Tail Intensity Research Articles

Antiproliferative, neurotoxic, genotoxic and mutagenic effects of toxic cyanobacterial extracts.

Cyanobacteria are the rich resource of various secondary metabolites including toxins with broad pharmaceutical significance. The aim of this work was to evaluate the antiproliferative, neurotoxic, genotoxic and mutagenic effects of cyanobacterial extracts containing Microcystin-LR (MCLR) in vitro. ELISA analysis results showed that MCLR contents of five cyanobacterial extracts were 2.07 ng/mL, 1.43 ng/mL, 1.41 ng/mL, 1.27 ng/mL, and 1.12 ng/mL for Leptolyngbya sp. SB1, Phormidium sp. SB4, Oscillatoria earlei SB5, Phormidium sp. SB2, Uncultured cyanobacterium, respectively. Phormidium sp. SB4 and Phormidium sp. SB2 extracts had the lowest neurotoxicity (86% and 79% cell viability, respectively) and Oscillatoria earlei SB5 extracts had the highest neurotoxicity (47% cell viability) on PC12 cell at 1000 µg/ml extract concentration. Leptolyngbya sp. SB1 and Phormidium sp. SB2 showed the highest antiproliferative effect (92% and 77% cell death) on HT29 cell. On the other hand, all concentrations of five toxic cyanobacterial extracts induced DNA damage between 3.0% and 1.3% of tail intensity and did not cause any direct mutagenic effect at the 1000 µg/plate cyanobacterial extracts. These results suggest that cyanobacteria-derived MCLR is a promising candidate for development of effective agents against colon cancer.

Consumption of a dark roast coffee blend reduces DNA damage in humans: results from a 4-week randomised controlled study.

To determine the DNA protective effects of a standard coffee beverage in comparison to water consumption. The single-blind, randomised controlled study with parallel design included healthy women (n = 50) and men (n = 50) recruited from the general Central European population. The subjects were randomised in a coffee and a control group, with stratification for sex and body mass index. The study comprised two periods of 4weeks: a preconditioning period, with daily consumption of at least 500ml water but no coffee, nor tea, nor any other caffeine-containing product. During the subsequent intervention period the coffee group consumed 500ml of freshly brewed dark roast coffee blend per day, the control group consumed water instead. On the last day of each period, blood was drawn and analysed by comet assay (single-cell gel electrophoresis) to assess the level of DNA damage (strand breakage). At the end of the intervention period the mean level of DNA strand breaks in the coffee group has decreased in comparison to the control group [difference in means 0.23% TI (tail intensity), p = 0.028]. The mean change from baseline (delta value) was - 23% in the coffee group (p = 0.0012). Effects of coffee intake were similar for men and women. During intervention, neither group showed any significant change in body weight or calorie intake. Our results indicate that regular consumption of a dark roast coffee blend has a beneficial protective effect on human DNA integrity in both, men and women.

The cytotoxic and genotoxic effects of daidzein on MIA PaCa-2 human pancreatic carcinoma cells and HT-29 human colon cancer cells

Daidzein (DZ) has anti-inflammatory and antioxidant effects, as well as the dose-dependent inhibition effect on cancer cells. In this study, the cytotoxic and genotoxic effects of DZ on HT-29 (human colorectal adenocarcinoma cells) and MIA PaCa-2 (human pancreatic cancer cells) cell lines were determined using the XTT method and Comet assay, respectively. IC50 concentrations of DZ were found to be 200 µM in both MIA PaCa-2 and HT-29 cells treated with DZ for 48 hours (h). When the cells were treated with 200 μM of DZ for 48 h, DNA damage was observed in both cell lines. DNA tail length (TL), tail moment (TM), and tail intensity (TI) increased more in MIA PaCa-2 cells treated with 200 μM of DZ than those in the control cell (untreated MIA PaCa-2 cell) group (p < 0.01). However, only DNA-TI and DNA-TM exhibited higher increases in HT-29 cells treated with 200 μM of DZ than those in the control cell (untreated HT-29 cell) group (p < 0.01). This shows that DZ has cytotoxic and genotoxic effects on both cell lines. The observed genotoxic effects of DZ still need to be confirmed in additional future studies.

