Alkaline Comet Assay Research Articles

3132 – IMPACT OF THE INFLAMED BONE MARROW NICHE ON THE PROGRESSION OF MYELOPROLIFERATIVE NEOPLASIA

Bcr/Abl-negative myeloproliferative neoplasia (MPN) is characterized by driver mutations in MPL, CALR, or JAK2, as well as an inflammatory microenvironment. The acquisition of clonal abnormalities and secondary mutations in MPN patients can progress the disease to Acute Myeloid Leukemia (AML), resulting in bone marrow (BM) fibrosis and BM failure. For these patients, treatment options fall short and do not include approaches to prevent leukemic transformation. Our group has developed an animal model for inflammation where loss of RBPJ/Notch signaling in the BM niche leads to upregulation of miR155 and an accumulation of proinflammatory cytokines. We propose to use this model (RBPJKO mouse) to study the influence of chronic inflammation on JAK2V617F-driven MPN progression. We hypothesize that chronic inflammation in the BM niche: i) promotes proliferation and genomic instability in hematopoietic cells; and ii) cooperates with preexisting genetic mutations such as JAK2V617F to promote clonal expansion and selection, accelerating disease progression. Our preliminary studies indicate that JAK2V617F hematopoietic cells drive a more aggressive disease when exposed to chronic inflammation following transplant into RBPJKO inflamed BM niches. Indeed, JAK2V617F/RBPJKO recipients exhibited accelerated neutrophilia, higher myeloid skewing and increased bone marrow cellularity and splenomegaly, when compared to JAK2V617F/RBPJWT recipients. By using alkaline comet assay and gH2Ax foci, we also demonstrate that JAK2V617F cooperates with the inflamed BM niche to promote more DNA single- and double-strand breaks in Lin- progenitors in vivo. To further study these events, we optimized an in vitro model in which WT and JAK2V617F BM Lin- progenitors are co-cultured with RBPJKO or RBPJWT BM-derived stroma. JAK2V617F cells showed increased proliferation, mitochondrial ROS accumulation, elevated 53BP1 foci and increased γH2AX MFI when exposed to the inflammatory conditions supported by RBPJKO stroma. Taken together, these data suggest that chronic inflammation can accentuate the MPN phenotype, promote replicative stress and lead to buildup of DNA-damaging ROS in JAK2V617F MPN cells. This work has the potential to provide us with new insights into the mechanisms of MPN progression and the opportunity to identify new therapeutic approaches.

Pooled analysis of genotoxicity markers in relation to exposure in the Flemish Environment and Health Studies (FLEHS) between 1999 and 2018

BackgroundThe Flemish Environment and Health Studies (FLEHS) are human biomonitoring surveys running in Flanders since 1999. Additionally to biomarkers of exposure, markers of genotoxicity and oxidative stress have been measured, including the alkaline comet and micronucleus assay in peripheral whole blood cells, and urinary concentrations of 8-oxo-2′-deoxyguanosine (8-oxodG). AimExposure-effect associations were explored in a pooled dataset of nine different cross-sectional FLEHS surveys. Data of adolescents collected in a time frame of about 20 years (1999–2018) were compiled. The aim of the study was to examine whether increased variation in exposure, lifestyle and environmental factors would lead to more powerful and robust exposure-effect associations. Materials & methodsThe biomarkers were measured in 2283 adolescents in the age range of 14–18 years. Exposure to polycyclic aromatic hydrocarbons [1-hydroxypyrene (1-OHP)], benzene (tt’-muconic acid), metals (arsenic, cadmium, copper, nickel, thallium, lead, chromium), persistent organochlorines and phthalates were assessed in blood or urine. Furthermore, outdoor air levels of particulate matter (PM10 and PM2.5) at the residences of the youngsters were calculated. Pooled statistical analysis was done using mixed models. Study-specific differences in the genotoxicity markers and in the strength/direction of the association were accounted for. This was done by incorporating the random factor ‘study’ and a random study slope (if possible). The exposure markers were centered around the study-specific mean in order to correct for protocol changes over time. ResultsA significant association was observed for the urinary oxidative stress marker 8-oxodG, which was positively associated with 1-OHP (5% increase for doubling of 1-OHP levels, p = 0.001), and with urinary copper (26% increase for doubling of copper levels, p = 0.001), a metal involved in the Fenton reaction in biological systems. 8-oxodG was also associated with the sum of the metabolites of the phthalate di(2-ethylhexyl) phthalate (DEHP) (3% increase for doubling of the DEHP levels, p = 0.02). For those associations, data pooling increased the statistical power. However, some of the associations in the individual surveys, were not confirmed in the pooled analysis (such as comet assay and 8-oxodG vs. atmospheric PM; and 8-oxodG vs. urinary nickel). This may be due to inconsistencies in exposure-effect relations and/or variations in the pollutant mix over time and regions. ConclusionPooled analysis including a large population of 2283 Flemish adolescents showed that 8-oxodG, a marker of oxidative DNA damage is a valuable marker to assess impact of daily life pollutants, such as PAHs, Cu and the phthalate DEHP.

