOCl, MTAD and citric acid

The aim of the present study was to evaluate the capacity of some root canal irrigants to induce genetic damage and/or cellular death in vitro. Murine fibroblast cells were exposed to ethylenediaminetetraacetic acid (EDTA), sodium hypochlorite (NaOCl), MTAD™ and citric acid in increasing concentrations for 3 h at 37ºC. The negative control group was treated with vehicle control (phosphate buffer solution - PBS) for 3 h at 37°C, and the positive control group was treated with methylmetanesulfonate, 1 μM. for 3 h at 37°C. Cytotoxicity was assessed by the trypan blue test and genotoxicity was evaluated by the single cell gel (comet) assay. The results showed that exposure to 2.5% and 5% NaOCl and 8.5% citric acid resulted in a significant cytotoxic effect. NaOCl, EDTA and citric acid did not produce genotoxic effects with respect to the comet assay data for all evaluated concentrations. Although MTAD was not a cytotoxic agent, it showed significant genotoxic effects at all tested concentrations (ANOVA and Tukey's test; p<0.05). NaOCl, EDTA and citric acid were found to be cytotoxic in a dose-dependent manner, but they were not genotoxic. MTAD did not cause cell death, but presented genotoxic effects.

Toxicological and mutagenic analysis of Artemisia dracunculus (tarragon) extract

Mutagenicity and liver toxicity of the herb tarragon (Artemisia dracunculus) were evaluated using single cell gel (comet) electrophoresis. Ten microlitres aliquots of peripheral venous human blood were incubated with tarragon extract, saline, or the mutagen sodium dichromate. Cell suspensions dispersed in low-melting agarose were electrophoresed in ethidium bromide. The resulting DNA migration trails were obtained using fluorescent microscopy at 400× magnification, and graded according to the mutagenicity index (MI) for each cell incubation condition. The in vivo liver toxicity of Artemisia dracunculus was assessed in the blood of mice treated orally with the extract of the herb, using alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as liver function indicators. Liver morphology was assessed using hematoxylin and eosin (HE) staining of liver tissue. The present study demonstrated a direct correlation between tarragon extract dosage and three major outcome variables: MI; serum liver enzyme activity; and liver histopathology. These outcomes are possibly due to the presence in tarragon of methylchavicol and other genotoxic compounds. These findings provide a preliminary guide for risk assessment of tarragon in diet and in possible therapeutic applications.