Effect of low-dose exposure of aluminium oxide nanoparticles in Swiss albino mice: Histopathological changes and oxidative damage.

Rapid growth in the use of aluminium oxide nanoparticles (Al2O3 NPs) in various fields such as medicine, pharmacy, cosmetic industries, and engineering creates concerns since the literature is replete with data regarding their toxicity in living organisms. The objective of the present study was to demonstrate the potential toxicological manifestations of repeated exposure to Al2O3 NP at low doses in vivo. In the present study, Al2O3 NP was orally administered at 15, 30 or 60 mg kg-1 body weight for 5 days to Swiss albino male mice. A battery of well-defined assays was undertaken to evaluate aluminium (Al) bioaccumulation, haematological and histological changes, oxidative damage and genotoxicity. Physico-chemical characterisation demonstrated increases in hydrodynamic diameter along the concentration gradient of Al2O3 NP dispersed in MilliQ water. Brain, liver, spleen, kidney and testes showed high Al retention levels. Histopathological lesions were prominent in the brain and liver. Al2O3 NP treatment increased levels of lipid peroxidation and decreased glutathione content in the test organs at all dose levels. The enzyme activities of catalase and superoxide dismutase were also significantly altered. DNA damage quantified using the comet assay was markedly increased in all the soft organs studied. Anatomical abnormalities, redox imbalance and DNA damage were positively correlated with Al retention in the respective organs. Size, zeta potential and colloidal state might have contributed to the bio-physico-chemical interactions of the NPs in vivo and were responsible for the non-linear dose response. The overall data indicate that Al2O3 NP exposure may result in adverse health consequences, inclusive of but not limited to disturbed redox homeostasis, hepatocellular toxicity, neurodegeneration and DNA damage.

Nanoscale zerovalent iron particles induce differential cytotoxicity, genotoxicity, oxidative stress and hemolytic responses in human lymphocytes and erythrocytes in vitro.

The growing usage of nanoscale zerovalent iron particles (nZVI) in the remediation of soil, ground/surface water has elicited large-scale environmental release triggering human exposure. The size of nanomaterials is a key regulator of toxicity. However, the effect of a variable size of nZVI on genotoxicity is unexplored in human cells. To the best of our knowledge, in this study, the cytotoxic, genotoxic and hemolytic potential of nZVI-1 (15 nm) and nZVI-2 (50 nm) at concentrations of 5, 10 and 20 µg/mL was evaluated for the first time in human lymphocytes and erythrocytes treated for 3 hours. In erythrocytes, spherocytosis and echinocytosis occurred upon exposure to nZVI-1 and nZVI-2, respectively, leading to hemolysis. Lymphocytes treated with 20 µg/mL nZVI-2 and 10 µg/mL nZVI-1, incurred maximum DNA damage, although nZVI-2 induced higher cyto-genotoxicity than nZVI-1. This can be attributed to higher Fe ion dissolution and time/concentration-dependent colloidal destabilization (lower zeta potential) of nZVI-2. Although nZVI-1 showed higher uptake, its lower genotoxicity can be due to lesser Fe content, Fe ion dissolution and superior colloidal stability (higher zeta potential) compared with nZVI-2. Substantial accumulation of Ca2+ , superoxide anions, hydroxyl radicals and H2 O2 leading to mitochondrial impairment and altered antioxidant enzyme activity was noted at the same concentrations. Pre-treatment with N-acetyl-cysteine modulated these parameters indicating the indirect action of reactive oxygen species in nZVI-induced DNA damage. The morphology of diffused nuclei implied the possible onset of apoptotic cell death. These results validate the synergistic role of size, ion dissolution, colloidal stability and reactive oxygen species on cyto-genotoxicity of nZVI and unlock further prospects in its environmental nano-safety evaluation.

Role of cerium oxide nanoparticle-induced autophagy as a safeguard to exogenous H2O2-mediated DNA damage in tobacco BY-2 cells.

