Evaluation of cytotoxic,genotoxic and inflammatory response in human alveolar and bronchial epithelial cells exposed to titanium dioxide nanoparticles

The toxicity of titanium dioxide nanoparticles (TiO₂‐NPs), used in several applications, seems to be influenced by their specific physicochemical characteristics. Cyto‐genotoxic and inflammatory effects induced by a mixture of 79% anatase/21% rutile TiO₂‐NPs were investigated in human alveolar (A549) and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1 30 min, 2 and 24 h to assess potential pulmonary toxicity. The specific physicochemical properties such as crystallinity, NP size and shape, agglomerate size, surface charge and specific surface area (SSA) were analysed. Cytotoxic effects were studied by evaluating cell viability using the WST1 assay and membrane damage using LDH analysis. Direct/oxidative DNA damage was assessed by the Fpg‐comet assay and the inflammatory potential was evaluated as interleukin (IL)‐6, IL‐8 and tumour necrosis factor (TNF)‐α release by enzyme‐linked immunosorbant assay (ELISA). In A549 cells no significant viability reduction and moderate membrane damage, only at the highest concentration, were detected, whereas BEAS‐2B cells showed a significant viability reduction and early membrane damage starting from 10 µg ml^–1. Direct/oxidative DNA damage at 40 µg ml^–1 and increased IL‐6 release at 5 µg ml^–1 were found only in A549 cells after 2 h. The secretion of pro‐inflammatory cytokine IL‐6, involved in the early acute inflammatory response, and oxidative DNA damage indicate the promotion of early and transient oxidative‐inflammatory effects of tested TiO₂‐NPs on human alveolar cells. The findings show a higher susceptibility of normal bronchial cells to cytotoxic effects and higher responsiveness of transformed alveolar cells to genotoxic, oxidative and early inflammatory effects induced by tested TiO₂‐NPs. This different cell behaviour after TiO₂‐NPs exposure suggests the use of both cell lines and multiple end‐points to elucidate NP toxicity on the respiratory system. Copyright © 2014 John Wiley & Sons, Ltd.     

Direct‐oxidative DNA damage and apoptosis induction in different human respiratory cells exposed to low concentrations of sodium chromate

The mechanism of Cr(VI) genotoxicity has still not been elucidated. We used Fpg‐modified comet assay to assess direct‐oxidative DNA damage on human lung (A549) and bronchial (BEAS‐2B) cells exposed to 0.1, 0.5, 1.0 and 10 μm sodium chromate for 0.5, 1 and 4 h. Moreover we evaluated apoptosis by morphological analysis and caspase‐3 activity, also after 24 h. On A549 cells a time‐dependent DNA damage, expressed as tail DNA%, beginning from 0.5 μm was found. For oxidative DNA damage an induction after 30 min to 0.5 μm decreasing with time, and a time‐dependent increase at 10 μm was found, indicating for low Cr(VI) concentration the oxidative stress as the first event followed by direct DNA damage and for the highest concentration a time‐dependent increase in oxidative DNA damage. On BEAS‐2B cells DNA damage was induced within 1 h at 0.5–10 μm , without changes with time, showing that BEAS‐2B cells are able to resist to Cr(VI) genotoxicity. Early oxidative DNA damage at 0.1 μm decreasing with time was also found. Significant apoptosis was observed by morphological analysis in A549 cells and to a lower extent in BEAS‐2B at 10 μm . The exposure to 10 μm induced caspase‐3 activity after 4 h in BEAS‐2B and after 24 h in A549 cells. The findings show a higher responsiveness of A549 cells to genotoxic effect of Cr(VI) and early transient oxidative DNA damage in BEAS‐2B. The results emphasize the suitability of this experimental model to evaluate the early genotoxic response of different cells to non‐cytotoxic concentrations of Cr(VI) on target organ. Copyright © 2009 John Wiley & Sons, Ltd.     