Genotoxicity and mutagenicity of particulate matter emitted from diesel, gas to liquid, biodiesel, and farnesane fuels: A toxicological risk assessment

The genotoxic and mutagenic potential of soluble organic material extracted from particulate matter emitted by a typical automotive diesel engine was evaluated from the combustion of different alternative and/or renewable fuels. One of these fuels, called Farnesane, is a novel renewable paraffinic hydrocarbon obtained from sugar cane using a biotechnology fermentation process. Tests were carried out under a steady state condition with high level of generated particulate matter, which was collected with a stainless-steel filter located 1.5 m downstream of the exhaust manifold. The mutagenicity index was evaluated using an Ames test, obtaining values for Farnesane and biodiesel fuels lower than those for fossil fuels. However, all fuels showed a mutagenicity index higher than the Phosphate Buffered Saline control. The soluble organic material was extracted from particulate matter to evaluate its genotoxicity activity using an alkaline comet assay in human lymphocytes. The obtained genotoxicity trends were similar to mutagenicity, biodiesel and Farnesane fuels showing the lowest toxicity impact. Additionally, the cell viability was studied and classified according to the type of damage. The lowest values of average Deoxyribonucleic Acid migration and cell damage associated to Farnesane, together with its larger particle size, makes this renewable fuel a potential substitute of the traditional diesel.

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.

To evaluate methods for analysis of genotoxic effects on mammalian cell lines, we tested the effect of three common genotoxic agents on Chinese hamster ovary (CHO) cells by single-cell gel electrophoresis (comet assay) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Suspension-grown CHO cells were separately incubated with etoposide, bleomycin, and ethyl methanesulfonate and analyzed by an alkaline comet assay and GC-MS/MS. Although DNA strand breaks were detected by the comet assay after treatment with all three agents, GC-MS/MS could only detect DNA nucleobase lesions oxidatively induced by bleomycin. This demonstrates that although GC-MS/MS has limitations in detection of genotoxic effects, it can be used for selected chemical genotoxins that contribute to oxidizing processes. The comet assay, used in combination with GC-MS/MS, can be a more useful approach to screen a wide range of chemical genotoxins as well as to monitor other DNA-damaging factors.

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.

Proteomic Analysis in Seminal Plasma of Fertile Donors and Infertile Patients with Sperm DNA Fragmentation.