The effect of cerium oxide nanoparticle (CeNP) in plants has elicited substantial controversy. While some investigators have reported that CeNP possesses antioxidant properties, others observed CeNP to induce reactive oxygen species (ROS). In spite of considerable research carried out on the effects of CeNP in metazoans, fundamental studies that can unveil its intracellular consequences linking ROS production, autophagy and DNA damage are lacking in plants. To elucidate the impact of CeNP within plant cells, tobacco BY-2 cells were treated with 10, 50 and 250 µg ml-1 CeNP (Ce10, Ce50 and Ce250), for 24 h. Results demonstrated concentration-dependent accumulation of Ca2+ and ROS at all CeNP treatment sets. However, significant DNA damage and alteration in antioxidant defence systems were noted prominently at Ce50 and Ce250. Moreover, Ce50 and Ce250 induced DNA damage, analysed by comet assay and DNA diffusion experiments, complied with the concomitant increase in ROS. Furthermore, to evaluate the antioxidant property of CeNP, treated cells were washed after 24 h (to minimise CeNP interference) and challenged with H2O2 for 3 h. Ce10 did not induce genotoxicity and H2O2 exposure to Ce10-treated cells showed lesser DNA breakage than cells treated with H2O2 only. Interestingly, Ce10 provided better protection over N-acetyl-L-cysteine against exogenous H2O2 in BY-2 cells. CeNP exposure to transgenic BY-2 cells expressing GFP-Atg8 fusion protein exhibited formation of autophagosomes at Ce10. Application of vacuolar protease inhibitor E-64c and fluorescent basic dye acridine orange, further demonstrated accumulation of particulate matters in the vacuole and occurrence of acidic compartments, the autophagolysosomes, respectively. BY-2 cells co-treated with CeNP and autophagy inhibitor 3-methyladenine exhibited increased DNA damage in Ce10 and cell death at all assessed treatment sets. Thus, current results substantiate an alternative autophagy-mediated, antioxidant and geno-protective role of CeNP, which will aid in deciphering novel phenomena of plant-nanoparticle interaction at cellular level.

In planta genotoxicity of nZVI: influence of colloidal stability on uptake, DNA damage, oxidative stress and cell death.

Nanoremediation of soil, ground and surface water using nanoscale zerovalent iron particles (nZVI) has facilitated their direct environmental exposure posing ecotoxicological concerns. Numerous studies elucidate their phytotoxicity in terms of growth and their fate within the plant system. However, their potential genotoxicity and cytotoxicity mechanisms are not known in plants. This study encompasses the physico-chemical characterisation of two forms of nZVI (nZVI-1 and nZVI-2) with different surface chemistries and their influence on uptake, root morphology, DNA damage, oxidative stress and cell death in Allium cepa roots after 24 h. To our knowledge, this is the first report on the cyto-genotoxicity of nZVI in plants. The adsorption of nZVI on root surfaces caused root tip, epidermal and root hair damage as assessed by Scanning Electron Microscopy. nZVI-1, due to its colloidal destabilisation (low zeta potential, conductivity and high polydispersity index), smaller size and high uptake imparted enhanced DNA damage, chromosome/nuclear aberrations (CAs/NAs) and micronuclei formation compared to nZVI-2. Although nZVI-2 exhibited high zeta potential and conductivity, its higher dissolution and substantial uptake induced genotoxicity. nZVI incited the generation of reactive oxygen species (ROS) (hydrogen peroxide, superoxide and hydroxyl radicals) leading to membrane lipid peroxidation, electrolyte leakage and mitochondrial depolarisation. The inactivation of catalase and insignificant glutathione levels marked the onset of oxidative stress. Increased superoxide dismutase and guaiacol peroxidase enzyme activities, and proline content indicated the activation of antioxidant defence machinery to alleviate ROS. Moreover, ROS-mediated apoptotic and necrotic cell death occurred in both nZVI-1 and nZVI-2-treated roots. Our results open up further possibilities in the environmental safety appraisal of bare and modified nZVI in correlation with their physico-chemical characters.

Genotoxicity of antiobesity drug orlistat and effect of caffeine intervention: an in vitro study.

CONTEXT: Obesity is a major global health problem associated with various adverse effects. Pharmacological interventions are often necessary for the management of obesity. Orlistat is an FDA-approved antiobesity drug which is a potent inhibitor of intestinal lipases. OBJECTIVE: In the current study, orlistat was evaluated for its genotoxic potential in human lymphocyte cells in vitro and was compared with that of another antiobesity drug sibutramine, presently withdrawn from market due its undesirable health effects. Caffeine intake may be an additional burden in people using anorectic drugs, therefore, further work is needed to be carried out to evaluate the possible effects of caffeine on orlistat-induced DNA damage. MATERIALS AND METHODS: Human lymphocytes were exposed to orlistat (250, 500 and 1000 µg/ml), sibutramine (250, 500 and 1000 µg/ml) and caffeine (25, 50, 75, 100, 125 and 150 µg/ml) to assess their genotoxicity by comet assay in vitro. In addition, lymphocytes were co-incubated with caffeine (50, 75 and 100 µg/ml) and a single concentration of orlistat (250 µg/ml). RESULTS: Orlistat and sibutramine were genotoxic at all concentrations tested, sibutramine being more genotoxic. Caffeine was found to be genotoxic at concentrations 125 µg/ml and above. Co-treatment of orlistat with non-genotoxic concentrations (50, 75 and 100 µg/ml) of caffeine lead to a decrease in DNA damage. DISCUSSION AND CONCLUSION: Orlistat can induce DNA damage in human lymphocytes in vitro and caffeine was found to reduce orlistat-induced genotoxicity.