Multi‐walled carbon nanotubes induce cytotoxicity and genotoxicity in human lung epithelial cells

The increasing use of nanomaterials in consumer products highlights the importance of understanding their potential toxic effects. We evaluated cytotoxic and genotoxic/oxidative effects induced by commercial multi‐walled carbon nanotubes (MWCNTs) on human lung epithelial (A549) cells treated with 5, 10, 40 and 100 µg ml^?1 for different exposure times. Scanning electron microscopy (SEM) analysis, MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays were performed to evaluate cytotoxicity. Fpg‐modified comet assay was used to evaluate direct‐oxidative DNA damage. LDH leakage was detected after 2, 4 and 24 h of exposure and viability reduction was revealed after 24 h. SEM analysis, performed after 4 and 24 h exposure, showed cell surface changes such as lower microvilli density, microvilli structure modifications and the presence of holes in plasma membrane. We found an induction of direct DNA damage after each exposure time and at all concentrations, statistically significant at 10 and 40 µg ml^?1 after 2 h, at 5, 10, 100 µg ml^?1 after 4 h and at 10 µg ml^?1 after 24 h exposure. However, oxidative DNA damage was not found. The results showed an induction of early cytotoxic effects such as loss of membrane integrity, surface morphological changes and MWCNT agglomerate entrance at all concentrations. We also demonstrated the ability of MWCNTs to induce early genotoxicity. This study emphasizes the suitability of our approach to evaluating simultaneously the early response of the cell membrane and DNA to different MWCNT concentrations and exposure times in cells of target organ. The findings contribute to elucidation of the mechanism by which MWCNTs cause toxic effects in an in vitro experimental model. Copyright © 2012 John Wiley & Sons, Ltd.     

On the mechanism of genotoxicity of ethephon on embryonic fibroblast cells

Ethephon is one of the most widely used plant growth regulator in agriculture that its application has been increased in recent years. Many reports have raised concern over the safety of this organophosphorus compound. The aim of the current study was to assess the potential genotoxic effect of ethephon on murine embryonic fibroblast (MEF) cell line, using two genotoxicity endpoints: γH2AX expression and comet assay. γH2AX served as an early and sensitive biomarker of genotoxic damage. Oxidative stress biomarkers, including reactive oxygen species (ROS), lipid peroxidation (LPO) and total antioxidant capacity were also examined. The results showed a significant increase in cell proliferation, 24?h post-treatment with 10, 40,160?μg/ml ethephon, while at the higher concentrations cytotoxic effect was observed. The γH2AX expression and γH2AX foci count per cell were significantly increased at non-cytotoxic concentrations of ethephon, accompanied with increased DNA damage as illustrated by comet assay. LPO and ROS levels were elevated only at 160?μg/ml and higher doses. The results interestingly showed that low non-cytotoxic doses of ethephon promoted DNA damage inducing cell proliferation, raising the possibility of ethephon mutagenicity. The genotoxic effect of ethephon at low doses might not relate to oxidative damage and that increased in the level of ROS and LPO generation at higher doses could account for the cytotoxic effect of ethephon. Taken together, our study provides strong in vitro evidence on potential genotoxicity of ethephon at low doses. More precise studies are needed to clarify the mutagenic effect of chronic exposure to ethephon.     

Ecotoxicity of single‐wall carbon nanotubes to freshwater snail Lymnaea luteola L.: Impacts on oxidative stress and genotoxicity

Mammalian studies have raised concerns about the toxicity of carbon nanotubes, but there is very limited data on ecogenotoxicity to aquatic organisms. The aim of this study was to determine eco‐geno toxic effects of single walled carbon nanotubes (SWCNTs) in fresh water snail, Lymnea luteola (L. luteola). A static test system was used to expose L. luteola to a freshwater control, 0.05, 0.15, 0.30, 0.46 mg/L SWCNTs for up to 4 days. SWCNTs changed a significant reduction in glutathione, glutathione‐S‐transferase, and glutathione peroxidase with in hepatopancreas of L. luteola. Lipid peroxidation (LPO) and catalase showed dose‐ and time‐dependent and statistically significant increase in hepatopancreas during SWCNTs exposure compared with control. However, a significant (p < 0.01) induction in DNA damage was observed by the comet assay in hepatopancreas cells treated with SWCNTs. These results demonstrate that SWCNTs are ecogenotoxic to freshwater snail L. luteola. The oxidative stress and comet assay can successfully be used as sensitive tools of aquatic pollution biomonitoring. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 674–682, 2015.     