Evaluation of the cytotoxic and genotoxic effects of mycotoxin fusaric acid

Fusaric acid (FA) is produced by several Fusarium species and is commonly found in grains. This investigation was performed to evaluate the cytotoxic and genotoxic effects of FA either in human cervix carcinoma (HeLa) cell line using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay and in human lymphocytes using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) as well as comet assay in vitro. The cells were treated with 0.78, 1.56, 3.125, 6.25, 12.50, 25, 50, 100, 200, and 400 µg/mL concentrations of FA. It has potent cytotoxic effect on HeLa cell line measured by MTT assay especially at higher concentrations (200, 400 µg/mL). The half of inhibitory concentration (IC50) evidenced by FA in the HeLa cells was 200 μg/mL at 24 h and between 200 and 400 μg/mL at 48 h. It was also observed that FA produced a significant decrease in mitotic index (MI) at 12.50 µg/mL compared to solvent control. Furthermore, it indicated a cytotoxic effect at the concentrations ranging from 25 to 400 μg/mL in human lymphocytes. The results of this research point out that being exposed to FA at high concentrations show cytotoxicity. Besides FA induced comet tail intensity at 3.125, 6.25, and 12.50 µg/mL concentrations in isolated human lymphocytes. On the other hand, no genotoxic effects were seen in human lymphocytes in vitro using CA, SCE and MN assays.

Assessment of cytotoxic and genotoxic effects of enniatin-A in vitro

ABSTRACTEnniatin A (EN-A) is a Fusarium mycotoxin which is a common contaminant in grains and especially in maize and it causes serious loss of product. The aim of this study was to investigate the cytotoxic effects using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay in human cervix carcinoma (HeLa) cell line, and genotoxic effects of EN-A using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) and comet assays in human lymphocytes. The cells were treated with 0.07, 0.14, 0.29, 0.57, 1.15, 2.29, 4.59 and 9.17 μM concentrations of EN-A. It exhibited cytotoxic effects in HeLa cell lines especially when the concentrations were increased. The half-inhibitory value (IC50) was determined as 1.15 μM concentration for 24 h and 0.57 μM concentration for 48 h. However, EN-A failed to affect the frequency of CAs, SCEs and MN in human lymphocytes. Only a slight increase was observed in the frequency of SCEs at 0.57 μM concentration over 48 h. The replication (RI) and nuclear division (NDI) indices were not affected. On the contrary, EN-A decreased the mitotic index (MI) significantly at all concentrations compared to the negative control and solvent control (except at 0.29 μM for 24 h, and except at 0.14, 0.29 and 0.57 μM for 48 h). Treatments over 2.29 μM showed toxic effects in human lymphocytes. EN-A significantly increased comet tail intensity (except at 0.07 and 0.57 μM) in isolated human lymphocytes. The results of this study demonstrate that EN-A has an obvious cytotoxic effect especially when the EN-A concentration was increased. In addition, EN-A could exhibit a mild genotoxic effect.

Phase II study of DFP-10917, a deoxycytidine analog, given by 14-day continuous intravenous infusion for chemotherapy-refractory advanced colorectal cancer

Background DFP-10917 is a cytotoxic deoxycytidine analogue that causes DNA fragmentation, G2/M-phase arrest, and apoptosis. This agent has been shown to have antitumor activity against colorectal cancer (CRC) in preclinical studies and to be tolerable in patients. The purpose of our phase II trial was to evaluate the safety, efficacy and pharmacogenomics of DFP-10917 as well as DNA damage studies in patients with advanced CRC refractory to cytotoxic chemotherapy. Methods In this single-arm, Simon two-stage, phase II trial, patients with chemotherapy-refractory advanced CRC received 2.0mg/m2/day DFP-10917 via 14-day continuous infusion. Enrollment criteria included age≥18years, Eastern Cooperative Oncology Group status of 0 or 1, and adequate organ function. The primary endpoint was 3-month progression-free survival, defined as the proportion of patients who did not have progressive disease or death within 3months of starting therapy. All patients who received any amount of DFP-10917 were included in the safety analysis. DNA damage study was assessed by comet assay. Results Of 28 patients initially enrolled, 26 received DFP-10917. Three patients (12%) were progression free at 3months. The median progression-free survival was 1.3months (95% confidence interval, 1.3-1.6months). There were no complete or partial responses. Most patients (n = 20, 77%) had progressive disease, and only six (23%) had stable disease at any time. The trial was terminated according to the pre-planned stopping rule. The most frequent (≥5%) medication-related grade 3 or higher adverse events were neutropenia (n = 10, 38%), fatigue (n = 4, 15%), anemia (n = 3, 12%), and leukopenia (n = 3, 12%). DNA strand-breaks were detected after infusion (medians of % tail intensity were 2.89 and 12.64 on day 1 and day 15, respectively, p < 0.001, sign test). Conclusion Overall, single-agent DFP-10917 did not show meaningful antitumor activity in chemotherapy-refractory advanced CRC. The safety profile of DFP-10917 was tolerable and similar to that observed in earlier clinical studies.