Seminal plasma proteomics studies could represent a new approach for the determination of molecular elements driving male infertility, resulting in a better male infertility characterization. The aim of this study is to investigate proteomic differences in seminal plasma samples from fertile and infertile individuals. For that, semen samples were selected according to semen analysis, clinical pathology, and values of sperm DNA fragmentation (alkaline and neutral Comet assay and Sperm Chromatin Dispersion test). A total of 24 seminal plasma samples classified in four groups were processed: fertile donors (FD), recurrent miscarriage patients (RM), asthenoteratozoospermic patients (ATZ), and asthenoteratozoospermic patients with varicocele (ATZ-VAR). Results obtained by 2D-differential gel electrophoresis (2D-DIGE) revealed 26 spots significantly increased in fertile donors when compared to patient groups. Also, eight spots in the ATZ group and two in the ATZ-VAR group were decreased compared to the other groups. Twenty-eight proteins were identified by mass spectrometry (MS), most of them involved in metabolic and cellular processes and with a catalytic or binding function. Protein–protein interactions through Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) tool suggest that a large part of them were associated with each other. Furthermore, most of them were associated with ubiquitin C, indicating that it could play an important regulation role, resulting in a potential male infertility biomarker.

Etomidate is devoid of genotoxicty and mutagenicity in human lymphocytes and in the Salmonella typhimurium/microsomal activation test

No carcinogenesis or mutagenesis studies have been carried out with etomidate. The current study showed that etomidate has weak cytotoxic potential after 48 h exposure in human lymphocytes and has no hemolytic activity. The weak cytotoxicity seems to be related with redox imbalance of etomidate (40.9 and 81.9 μM) treated lymphocytes. At both etomidate concentrations, a slight decrease of the levels of GSH intracellular content and a significant increase in the amount of carbonylated proteins were observed after 48 h. The contribution of oxidative stress to genetic toxicity was only perceived when the enzyme Fpg was applied in the comet assay. Etomidate (40.9 and 81.9 μM) is a weak generator of oxidative DNA damage in lymphocytes. These damages to DNA probably were repaired, since no DNA strand breaks were detected in the standard alkaline comet assay (in the presence or absence of hepatic S9 microsomal fraction) without Fpg. Also, no micronucleated lymphocytes or carrying chromosomal aberrations were observed. Finally, etomidate (2046.8 and 4093.5 μM) was not mutagenic in the Salmonella/microsome mutagenicity assay, which used four Salmonella typhimurium strains (TA97a, TA98, TA100, and TA102) to detect frameshift and base-substitution mutations. In summary, etomidate is a weak oxidative DNA damaging anesthetic and is devoid of mutagenic properties in eukaryotic and prokaryotic models.

Impact of single nucleotide polymorphisms in the OGG1 and XRCC1 genes on modulation of DNA damage in pesticide-exposed agricultural workers in Punjab, North-West India

Pesticide-induced DNA damage is primarily repaired by base excision repair (BER) pathway. However, polymorphism in DNA repair genes may modulate individual’s DNA repair capacity (DRC) leading to increased genotoxicity and adverse health effects. Our first study in North-West Indian population aimed to evaluate the impact of OGG1 rs1052133 (Ser326Cys; C1245G), XRCC1 rs1799782 (Arg194Trp; C26304T) and XRCC1 rs25487 (Arg399Gln; G28152A) polymorphisms on the modulation of pesticide-induced DNA damage in a total of 450 subjects (225 pesticide-exposed agricultural workers and 225 age- and sex-matched controls). DNA damage was estimated by alkaline comet assay using silver-staining method. Genotyping was carried out by PCR-RFLP using site-specific restriction enzymes. Mann-Whitney U-test revealed elevation in DNA damage parameters (p < 0.01) in pesticide-exposed agricultural workers than controls. Chi-square test showed significant (p < 0.05) differences in the XRCC1 Arg194Trp (C26304T) and Arg399Gln (G28152A) genotypes among two groups. Multivariate logistic-regression analysis revealed that heterozygous genotypes of OGG1 rs1052133 (326Ser/Cys; 1245CA), XRCC1 rs1799782 (194Arg/Trp; 26304CT) and XRCC1 rs25487 (399Arg/Gln; 2815GA) were positively associated (p < 0.05) with elevated DNA damage parameters in pesticide-exposed agricultural workers. Our results strongly indicate significant positive association of variant OGG1 and XRCC1 genotypes with reduced DRC and higher pesticide-induced DNA damage in North-West Indian agricultural workers.