Protection of DNA and membranes from gamma‐radiation induced damages by Centella asiatica

Objectives The objective of the present study was to examine the ability of Centella asiatica extract to offer protection to DNA and membranes against the deleterious effects of ionizing radiation exposure. Methods Protection of DNA under in‐vitro conditions of irradiation was estimated using plasmid relaxation assay. For in‐vivo studies the extract was administered orally to mice exposed to whole‐body γ‐radiation. The ability of the extract to offer protection against whole‐body γ‐radiation exposure was analysed by performing an alkaline comet assay on mouse bone marrow cells. The extent of lipid peroxidation was estimated using the TBARS (thio‐barbituric acid reacting substances) method, in order to monitor membrane damage. Radiation‐induced mortality of the animals following a lethal dose of γ‐radiation was also examined. Key findings Centella asiatica extract significantly reduced radiation‐induced damage to DNA. The extent of radiation‐induced mortality and lipid peroxidation was also found to be considerably reduced in animals administered with the extract. Conclusions Centella asiatica rendered radioprotection to DNA and membranes against radiation exposure, both in vitro and in vivo. We have earlier reported that administration of the extract can prevent a radiation‐induced decline in antioxidant enzyme levels. This suggests that radioprotection by Centella asiatica extract could be mediated by mechanisms that act in a synergistic manner, especially involving antioxidant activity.     

Gallic acid provokes DNA damage and suppresses DNA repair gene expression in human prostate cancer PC‐3 cells

Our earlier studies have demonstrated that gallic acid (GA) induced cytotoxic effects including induction of apoptosis and DNA damage and inhibited the cell migration and invasion in human cancer cells. However, GA‐affected DNA damage and repair gene expressions in human prostate cancer cells are still unclear. In this study, we investigated whether or not GA induces DNA damage and inhibits DNA repair gene expression in a human prostate cancer cell line (PC‐3). The results from flow cytometric assay indicated that GA decreased the percentage of viable PC‐3 cells in a dose‐ and time‐dependent manner. PC‐3 cells after exposure to different doses (50, 100, and 200 μM) of GA and various periods of time (12, 24, and 48 h) led to a longer DNA migration smear (comet tail) occurred based on the single cell gel electrophoresis (comet assay). These observations indicated that GA‐induced DNA damage in PC‐3 cells, which also confirmed by 4,6‐diamidino‐2‐phenylindole dihydrochloride staining and DNA agarose gel electrophoresis. Alternatively, results from real‐time polymerase chain reaction assay also indicated that GA inhibited ataxia telangiectasia mutated, ataxia‐telangiectasia and Rad3‐related, O⁶‐methylguanine‐DNA methyltransferase, DNA‐dependent serine/threonine protein kinase, and p53 mRNA expressions in PC‐3 cells. Taken together, the present study showed that GA caused DNA damage and inhibited DNA repair genes as well as both effects may be the critical factors for GA‐inhibited growth of PC‐3 cells in vitro. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 579–587, 2013.     

Cyto‐genotoxicity and oxidative stress in common carp (Cyprinus carpio) exposed to a mixture of ibuprofen and diclofenac