Zinc Oxide Nanoparticles Induced Oxidative DNA Damage, Inflammation and Apoptosis in Rat's Brain after Oral Exposure.

Growing evidences demonstrated that zinc oxide nanoparticles (ZnONPs) could reach the brain after oral ingestion; however, the “neurotoxicity of” ZnONPs after oral exposure has not been fully investigated. This study aimed to explore the “neurotoxicity of” ZnONPs (<100 nm) after oral exposure to two doses; 40 and 100 mg/kg for 24 h and 7 days. The exposure to 40 and 100 mg/kg of ZnONPs for 24 h did not elicit “neurotoxicity” compared to normal control. However, the daily exposure to both doses for 7 days caused oxidative stress in brain tissue as detected by the elevation of the levels of malondialdehyde, the main product of lipid peroxidation and nitrite as an index of nitric oxide with concomitant decline in the concentrations of antioxidants. In addition, both doses resulted in DNA fragmentation which was confirmed by increased percentage of tailed DNA, DNA tail intensity and length and tail moment particularly with the dose 100 mg/kg. Moreover, both doses led to the elevation of the inflammatory cytokines along with increased apoptotic markers including caspase-3 and Fas. Heat shock protein-70 levels were also elevated possibly as a compensatory mechanism to counteract the ZnONPs-induced oxidative stress and apoptosis. The present results indicate the “neurotoxicity of” ZnONPs after recurrent oral exposure via oxidative stress, genotoxicity, inflammatory response and apoptosis.

The metabolism of imidacloprid by aldehyde oxidase contributes to its clastogenic effect in New Zealand rabbits

Imidacloprid (IMI) is a systemic, chloro-nicotinyl insecticide classified in Regulation N° 1272/2008 of the European Commision as “harmful if swallowed and very toxic to aquatic life, with long-lasting effects”. IMI is metabolized in vitro both by aldehyde oxidase (AOX) (reduction) and by cytochrome P450s enzymes (CYPs). In the present study, the AOX inhibitor sodium tungstate dihydrate (ST) was used to elucidate the relative contribution of CYP 450 and AOX metabolic pathways on IMI metabolism, in male rabbits exposed to IMI for two months. To evaluate the inhibition effectiveness, various metabolite concentrations in the IMI and IMI + ST exposed groups were monitored. DNA damage was also evaluated in micronucleus (MN) and single cell electrophoresis (SCGC) assays in both groups, along with oxidative stress (OS) with the inflammatory status of the exposed animals, in order to clarify which metabolic pathway is more detrimental in this experimental setting. A significant increase in the frequency of binucleated cells with MN (BNMN, 105%) and micronuclei (MN, 142%) was observed after exposure to IMI (p < 0.001). The increase in the ST co-exposed animals was less pronounced (BNMN 75%, MN 95%). The Cytokinesis Block Proliferation Index (CBPI) showed no significant difference between controls and exposed animals at any time of exposure (p > 0.05), which indicates no cytotoxic effect. Similarly, comet results show that the IMI group exhibited the highest achieved tail intensity, which reached 70.7% over the control groups, whereas in the IMI + ST groups the increase remained at 48.5%. No differences were observed between all groups for oxidative-stress biomarkers. The results indicate that the AOX metabolic pathway plays a more important role in the systemic toxicity of IMI.