Malathion and dithane induce DNA damage in Vicia faba.

The increasing use of pesticides such as malathion and dithane in agriculture causes environmental mutagenicity. However, their genotoxicity in edible crops is seldom assessed. In this study, the genotoxic potential of malathion and dithane was evaluated in the roots of Vicia faba L. All three concentrations (0.05, 0.1, and 0.2%) of malathion and dithane tested resulted in a significant decrease in root length and inhibited seed germination. Cytological observations showed that the mitotic frequency in the root meristematic cells decreased parallel to the increase in concentrations, and the increase in chromosome aberrations and micronuclei frequency was concentration dependent. Alkaline comet assay revealed significant onset of DNA damage at all tested concentrations. For the randomly amplified polymorphic (RAPD)-polymerase chain reaction (PCR) analyses, 10 random RAPD primers were found to produce 116 unique polymorphic RAPD band fragments of 223-3139 bp. Each primer generated 3-15 RAPD bands on an average. The percentage of polymorphic DNA fragments was higher in malathion-exposed plants than dithane ones. The changes in RAPD profiles included disappearance and/or appearance of DNA bands in malathion and dithane treatment. Hence, DNA damage observed by the cytogenetic endpoints and comet assay corroborated with RAPD-PCR analysis. A total of 15 new protein bands of molecular weight ranging 11.894-226.669 kDa were observed in roots of Vicia plants that were exposed to the pesticides. The number of new protein bands was higher in malathion-treated DNA samples than in dithane-treated ones. Based on the results, we conclude that the pesticides can alter genomic template stability and change protein profiles. Malathion was more genotoxic than dithane. Therefore, RAPD assays can be useful in determining genotoxicity of pesticides in V. faba and other crops along with other quantitative parameters.

Argemone oil induces genotoxicity in mice.

CONTEXT: Argemone mexicana L. is native to Mexico and the plant extracts are used in traditional medicine in India and South American countries. Argemone oil (AO) is a common adulterant of mustard oil in India and causes serious pathophysiological consequences leading to outbreaks of epidemic dropsy among consumers. In vivo cytogenetic studies on the toxicological effects of AO and its component alkaloids are limited. OBJECTIVE: The present study was undertaken to evaluate the safety of AO by assessment of their in vivo genotoxic potential in bone marrow cells of mice. MATERIALS AND METHODS: AO mixed in corn oil in the proportions of 0.01, 0.1, and 1 ml AO/kg body weight in a total volume of 10 ml/kg body weight and a single undiluted dose of AO (10 ml/kg body weight) were administered intraperitoneally in separate groups of male Swiss Albino mice for 24 h. In addition, a single concentration of sanguinarine (SG) (50 mg/kg body weight) was also administered. Genotoxicity was evaluated by chromosome aberration (CA) and sister chromatid exchange (SCE) tests. Bromodeoxyuridine (BrdU) differential technique was used to study the effect on cell replication by the calculation of average generation time (AGT). RESULTS: The minimum effective concentrations that produced significant frequencies of CA and SCE were 0.1 and 0.01 ml/kg, respectively. AO and SG induced an insignificant increase of AGT indicating that they are non-cytotoxic in the concentrations tested. DISCUSSION AND CONCLUSION: Our results confirm that AO is genotoxic even at low concentrations and its usage should be checked.

Synaptically-targeted long non-coding RNA SLAMR promotes structural plasticity by increasing translation and CaMKII activity.

Long noncoding RNAs (lncRNAs) play crucial roles in maintaining cell homeostasis and function. However, it remains largely unknown whether and how neuronal activity impacts the transcriptional regulation of lncRNAs, or if this leads to synapse-related changes and contributes to the formation of long-term memories. Here, we report the identification of a lncRNA, SLAMR, which becomes enriched in CA1-hippocampal neurons upon contextual fear conditioning but not in CA3 neurons. SLAMR is transported along dendrites via the molecular motor KIF5C and is recruited to the synapse upon stimulation. Loss of function of SLAMR reduces dendritic complexity and impairs activity-dependent changes in spine structural plasticity and translation. Gain of function of SLAMR, in contrast, enhances dendritic complexity, spine density, and translation. Analyses of the SLAMR interactome reveal its association with CaMKIIα protein through a 220-nucleotide element also involved in SLAMR transport. A CaMKII reporter reveals a basal reduction in CaMKII activity with SLAMR loss-of-function. Furthermore, the selective loss of SLAMR function in CA1 disrupts the consolidation of fear memory in male mice, without affecting their acquisition, recall, or extinction, or spatial memory. Together, these results provide new molecular and functional insight into activity-dependent changes at the synapse and consolidation of contextual fear.