Thirty million people worldwide consume each day nonsteroidal anti‐inflammatory drugs (NSAIDs), a heterogeneous group of pharmaceuticals used for its analgesic, antipyretic, and anti‐inflammatory properties. Recent studies report high NSAID concentrations in wastewater treatment plant effluents, in surface, ground, and drinking water, and in sediments. NSAIDs are also known to induce toxicity on aquatic organisms. However, toxicity in natural ecosystems is not usually the result of exposure to a single substance but to a mixture of toxic agents, yet only a few studies have evaluated the toxicity of mixtures. The aim of this study was to evaluate the toxicity induced by diclofenac (DCF), ibuprofen (IBP), and their mixture on a species of commercial interest, the common carp Cyprinus carpio. The median lethal concentration of IBP and DCF was determined, and oxidative stress was evaluated using the following biomarkers: lipid peroxidation and activity of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase. Cyto‐genotoxicity was evaluated by micronucleus test, comet assay, and the specific activity of caspase‐3. Results show that DCF, IBP, and a mixture of these pharmaceuticals induced free radical production, oxidative stress and cyto‐genotoxicity in tissues of C. carpio. However, a greater effect was elicited by the mixture than by either pharmaceutical alone in some biomarkers evaluated, particularly in gill. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1637–1650, 2017.     

Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing

Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2?h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2?h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement).     

Cantharidin induces DNA damage and inhibits DNA repair‐associated protein levels in NCI‐H460 human lung cancer cells

Cantharidin is one of the major compounds from mylabris and it has cytotoxic effects in many different types of human cancer cells. Previously, we found that cantharidin induced cell death through cell cycle arrest and apoptosis induction in human lung cancer NCI‐H460 cells. However, cantharidin‐affected DNA damage, repair, and associated protein levels in NCI‐H460 cells have not been examined. In this study, we determined whether cantharidin induced DNA damage and condensation and altered levels of proteins in NCI‐H460 cells in vitro. Incubation of NCI‐H460 cells with 0, 2.5, 5, 10, and 15 μM of cantharidin caused a longer DNA migration smear (comet tail). Cantharidin also increased DNA condensation. These effects were dose‐dependent. Cantharidin (5, 10, and 15 μM) treatment of NCI‐H460 cells reduced protein levels of ataxia telangiectasia mutated (ATM), breast cancer 1, early onset (BRCA‐1), 14‐3‐3 proteins sigma (14‐3‐3σ), DNA‐dependent serine/threonine protein kinase (DNA‐PK), O⁶‐methylguanine‐DNA methyltransferase (MGMT), and mediator of DNA damage checkpoint protein 1 (MDC1). Protein translocation of p‐p53, p‐H2A.X (S140), and MDC1 from cytoplasm to nucleus was induced by cantharidin in NCI‐H460 cells. Taken together, this study showed that cantharidin caused DNA damage and inhibited levels of DNA repair‐associated proteins. These effects may contribute to cantharidin‐induced cell death in vitro. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1135–1143, 2015.     

Involvement of oxidative stress in methyl parathion and parathion‐induced toxicity and genotoxicity to human liver carcinoma (HepG2) cells

Methyl parathion (C₈H₁₀NO₅PS) and parathion (C₁₀H₁₄NO₅PS) are both organophosphate insecticides (OPI) widely used for household and agricultural applications. They are known for their ability to irreversibly inhibit acetylcholinesterase which often leads to a profound effect on the nervous system of exposed organisms. Many recently published studies have indicated that human exposure to OPI may be associated with neurologic, hematopoietic, cardiovascular, and reproductive adverse effects. Studies have also linked OPI exposure to a number of degenerative diseases including Parkinson's, Alzheimer's, and amyotrophic lateral sclerosis. Also, oxidative stress (OS) has been reported as a possible mechanism of OPI toxicity in humans. Hence, the aim of the present investigation was to use human liver carcinoma (HepG₂) cells as a test model to evaluate the role of OS in methyl parathion‐ and parathion‐induced toxicity. To achieve this goal, we performed the MTT [3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide] assay for cell viability, lipid peroxidation assay for malondialdehyde (MDA) production, and Comet assay for DNA damage, respectively. Results from MTT assay indicated that methyl parathion and parathion gradually reduce the viability of HepG₂ cells in a dose‐dependent manner, showing 48 h‐LD₅₀ values of 26.20 mM and 23.58 mM, respectively. Lipid peroxidation assay resulted in a significant increase (P < 0.05) of MDA level in methyl parathion‐ and parathion‐treated HepG₂ cells compared with controls, suggesting that OS plays a key role in OPI‐induced toxicity. Comet assay indicated a significant increase in genotoxicity at higher concentrations of OPI exposure. Overall, we found that methyl‐parathion is slightly less toxic than parathion to HepG₂ cells. The cytotoxic effect of these OPI was found to be associated, at least in part, with oxidative cell/tissue damage. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Effects of (+)catechin and (−)epicatechin on heterocyclic amines‐induced oxidative DNA damage