Effect of low-dose fast neutrons on the protein components of peripheral blood mononuclear cells of whole-body irradiated Wistar rats.

The immune system is exposed to extremely low doses of neutrons under different circumstances, such as through exposure to cosmic rays, nuclear accidents, and neutron therapy. Peripheral blood mononuclear cells (PBMCs) are the primary immune cells that exhibit selective immune responses. Changes in the functions of the protein components of PBMC can be induced by structural modifications of these proteins themselves. Herein, we have investigated the effect of low-dose fast neutrons on PBMC proteins at 0, 2, 4, and 8days post-whole body irradiation. 64 Wistar rats were used in this study of which, 32 were exposed to fast neutrons at a total dose of 10mGy (241Am-Be, 0.2mGy/h), and the other 32 were used as controls. Blood samples were drawn, and PBMCs were isolated from whole blood. Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy were used to estimate the changes in the proteins of PBMCs. An alkaline comet assay was performed to assess DNA damage. Hierarchical cluster analysis (HCA) and principal components analysis (PCA) were utilized to discriminate between irradiated and non-irradiated samples. FTIR and fluorescence spectra of the tested samples revealed alterations in the amides and tryptophan, and therefore protein structure at time intervals of 2 and 4days post-irradiation. No changes were recorded in samples tested at time intervals of 0 and 8days post-irradiation. The FTIR band intensities of the PBMC proteins of the irradiated samples decreased slightly and were statistically significant. Curve fitting of the amide I band in the FTIR spectra showed changes in the secondary structure of the proteins. At 2days post-irradiation, fluorescence spectra of the tested samples revealed decreases in the band tryptophan. The comet assay revealed low levels of DNA damage. In conclusion, low-dose fast neutrons can affect the proteins of PBMC.

DNA damage analysis concerning GSTM1 and GSTT1 gene polymorphism in gold jewellery workers from Peshawar Pakistan

Purpose To evaluate the genotoxic effects of gold jewellery fumes and its association with GSTM1 and GSTT1 genetic polymorphisms. Materials and methods We examined 94 subjects including 54 gold jewellery workers and 40 controls. The DNA damage was evaluated by alkaline comet assay and genotyping by PCR. Results The mean total comet score (TCS) in gold jewellery workers was significantly higher as compared to the control subjects (128.0 ± 60.6 versus 47.7 ± 21.4; p = 0.0001). Duration of occupational exposure had positive correlation (r = 0.453, p < 0.01) with DNA damage. Age and tobacco use had significant effects on the TCS of the exposed group as compared to the control group (p < 0.05). The frequency of the GSTM1-null genotype in the exposed group was significant (p = 0.004) as compared to the control group. No significant association (p > 0.05) between the GSTM1 and GSTT1 genotypes and DNA damage was found. Conclusions Our results suggest that there is increased DNA damage in gold jewellery workers due to their occupational surroundings. Hence there is a strong need to educate the workers about the adverse health effects of potentially hazardous chemicals and highlight the importance of using protective measures.

In vitro and in vivo assessment of the genotoxic effects of ceric ammonium nitrate and 1,3-propane sultone

A variety of methods have been developed for accurate and systematic evaluation of chemical genotoxicity. Ceric ammonium nitrate (CAN) and 1,3-propane sultone (1,3-PS) have been extensively applied in industrial fields. Although 1,3-PS, but not CAN, has been reported as a potent carcinogen, systematic assessment of the genotoxic properties of these chemicals has not been conducted. The purpose of this study was to establish a decision tree for evaluating genotoxicity based on the good laboratory practices (GLP) system using 1,3-PS and CAN as test chemicals. In vitro studies were performed including the bacterial reverse mutation assay, chromosomal aberration assay, and micronucleus assay. We conducted in vivo studies using a combined micronucleus and alkaline comet (MN-CMT) assay and the Pig-a gene mutation assay, which is a promising method for detecting gene mutations in vivo. CAN showed negative responses in all in vitro genotoxicity assays and the in vivo combined MN-CMT assay. Meanwhile, 1,3-PS had positive results in all in vitro and in vivo genotoxicity assays. In this study, we confirmed the genotoxicity of 1,3-PS and CAN using both in vitro and in vivo assays. We propose a decision tree for evaluating chemical-induced genotoxicity.