Genotoxicity study of Ethiopian medicinal plant extracts on HepG2 cells

BackgroundMost of herbal medicines are used without any standard safety and toxicological trials although common assumption is that these products are nontoxic. However, this assumption is incorrect and dangerous, so toxicological studies should be done for herbal drugs. Although Pterolobium stellatum, Otostegia integrifolia and Vernonia amygdalina root extracts are frequently used in Ethiopian traditional medicine, there are no evidences of their active toxic compounds. Therefore, we made an effort to assess probable genotoxic effect of these plant extracts on DNA of human hematoma (HepG2) cells using alkaline comet assay.MethodsGenotoxic effects of extracts were evaluated using single cell gel electrophoresis (SCGE) method on HepG2 cell. Regarding comet data, the average mean tail intensities (TI) from each individual experiment and treatment (usually at least 3 cultures/treatment) were pooled and the average mean TI was used as an indicator of DNA damage and the standard error of mean (SEM) as the measure of variance.ResultsDNA damage in the form of comet tail has been observed for 1 and 0.5 mg/ml P. stellatum chloroform and 80% methanol extracts on HepG2 cells, respectively. The chloroform extract of P. stellatum showed increased tail DNA percentage in a concentration dependent manner. Comet tail length in the chloroform P. stellatum extract treated cells (1 mg/ml) was significantly higher by 89% (p < 0.05) compared to vehicle treated controls. The rest of test extracts seemed to be without genotoxic effect up to a concentration of 0.5 mg/ml.ConclusionsOur findings show that two extracts from one plant evaluated have a genotoxic potential in vitro which calls for a more thorough safety evaluation. Such evaluation should include other end-points of genotoxicity apart from DNA damage, and possibly also pure compounds.

In vitro cytotoxicity and genotoxicity of Furia®180 SC (zeta-cypermethrin) and Bulldock 125®SC (β-cyfluthrin) pyrethroid insecticides in human peripheral blood lymphocytes

In the present study, human peripheral blood lymphocytes were exposed in vitro to 0, 6, 12, 18, 24, and 30 μg/mL Furia®180 SC (zeta-cypermethrin) and 0, 6.3, 12.5, 18.8, 25, and 31.3 μg/mL Bulldock®125 SC (β-cyfluthrin). Exposure to 32 µg/mL bleomycin for 24 h served as a positive control. The cytotoxic and genotoxic effects of each insecticide were analyzed using alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated through three genotoxicity parameters: tail length (TL), tail moment (TM) and tail intensity (TI). Furia®180 SC and Bulldock®125 SC pyrethroid insecticides and bleomycin significantly increased DNA damage in a concentration-dependent manner. Bulldock®125 SC induced more DNA damage than Furia. Lymphocyte viability did not change after exposure to different concentrations of the two pyrethroid insecticides and bleomycin. Moreover, genotoxic results demonstrated that Furia®180 SC and Bulldock®125 SC insecticides caused in vitro DNA damage in human peripheral lymphocytes.

Investigation of in vitro genotoxic effects of an anti-diabetic drug sitagliptin

Sitagliptin is an active ingredient of antidiabetic drug used in the treatment of type 2 diabetes mellitus (T2DM). In this study, the genotoxic effects of sitagliptin were determined in human lymphocytes by using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN) and comet assays. 31.25–1000 μg/mL concentrations of sitagliptin were used. Sitagliptin significantly increased the frequency of CAs and SCEs at the highest concentration at 24 h treatment and all concentrations (except 250 μg/mL for CA, except 31.25 and 62.50 μg/mL for SCE) at 48 h treatment compared with solvent control (DMSO). This compound increased the MN at only the highest concentration compared with the solvent control. Mitotic index (MI) significantly decreased at the three highest concentrations of sitagliptin at 48 h treatment. However, replication (RI) and nuclear division (NDI) indices were not affected at all the treatments. Comet assay results indicated that sitagliptin significantly increased mean comet tail intensity and tail moment at only two concentrations (62.50 and 1000 μg/mL for intensity, 125 and 1000 μg/mL for tail moment), and tail length at all concentrations (except 125 and 500 μg/mL). It was concluded that higher concentration of sitagliptin had genotoxic effects in the human lymphocytes in vitro.