The aim of the present study was to evaluate the protective effect of (+)‐catechin and (?)‐epicatechin against 2‐amino‐3,8‐ dimethylimidazo[4,5‐f]quinoxaline (8‐MeIQx), 2‐amino‐3,4,8‐trimethylimidazo[4,5‐f]‐quinoxaline (4,8‐diMeIQx) and 2‐amino‐1‐methyl‐6‐phenyl‐imidazo[4,5‐b]pyridine (PhIP)‐induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single‐cell gel electrophoresis or comet assay. Increasing concentrations of 8‐MeIQx, 4,8‐diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose‐dependent manner. Among those, PhIP (300 µm ) exerted the highest genotoxicity. (+)‐Catechin exerted protection against oxidized purines induced by 8‐MeIQx, 4,8‐diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)‐catechin. Otherwise, (?)‐epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8‐MeIQx and 4,8‐diMeIQx. One feasible mechanism by which (+)‐catechin and (?)‐epicatechin exert their protective effect towards heterocyclic amines‐induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O‐deethylation (CYP1A1) activity was moderately inhibited by (+)‐catechin, while little effect was observed by (?)‐epicatechin. However, (+)‐catechin showed the greatest increase in UDP‐glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)‐catechin was more efficient than (?)‐epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8‐MeIQx and 4,8‐diMeIQx. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative study of cyto‐ and genotoxic potential with mechanistic insights of tungsten oxide nano‐ and microparticles in lung carcinoma cells

The exigency of semiconductor and super capacitor tungsten oxide nanoparticles (WO₃ NPs) is increasing in various sectors. However, limited information on their toxicity and biological interactions are available. Hence, we explored the underlying mechanisms of toxicity induced by WO₃ NPs and their microparticles (MPs) using different concentrations (0–300 μg ml^–1) in human lung carcinoma (A549) cells. The mean size of WO₃ NPs and MPs by transmission electron microscopy was 53.84 nm and 3.88 μm, respectively. WO₃ NPs induced reduction in cell viability, membrane damage and the degree of induction was size‐ and dose‐dependent. There was a significant increase in the percentage tail DNA and micronuclei formation at 200 and 300 μg ml^–1 after 24 hours of exposure. The DNA damage induced by WO₃ NPs could be attributed to increased oxidative stress and inflammation through reactive oxygen species generation, which correlated with the depletion of reduced glutathione content, catalase and an increase in malondialdehyde levels. Cellular uptake studies unveiled that both the particles were attached/surrounded to the cell membrane according to their size. In addition, NP inhibited the progression of the cell cycle in the G₂/M phase. Other studies such as caspase‐9 and ‐3 and Annexin‐V‐fluorescein isothiocyanate revealed that NPs induced intrinsic apoptotic cell death at 200 and 300 μg ml^–1 concentrations. However, in comparison to NPs, WO₃ MPs did not incite any toxic effects at the tested concentrations. Under these experimental conditions, the no‐observed‐significant‐effect level of WO₃ NPs was determined to be ≤200 μg ml^–1 in A549 cells.     

Platinum nanoparticles induced genotoxicity and apoptotic activity in human normal and cancer hepatic cells via oxidative stress‐mediated Bax/Bcl‐2 and caspase‐3 expression