Abstract IA20: Novel cell microarray technologies for studies of DNA damage and cell survival and applications for studies of microbe-induced DNA damage

Abstract Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The comet assay is a commonly used approach for detecting DNA damage that is based on the underlying principle that damaged DNA migrates more readily than undamaged DNA when electrophoresed in agarose. We previously developed a higher-throughput version of the comet assay (Wood DK, PNAS 2010). For the “CometChip” assay, cells are loaded into an array of agarose microwells to create a cell microarray. The CometChip can be fully automated, making the assay more than 1000X faster, while improving reproducibility. While effective for detecting single-strand breaks, abasic sites, and alkali-sensitive sites, the alkaline comet assay is not effective for detection of carcinogenic bulky DNA lesions that do not impact DNA mobility and that require metabolic activation. To overcome this, we use DNA replication inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) shown to trap single-strand breaks that are formed during nucleotide excision repair, which normally removes bulky lesions. Thus, comet-undetectable bulky lesions are converted into comet-detectable single-strand breaks. Together with HepaRG™ cells that recapitulate in vivo metabolic capacity, we have thus created the “HepaCometChip,” which provides a more broadly effective approach for detection of DNA damage. In addition to genotoxicity testing, the microwell array has also been harnessed for use in a cell survival assay. For the “MicroColonyChip” (Ngo LP, Cell Reports 2019), cells are loaded into the agarose microwell array and allowed to grow to form microcolonies. Differences in cell survival can be detected by changes in the distribution of colony sizes, a novel metric for cell survival quantitation. The assay is as sensitive as the traditional “gold standard” colony-forming assay, yet it takes days instead of weeks to complete. Parallel studies of genotoxicity and cytotoxicity can thus be performed using a shared microwell platform. These tools have broad utility, including studies of microbe-induced DNA damage. Examples of S. pneumoniae and E. coli microbial product-induced double-strand breaks and interstrand crosslinks contribute to an emerging paradigm wherein pathogen-induced DNA damage has the potential to promote disease. Citation Format: Le P. Ngo, Prashant Rai, Matthew R. Wilson, Carole Swartz, John Winters, Yang Su, Jing Ge, Tze-Khee Chan, Emily P. Balskus, Vincent Chow, Les Recio, Leona D. Samson, Bevin P. Engelward. Novel cell microarray technologies for studies of DNA damage and cell survival and applications for studies of microbe-induced DNA damage [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr IA20.

2-Monokloropropandiol (2-MCPD)’nin Sitotoksik Etkilerinin Fare TM3 Leydig ve TM4 Sertoli Hücreleri Üzerinde in vitro Değerlendirilmesi