EFFECTS OF LONG-TERM AEROBIC EXERCISE ON THE ANTIOXIDANT SYSTEM AND LYMPHOCYTE DNA DAMAGE BY TRIATHLON DISTANCE

Background and Objective&#x0D; This study aimed to investigate the effects of long-term aerobic exercise on muscle damage markers, lymphocyte DNA damage, and antioxidant system in amateur athletes.&#x0D; Material and Methods&#x0D; Eleven healthy men in their 30s and 40s without any medical illness, who did not smoke or drink, and had completed at least two amateur triathlon races (O2 and Olympic courses) were enrolled. They underwent physical examination and four blood sampling sessions: at rest, immediately after a race, during recovery (3 and 6 days after the race), and after completing an Olympic course. Blood sampling was performed using the same method one month later. Weight (kg) and saturation of peripheral oxygen (SpO2) were measured. Tail intensity, tail moment, and tail length, and levels of superoxide dismutase (SOD), creatine kinase (CK), and lactate dehydrogenase (LDH) were analyzed.&#x0D; Results&#x0D; First, the study found significant changes between the body weight at rest and immediately after the race (p&lt;.001) and between those immediately after the race and 3 and 6 days after the race (p&lt;.001) for both courses. Second, for both courses, SpO2 declined immediately after the race and tended to rise again during recovery, but the difference was not significant. Third, in the Olympic course, significant differences were found between lymphocyte tail moment ™ at rest and that immediately after the race (p&lt;.01) and between those immediately after the race and 3 and 6 days after the race (p&lt;.05, p&lt;.01). In the O2 course, significant differences were found between lymphocyte TM at rest and that immediately after the race (p&lt;.01), between those at rest and 3 days of recovery (p&lt;.001), between those immediately after the race and 3 days of recovery (p&lt;.001), between those at rest and 6 days of recovery (p&lt;.01), and between those at 3 and 6 days after the race (p&lt;.01). Both courses significantly differed in lymphocyte TM immediately after the race (p&lt;.05). Fourth, significant differences were observed between serum SOD at rest and that immediately after the race (p&lt;.05), between those at rest and 3 days after the race (p&lt;.01) and in serum SOD between that immediately after the race and 6 days after the race (p&lt;.05) in the Olympic course. In the O2 course, serum SOD at rest and those at 3 and 6 days after the race significantly differed (p&lt;.05). The two courses differed in serum SOD at 3 days after the race (p&lt;.05). Fifth, in both courses, compared with the levels at rest, serum CK concentrations immediately after the race (p&lt;.001) and 3 and 6 days after the race significantly differed (p&lt;.01, p&lt;.001). In both courses, significant differences were observed between serum CK concentrations immediately after the race and those at 3 and 6 days after the race (p&lt;.01, p&lt;.001) and between those at 3 and 6 days after the race (p&lt;.001). Both courses significantly differed in serum CK concentrations immediately after the race (p&lt;.001) and those at 3 and 6 days after the race (p&lt;.05). In the Olympic course, serum LDH concentrations between those at rest and immediately after the race (p&lt;.001), between those at rest and 3 days of recovery (p&lt;.01), and between those immediately after the race and 3 and 6 days after the race showed significant differences (p&lt;.001). In the O2 course, significant differences were found between serum LDH at rest and that immediately after the race (p&lt;.001), between those at rest and 3 and 6 days after the race (p&lt;.01, p&lt;.001), between those immediately after the race and 3 and 6 days after the race (p&lt;.001), and between those at 3 and 6 days after the race (p&lt;.001). The two courses significantly differed in serum LDH levels immediately after the race (p&lt;.001) and those at 3 and 6 days after the race (p&lt;.05).&#x0D; Conclusion&#x0D; Triathlon, which involves long-term high-intensity aerobic exercise, leads to temporary weight loss, DNA damage, and muscle damage after the race, and such changes are affected by exercise duration and intensity. During this change, defense mechanisms, including the antioxidant defense mechanism, are thought to protect the body from DNA and muscle damage.

Genotoxic, histologic, immunohistochemical, morphometric and hormonal effects of di-(2-ethylhexyl)-phthalate (DEHP) on reproductive systems in pre-pubertal male rats.

Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer and people are exposed to various amounts on a daily basis. This study was designed to evaluate the genotoxic, histologic, immunohistochemical, morphometric and hormonal effects of DEHP (100, 200 and 400 mg kg-1 per day DEHP) administered daily to rats by oral gavage for 28 days. The rats were divided into five groups including oil control, positive control (MMS) and treatment groups (100, 200 and 400 mg kg-1 per day DEHP). They were euthanized at the end of the experiment, organ and body weights were recorded and serum was collected for biochemical and hormone analysis. The genotoxic effect was measured in blood and sperm using the Comet assay. The testes, epididymis, prostate gland and seminal vesicle were collected and stained with hematoxylin and eosin for histopathologic analysis. Epithelial height, luminal and tubular diameters (μM) in seminiferous tubules were also measured. Moreover, the study revealed an increase in the DNA damage level in both blood lymphocytes and sperm. At the end of the experiment, the tail intensity showed a significant increase in the 100 mg kg-1 per day (p = 0.032), 200 mg kg-1 per day (p = 0.019) and 400 mg kg-1 per day (p = 0.012) dose groups compared to the control group in blood. Furthermore, testosterone was decreased in all treatment groups compared to the control group. Besides, DEHP caused a significant decrease in the leukocyte levels (p = 0.017) and hemoglobin content, as well as an increased mean cell volume (MCV) count (p = 0.029) in the 400 mg kg-1 per day group when compared to the control values. It is important to indicate that there were apoptotic cells seen in the lumen of testes in the 200 and 400 mg kg-1 per day dose groups using the Tunel method. Therefore, with this study, it has been illustrated that DEHP caused DNA damage in blood and sperm and concrete negative effects on the reproductive system in rats from the pre-pubertal period to the pubertal period. This is a unique study since there has not been any other study that presents the indicated level of DNA damage while considering the genotoxic, histologic, immunohistochemical, morphometric and hormonal effects of DEHP.

Effect on oxidative stress, hepatic chemical metabolizing parameters, and genotoxic damage of mad honey intake in rats.

A total of 66 male Wistar rats were used and six groups (control: 10 animals and experimental: 12 animals) were formed. While a separate control group was established for each study period, mad honey application to the animals in the experimental group was carried out with a single dose (12.5 g kg-1 body weight (b.w.); acute stage), at a dose of 7.5 g kg-1 b.w. for 21 days (subacute stage), and at a dose of 5 g kg-1 b.w. for 60 days (chronic stage). Tissue and blood oxidative stress markers (malondialdehyde (MDA), nitric oxide (NO), 4-hydroxynonenal (HNE), superoxide dismutase, catalase, glutathione (GSH) peroxidase, and glucose-6-phosphate dehydrogenase), hepatic chemical metabolizing parameters in the liver (cytochrome P450 2E1, nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase, nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase (CYTC), GSH S-transferase (GST), and GSH), and micronucleus and comet test in some samples were examined. Findings from the study showed that single and repeated doses given over the period increased MDA, NO, and HNE levels while decreasing/increasing tissue and blood antioxidant enzyme activities. From hepatic chemical metabolizing parameters, GST activity increased in the subacute and chronic stages and CYTC activity increased in the acute period, whereas GSH level decreased in the subacute stage. Changes in tail and head intensities were found in most of the comet results. Mad honey caused oxidative stresses for each exposure period and made some significant changes on the comet test in certain periods for some samples obtained. In other words, according to the available research results obtained, careless consumption of mad honey for different medical purposes is not appropriate.

ROS generation and JNK activation contribute to 4-methoxy-TEMPO-induced cytotoxicity, autophagy, and DNA damage in HepG2 cells.

4-Methoxy-TEMPO, a derivative of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), is a stable nitroxide radical and is generally used in organic and pharmaceutical syntheses for the oxidation of alcohols. Previously, we reported the involvement of reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) in TEMPO-induced apoptosis in mouse L5178Y cells. In this study, we investigated 4-methoxy-TEMPO induced toxicity in human HepG2 hepatoma cells and its underlying mechanisms. Treatments with 4-methoxy-TEMPO (0.5-5mM for 2-6h) caused oxidative stress as demonstrated by increased intensity of the ROS indicator H2DCF-DA, decreased levels of glutathione. 4-Methoxy-TEMPO treatment also induced DNA damage as characterized by increased levels of DNA tail intensity in the Comet assay, increased phosphorylation of related proteins including γ-H2A.X, p-Chk1, and p-Chk2, and activation of MAPK signaling pathways. In addition, 4-methoxy-TEMPO also induced autophagy as demonstrated by the conversion of LC3B-I to II, decreased level of p62, and the appearance of GFP-LC3B punctae. To investigate the crosstalk between different signaling pathways, pretreatment of HepG2 with N-acetylcysteine, an ROS scavenger, attenuated 4-methoxy-TEMPO-induced DNA damage, suppressed JNK activation, and diminished autophagy induction. Furthermore, inhibiting JNK activation by a JNK-specific inhibitor, SP600125, decreased DNA damage levels induced by 4-methoxy-TEMPO. These results suggest that multiple mechanisms including ROS generation, DNA damage, and MAPK activation contribute to 4-methoxy-TEMPO-induced toxicity.