Platinum nanoparticles (PtNPs) attract much attention due to their excellent biocompatibility and catalytic properties, but their toxic effects on normal (CHANG) and cancerous (HuH‐7) human liver cells are meagre. The cytotoxic and apoptotic effects of PtNPs (average size, 3 nm) were determined in CHANG and HuH‐7 cells. After treating these cells were with PtNPs (10, 50, 100, 200, and 300 μg/mL) for 24 and 48 hours, we observed dose‐ and time‐dependent cytotoxicity, as evaluated by using (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide, a tetrazole) (MTT) and neutral red uptake (NRU) assays. The production of reactive oxygen species (ROS) was increased in both cells after treatment with the above dose of PtNPs for 24 and 48 hours. Determination of morphological changes of cells, chromosome condensation, mitochondrial membrane potential, and caspase‐3 assays showed that PtNPs induce cytotoxicity and apoptosis in CHANG and HuH‐7 cells by altering the cell morphology and density, increasing cell population in apoptosis, and causing chromosome condensation. Furthermore, we have studied fragmentation of DNA using alkaline single cell gel electrophoresis and expression of apoptotic genes by real‐time PCR (RT‐PCR). The percentage of DNA fragmentation was more at 300 μg/mL for 48 hours in both cells, but slightly more fragmentation was found in HuH‐7 relative to CHANG cells. Considering all of the above parameters, PtNPs elicited cytotoxicity on CHANG and HuH‐7 cells by blocking cell proliferation and inducing apoptosis. Thus this study may be useful in in vitro laboratory studies using cell lines for screening the genotoxic and apoptotic potential of nanoparticles.     

Chromium oxide nanoparticle‐induced genotoxicity and p53‐dependent apoptosis in human lung alveolar cells

Chromium oxide (Cr₂O₃) nanoparticles (NPs) are being increasingly used as a catalyst for aromatic compound manufacture, abrading agents and as pigments (e.g., Viridian). Owing to increased applications, it is important to study the biological effects of Cr₂O₃ NPs on human health. The lung is one of the main exposure routes to nanomaterials; therefore, the present study was designed to determine the genotoxic and apoptotic effect of Cr₂O₃ NPs in human lung epithelial cells (A549). The study also elucidated the molecular mechanism of its toxicity. Cr₂O₃ NPs led to DNA damage, which was deduced by comet assay and cytokinesis block micronucleus assay. The damage could be mediated by the increased levels of reactive oxygen species. Further, the oxygen species led to a decrease in mitochondrial membrane potential and an increase in the ratio of BAX/Bcl‐2 leading to mitochondria‐mediated apoptosis induced by Cr₂O₃ NPs, which ultimately leads to cell death. Hence, there is a need of regulations to be imposed in NP usage. The study provided insight into the caspase‐dependent mechanistic pathway of apoptosis. Copyright © 2015 John Wiley & Sons, Ltd.     

Enantiomer‐specific profenofos‐induced cytotoxicity and DNA damage mediated by oxidative stress in rat adrenal pheochromocytoma (PC12) cells

Recent studies have shown that chiral pesticides could enantioselectively induce cytotoxicity and genotoxicity. However, investigations on molecular mechanisms of enantioselective toxicity of pesticides are limited. In this study, the role of oxidative stress in enantiomer‐specific, profenofos (PFF)‐induced cytotoxicity and genotoxicity was investigated using PC12 cells. The results demonstrated that PFF enantioselectively reduced cell viability and induced DNA damage in PC12 cells. A concentration‐ and time‐dependent significant induction of reactive oxygen species (ROS), malondialdehyde and gene expression encoding antioxidant enzyme (Cu‐ZnSOD, GST and CAT) and stress protein (HSP 70 and HSP 90) was observed in (?)­PFF, whereas (+)­PFF and rac‐PFF exhibited these effects to lesser degrees. Pre‐treatment with vitamin E (600 μM) caused a significant attenuation in the toxic effect; reversing subsequent PFF‐induced elevation of ROS and malondialdehyde (MDA) levels, further strengthening the involvement of oxidative stress in PFF‐mediated toxicity. In addition, the results also showed that PFF‐dependent ROS accumulation, MDA release and oxidative stress gene expression preceded the loss of cell viability and induction of DNA damage, and already significantly changed at concentrations which are not yet cytotoxic or genotoxic. These results indicate that oxidative stress may contribute to PFF‐induced toxicity and that it was not a consequence of it. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of inhibition of hepatic sulfotransferase activity on renal genotoxicity induced by lucidin‐3‐O‐primeveroside