Amaç: 2-monokloropropandiol (2-MCPD), 3-monokloropropandiol (3-MCPD) ve esterleri ile glisidil yağ asidi esteri (GE), gıda veya gıda işlem kaynaklı kontaminantlar olarak adlandırılmaktadır. Bu gıda kontaminantlarının başta palmiye yağı olmak üzere bitkisel yağlar, margarinler, pasta ve kek gibi işlem görmüş gıdalarda ve bebek mamalarında tespit edilmesi, güvenlikleri konusundaki ilgiyi arttırmıştır. Avrupa Gıda Güvenliği Otoritesi’nin (European Food Safety Authority, EFSA) Mayıs 2016 yılında yayınlamış olduğu bilimsel görüşe göre bitkisel yağ ve işlenmiş gıdalarda bulunan GE, 3-MCPD, 2-MCPD ve bunların yağ asidi esterlerinin insan sağlığı için zararlı olduğu raporlanmıştır. Ancak 2-MCPD toksisitesi hakkında yeterli veri olmadığı için tam bir değerlendirme yapılamamıştır. Bu amaçla 2-MCPD’nin, TM3 Leydig ve TM4 Sertoli hücre hatları üzerinde in vitro sitotoksik, apoptotik etkileri ve deoksiribonükleik asit (DNA) hasarı yapma potansiyeli değerlendirilmiştir.&#x0D; Gereç ve Yöntem: Hücre canlılığı, 3-(4,5-dimetiltiyazol-2-il)-2,5-difeniltetrazolyum bromid (MTT) ve nötral kırmızısı alım (NRU) yöntemleri ile değerlendirilmiştir. Akış sitometrisi ile hücre ölüm tipinin belirlenmesinde aneksin V-FITC/PI ikili boyama kiti kullanılmıştır. Genotoksisite değerlendirmesinde komet yöntemi uygulanmıştır. &#x0D; Bulgular: Hücre canlılık yöntemleri sonucu 2-MCPD’nin test edilen konsantrasyonlarda 1000 µM’a kadar sitotoksik olmadığı tespit edilmiştir. Aynı zamanda 2-MCPD’nin, sub-toksik konsantrasyonlarda (1-1000 µM) her iki hücre hattında da (TM3 Leydig ve TM4 Sertoli hücreleri) DNA hasarını indüklemediği bulunmuştur. &#x0D; Sonuç: 2-MCPD, her ne kadar test edilen konsantrasyonlarda (1-1000 µM) sitotoksik ve apoptotik etki göstermese de daha yüksek konsantrasyonlarda hücre ölümüne ve DNA hasarına neden olabilir. Ancak bu yüksek konsantrasyonlar günlük maruziyet dozlarından çok uzaktır.

Changes in the levels of comet parameters before and after fluoxetine therapy in major depression patients.

Major depression belongs to mood disorders and characterized by worthlessness, no interest or happiness in any activity; lasting for atleast two weeks. Etio-pathological changes of major depression include oxidative stress leading to free radical synthesis which causes damage to carbohydrates, proteins, lipids and nucleic acids. Nucleic acid damage can be identified by either single or double strand breaks and for quantitative estimation of the same, neutral or alkaline comet assay is performed. Fluoxetine is the drug of choice for treatment of major depression having antioxidant function. In the current study eighty drug naïve major depression patients were recruited and comet parameters namely total comet length, head diameter and tail length were measured before starting the treatment and after completion of eight week fluoxetine therapy. The levels of comet parameters were higher in females than males suggesting higher prevalence of major depression among females. On categorizing into three age groups, the numbers of major depression patients belonging to 18–30 year age group were higher than 31–40 and 41–50 year age groups. All the parameters of deoxyribonucleic acid damage were reduced after eight week of fluoxetine therapy indicating that fluoxetine has anti-oxidant action along with its antidepressant properties, which cause reversal of oxidative stress induced damage occurring during major depression.

Assessment of genoprotective effects of Gentiana lutea extracts prepared from plants grown in field and in vitro

Medicinal plant Gentiana lutea is considered as an official drug for gastrointestinal disorders in many pharmacopoeias. Its uncontrolled overexploitation led to the protection regime and encouraged development of in vitro culture. The aim of this work was to chemically characterize root and leaf methanol extracts of plant grown in plantation and in vitro conditions, and to determine their antigenotoxicity against heterocyclic aromatic amines IQ and PhIP, the food-borne mutagens that are formed during high temperature cooking of protein-reach foods.High yields and growth ratios were determined for in vitro plants. Higher content of polyphenols and flavonoids were determined in aerial parts, while UPLC-MS/MS analysis pointed at the richness of in vitro grown shoot extract (GLvS) in active constituents, and high amounts of gentiopicroside in all tested extracts. Notable amounts of sweroside were detected in plantation root extract (GLR) and in GLvS, and of loganic acid in plantation leaf extract (GLL) and GLvS. Two experimental models for genotoxicity/antigenotoxicity study, i.e. SOS/umuC test with metabolic activation (addition of rat liver S9 fraction) and alkaline comet assay on hepatocellur carcinoma HepG2 cells indicated dual effects: genotoxic of high and antigenotoxic of lower non-genotoxic doses. While in vitro extracts were genotoxic in both models, plantation extracts were active only in SOS/umuC with S9. In contrast, inhibition of genotoxicity was observed in both models for all extracts (up to 83 %). Results of DPPH assay indicated that antioxidativity could be responsible for antigenotoxic properties. In conclusion, genoprotective effect encourages further investigation of antigenotoxicity, while high growth ratio and richness of GLvS in active compounds justify the use of in vitro cultivation method.