Diclofop-methyl: A phenoxy propionate herbicide with multiple toxic effects in mouse embyro fibroblast (NIH/3T3) cell line

Diclofop-methyl is a selective post-emergence graminicide from the phenoxy propionate group of herbicides to be developed for control of wild oats, millets, and other annual grass weeds. Diclofop-methyl usage is limited in various grass weed species due to its toxic effect and exposure risks. However, total annual usage of is approximately 750.000 pounds in United States, and more in Asia. Therefore, we aimed to investigate diclofopmethyl’s toxic potentials in vitro and the following assays were used; MTT assay for cytotoxicity, comet assay for genotoxicity, generation of reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) for the potential of oxidative damage in mouse embryo fibroblast (NIH/3T3) cell line. Diclofop-methyl was observed to reduce the cell viability in a concentration manner and the half maximal inhibitory concentration (IC50) value was 301.7 μM. Diclofop-methyl caused DNA damage and oxidative stress at the concentrations between 12.5-400 μM. Tail intensities were at the range of 1.24- 58.21% with increasing concentrations, which are approximately ≤ 1.63-fold of the negative control. Also, MDA levels were increased ≥ ³11.4-fold of the negative control that denotes lipid peroxidation was induced. However, there was no significant increment in the ROS and GSH levels at all concentrations. In view of the fact that ROS has not been detected, despite its level of MDA proffers, the idea that oxidative damage may have been caused by other mechanisms. Our results indicate that diclofopmethyl was cytotoxic, genotoxic and might have oxidative damage potential in vitro conditions.

Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays

Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H2O2) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell’s response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H2O2, less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage.

Impact of surface functionalization on the uptake mechanism and toxicity effects of silver nanoparticles in HepG2 cells

Safe and successful bioapplications of metallic nanoparticles depend on their physicochemical characteristics, in particular their surface properties. This study aimed to investigate how different surface functionalization of silver nanoparticles (AgNP) affect their interaction with mammalian liver cells with regard to cytotoxicity, genotoxicity and mechanism of cellular uptake. Differentially coated AgNP were prepared by surface functionalization using sodium bis(2-ethylhexyl)-sulfosuccinate (AOTAgNP), cetyltrimethylammonium bromide (CTABAgNP), poly(vinylpyrrolidone) (PVPAgNP), poly-l-lysine (PLLAgNP), and bovine serum albumin (BSAAgNP). Data showed varying toxic potential of differentially coated AgNP. All AgNP types demonstrated concentration dependent effects on cytotoxicity and genotoxicity in HepG2 cells. Cytotoxic potential of differentially coated AgNP followed the order of BSAAgNP > PLLAgNP > CTABAgNP > AOTAgNP > PVPAgNP. Exposure of HepG2 cells to non-cytotoxic concentrations (up to 10 mg Ag/L) of AgNP for 24 h induced primary DNA damage as evaluated by alkaline comet assay. The highest increase in both comet tail length and tail intensity was produced by PLLAgNP followed by AOTAgNP, while CTABAgNP appeared to be least damaging. The main uptake mechanisms of AgNP were macropinocytosis and clathrin-mediated endocytosis. The study findings contribute to the criteria that should be considered in evaluating the biocompatibility and safety of novel nanomaterials.

Evaluation of Relationship Between Lymphocyte DNA Damages and Blood Arsenic Levels in Silver Mining Workers using Alkaline Comet Assay

The purpose of this study is to investigate the association between DNA damage and blood arsenic levels in individuals occupationally exposed to arsenic (As). A hundred-twenty exposed individuals from silver mining plate workers were monitored in the way of DNA damage in blood lymphocytes using comet assay (single cell gel electrophoresis, SSGE). The DNA damage levels were measured by BAB Bs Comet Assay system and As levels were analyzed by the atomic absorption spectrometer (AAS) system. The mean blood arsenic level was 17.03±10.85 μg/L. A statistically significant positive correlations were found between the arsenic levels and comet parameters (tail intensity, comet length and tail length) (r=0.360, r=0.334, r=0.259, respectively, p<0.01). Our results showed that exposure to arsenic may cause DNA damage in peripheral

Dynamic In Vivo Profiling of DNA Damage and Repair after Radiotherapy Using Canine Patients as a Model.

Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX) immunohistochemistry. In vivo results were then compared (in silico) with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15–60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.