Sulfotransferase 1A (SULT1A) expression is lower in the liver of humans than that of rodents. Therefore, species differences should be taken into consideration when assessing the risk of rodent hepatocarcinogens metabolically activated by SULT1A in humans. Although some renal carcinogens require SULT1A‐mediated activation, it is unclear how SULT1A activity in the liver affects renal carcinogens. To explore the effects of SULT1A activity in the liver on genotoxicity induced by SULT1A‐activated renal carcinogens, B6C3F₁ mice or gpt delta mice of the same strain background were given lucidin‐3‐O‐primeveroside (LuP), a hepatic and renal carcinogen of rodents, for 4 or 13 weeks, respectively, and pentachlorophenol (PCP) as a liver‐specific SULT inhibitor, was given from 1 week before LuP treatment to the end of the experiment. A 4 week exposure of LuP induced lucidin‐specific DNA adduct formation. The suppression of Sult1a expression was observed only in the liver but not in the kidneys of PCP‐treated mice, but co‐administration of PCP suppressed LuP‐induced DNA adduct formation in both organs. Thirteen‐week exposure of LuP increased mutation frequencies and cotreatment with PCP suppressed these increases in both organs. Given that intact levels of SULT activity in the liver were much higher than in the kidneys of rodents, SULT1A may predominantly activate LuP in the liver, consequently leading to genotoxicity not only in the liver but also in the kidney. Thus, species differences should be considered in human risk assessment of renal carcinogens activated by SULT1A as in the case of the corresponding liver carcinogens.     

Detection and quantification of genotoxicity in wastewater-treated Tetrahymena thermophila using the comet assay

In the present study, the comet, or single-cell, gel electrophoresis assay was adapted for use with the ubiquitous unicellular protozoan Tetrahymena thermophila, and the method was evaluated for its ability to detect DNA damage induced by known genotoxins and wastewater samples. The original comet assay protocol was substantially modified (e.g., lower concentrations of detergents were used in the lysis buffer; electrophoresis time was reduced). Using the modified method, T. thermophila were subjected to short exposures of phenol, hydrogen peroxide, and formaldehyde, leading to concentration-dependent increases in DNA damage. The genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant was evaluated. The results indicated that the influent wastewater was genotoxic and that the genotoxicity in the effluent water was substantially reduced. We assume employing T. thermophila in the use of the comet assay may become a cost-effective and reliable tool for genotoxicity screening and monitoring of wastewater and similar systems.     

Rhamnose‐coated superparamagnetic iron‐oxide nanoparticles: an evaluation of their in vitro cytotoxicity,genotoxicity and carcinogenicity

Tumor recurrence after the incomplete removal of a tumor mass inside brain tissue is the main reason that scientists are working to identify new strategies in brain oncologic therapy. In particular, in the treatment of the most malignant astrocytic tumor glioblastoma, the use of magnetic nanoparticles seems to be one of the most promising keys in overcoming this problem, namely by means of magnetic fluid hyperthermia (MFH) treatment. However, the major unknown issue related to the use of nanoparticles is their toxicological behavior when they are in contact with biological tissues. In the present study, we investigated the interaction of glioblastoma and other tumor cell lines with superparamagnetic iron‐oxide nanoparticles covalently coated with a rhamnose derivative, using proper cytotoxic assays. In the present study, we focused our attention on different strategies of toxicity evaluation comparing different cytotoxicological approaches in order to identify the biological damages induced by the nanoparticles. The data show an intensive internalization process of rhamnose‐coated iron oxide nanoparticles by the cells, suggesting that rhamnose moiety is a promising biocompatible coating in favoring cells’ uptake. With regards to cytotoxicity, a 35% cell death at a maximum concentration, mainly as a result of mitochondrial damages, was found. This cytotoxic behavior, along with the high uptake ability, could facilitate the use of these rhamnose‐coated iron‐oxide nanoparticles for future MFH therapeutic treatments. Copyright © 2015 John Wiley & Sons, Ltd.