Genotoxicity and cellular uptake of nanosized and fine copper oxide particles in human bronchial epithelial cells in vitro

We studied the genotoxicity and cellular uptake of nanosized (<50 nm) and fine (<10 μm) copper oxide (CuO) particles in vitro in human bronchial epithelial (BEAS-2B) cells. In addition, the effect of dispersing the particles using bovine serum albumin (BSA) on DNA damage induction was investigated. DNA damage was assessed by the alkaline comet (single cell gel electrophoresis) assay after 3-h, 6-h and 24-h exposures. The cytokinesis-block micronucleus assay was applied to study chromosome damage. Both fine- and nanosized CuO particles induced a dose-dependent increase in DNA damage at all timepoints tested. However, nanosized CuO damaged DNA at lower doses and higher levels compared with fine CuO. Dispersing the nanoparticles in the presence of BSA (0.6 mg/mL) resulted in a small and inconsistent decrease in DNA damage compared with dispersions in serum-free cell culture medium only. CuO nanoparticles induced a clear dose-dependent increase in micronucleated cells at doses that strongly increased cytostasis and were markedly cytotoxic at 24 and 48 h. Fine CuO showed a slight induction of micronuclei. Hyperspectral microscopy indicated a substantial cellular uptake of both types of particles after a 3-h exposure to a dose of 20 μg/cm2. The number of particles internalized by the cells was higher for nanosized than fine CuO, as quantified by the frequency of spectral matches in the total cell area and by the number of spectrally matched visible particles or agglomerates per cell. The particle uptake was limited by particle size. The stronger genotoxic activity of nanosized than fine CuO particles is likely to derive from the higher cellular uptake and more effective intracellular dissolution of nanoparticles.

Polymorphisms in XPC and XPD genes modulate DNA damage in pesticide-exposed agricultural workers of Punjab, North-West India.

The genetic susceptibility of individuals to the genotoxic effect of pesticides may be modulated by variations in genes involved in nucleotide excision repair (NER) pathway and therefore plays an important role in the evaluation of occupational risk. We aimed to evaluate the role of xeroderma pigmentosum complementation group C (XPC) Lys939Gln (A2920C, rs2228001), XPC Ala499Val (C2151T, rs2228000), xeroderma pigmentosum complementation group D (XPD) Asp312Asn (G23591A, rs1799793) and XPD Lys751Gln (A35931C, rs13181) in the modulation of DNA damage. A total of 450 subjects (225 pesticide-exposed agricultural workers and 225 age- and sex-matched controls) from Punjab, North-West India were recruited to study DNA damage by alkaline comet assay. Genotyping was carried out by PCR-RFLP using site-specific restriction enzymes. We found significant elevation in DNA damage parameters in pesticide-exposed agricultural workers as compared to the controls (p < 0.01). Association of comet tail length with XPC 939Gln/Gln (CC), XPD 312Asp/Asn (GA) and XPD 312Asn/Asn (AA) genotypes was observed. Frequency of cells showing DNA migration was significantly higher in exposed workers with variant XPC 939Gln/Gln (CC), XPD 312Asp/Asn (GA) and XPD 312Asn/Asn (AA) genotypes. Mean tail length was significantly increased in agricultural workers carrying XPD 312Asn/Asn (AA) genotype. Elevation in total comet DNA migration was also observed in exposed workers carrying variant XPC 939Lys/Gln (AC), XPC 939Gln/Gln (CC), XPC 499Val/Val (TT) and XPD 312Asn/Asn (AA) genotypes. Our results strongly indicate significant positive association of variant XPC and XPD genotypes with higher pesticide-induced DNA damage in North-West Indian agricultural workers.

Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

In vitro toxicological assessment of gadolinium (III) chloride in V79\u20134 fibroblasts

BackgroundRare earth minerals of the lanthanide series are widely used in the field of medical and clinical application. Gadolinium (Gd), the most preferred rare earth mineral is frequently used as magnets, superconductors and magnetic resonance imaging (MRI) contrast agent. Increasing production of gadolinium waste, known potent toxicity of this element and lack of information on its Material Safety Data Sheet (MSDS) prompts health risk assessment on gadolinium. In this study, cytotoxicity and genotoxicity of Gadolinium (III) chloride (GdCl3) were investigated using MTT assay, Alkaline Comet assay and Micronucleus assay, respectively.ResultsOur results demonstrated that the viability of GdCl3 treated V79–4 cells was significantly (p < 0.05) reduced at 1.0 mM after 24 h of incubation. However, no IC50 values were obtained. GdCl3 showed no significant (p > 0.05) DNA damage both in the presence and absence of metabolic activation. However, it induced significant (p < 0.05) clastogenic effect in V79–4 cells at 1.0 mM in the absence of metabolic activation. The clastogenic effect was also seen in the presence of metabolic activation at 0.25 mM, 0.5 mM and 1.0 mM.ConclusionTaken together, our study indicated that GdCl3 had no cytotoxic effect and does not induce DNA damage. However, this study supports that GdCl3 is a probable clastogen. Further studies are needed to investigate the effect of free gadolinium ion (Gd3+) for risk assessment on human health.

Assessment of Dog Testis Perfusion by Colour and Pulsed-Doppler Ultrasonography and Correlation With Sperm Oxidative DNA Damage

The assessment of testicular artery blood flow by colour and pulsed-Doppler ultrasonography is an important diagnostic technique to assess vascular perfusion. Recently, it has been suggested as a good predictor of sperm quality. On the other hand, through the alkaline Comet Assay, it is possible to quantify sperm oxidative DNA damage. The aim of this study was to evaluate the relationship between routine sperm parameters, testicular artery blood flow and oxidative DNA damage in canine sperm. Testicular ultrasonography and sperm collection were performed on 12 male dogs, with the animals being allocated into 2 groups, according to the classification of the ejaculates’ quality, as normozoospermic (N; n = 7) or non-normozoospermic (OAT; n = 5). Seven dogs aged between 1.5 and 8.0 years old were included in group N and 5 dogs, aged between 2.0 and 11.0 years old, were included in group OAT. The sperm-rich fraction of the ejaculates was evaluated for sperm routine parameters and DNA damage by comet assay. Colour and pulsed-Doppler ultrasonography were used to evaluate the blood flow of the supratesticular and marginal arteries of right and left testis. Group OAT presented higher levels of sperm oxidative DNA damage (A.U.) in comparison to group N (N:11.7 ± 9.9; OAT:34.2 ± 6.1; P< .001). The peak of systolic velocity was positively correlated with sperm concentration (r = 0.685; P= .005). The resistive and pulsatility indexes (RI and PI) of the supratesticular artery were negatively correlated with sperm membrane integrity (HOST+) (r = −0.594; P = .042; r = −0.612; P = .035, respectively). The end diastolic velocity (EDV) of the supratesticular artery was positively correlated with sperm concentration (r = 0.748; P = .005) and negatively correlated with sperm oxidative DNA damage (r = −0.766; P = .004). Our results suggest that the assessment of the testicular artery blood flow by colour and pulsed-Doppler ultrasonography could be a good predictor of sperm quality in dogs in terms of sperm concentration, membrane integrity and sperm oxidative DNA damage.