SLAMR, a synaptically targeted lncRNA, facilitates the consolidation of contextual fear memory.

LncRNAs are involved in critical processes for cell homeostasis and function. However, it remains largely unknown whether and how the transcriptional regulation of long noncoding RNAs results in activity-dependent changes at the synapse and facilitate formation of long-term memories. Here, we report the identification of a novel lncRNA, SLAMR, that becomes enriched in CA1- but not in CA3-hippocampal neurons upon contextual fear conditioning. SLAMR is transported to dendrites via the molecular motor KIF5C and recruited to the synapse in response to stimulation. Loss of function of SLAMR reduced dendritic complexity and impaired activity dependent changes in spine structural plasticity. Interestingly, gain of function of SLAMR enhanced dendritic complexity, and spine density through enhanced translation. Analyses of the SLAMR interactome revealed its association with CaMKIIα protein through a 220-nucleotide element and its modulation of CaMKIIα activity. Furthermore, loss-of-function of SLAMR in CA1 selectively impairs consolidation but neither acquisition, recall, nor extinction of fear memory and spatial memory. Together, these results establish a new mechanism for activity dependent changes at the synapse and consolidation of contextual fear.

TEM observation of compacted DNA of Synechococcus elongatus PCC 7942 using DRAQ5 labeling with DAB photooxidation and osmium black.

To visualize the fine structure of compacted DNA of Synechococcus elongatus PCC 7942, which appears at a specific time in the regular light/dark cycle prior to cell division, ChromEM with some modifications was applied. After staining DNA with DRAQ5, the cells were fixed and irradiated by red laser in the presence of 3,3'-diaminobenzidine and subsequently fixed with OsO4. A system with He-Ne laser (633 nm) was set up for efficient irradiation of the bacterial cells in aqueous solution. The compacted DNA was visualized by transmission electron microscopy, in ultrathin sections as electron dense staining by osmium black.

Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles: Influence of surface modification on biodistribution, retention, DNA damage and oxidative stress.

Superparamagnetic iron oxide nanoparticles (SPION) require stable surface modifications to render safe nanocapsules for biomedical applications. Herein, two types of surface modified poly(lactic-co-glycolic acid)-encapsulated SPION were synthesized using either α-tocopheryl-polyetheleneglycol-succinate (TPGS) or didodecyl-dimethyl-ammonium-bromide (DMAB) as surfactants by emulsification. SPION-TPGS (180 nm) was larger than SPION-DMAB (25 nm) and uncoated SPION (10 nm). Both formulations were positively charged and induced lower cyto-genotoxicity and ROS generation than uncoated SPION in human lymphocytes. SPION-DMAB was least cyto-genotoxic among the three. Based on these results, mice were gavaged with the formulations for 5 consecutive days and biocompatibility studies were performed on the 7th and 21st days. ICP-AES and Prussian blue staining revealed the internalization of SPION-DMAB in brain and spleen, and SPION-TPGS in liver and kidney on day 7. This was correlated with high DNA damage and oxidative stress in the same organs. Substantial clearance of Fe was accompanied by reduced genotoxicity and oxidative stress on day 21. Therefore, SPION-DMAB can be further studied for oral drug delivery to the brain and imaging of cerebral tissue without any functional ligand or external magnetic field.

Manganese oxide nanoparticles induce genotoxicity and DNA hypomethylation in the moss Physcomitrella patens.

The genotoxicity of nanoparticles is a major concern for nano-safety appraisal in the bryophytes as they are the primary colonizers of bare land, indicators of atmospheric pollution and excellent accumulators of trace metals. The present study for the first time evince the in planta genotoxicity of MnONP in Physcomitrella patens a model plant system utilized for evolutionary developmental genetics. The induction of DNA strand breaks was confirmed by comet assay at all tested concentrations corroborated with the enhanced generation of ROS, increase in Mn dissolution, uptake and internalization. Genotoxicity is often coupled with epigenetic alterations. In the present study, global DNA methylation pattern at the level of single cells was studied by the methylation sensitive comet assay using the isochizomeric restriction endonucleases HpaII (digests unmethylated and hemimethylated DNA) and MspI (digests methylated DNA at 5'-CmCGG-3'). MnONP incited DNA hypomethylation in P. patens gametophores treated with the highest concentration of MnONP (20 µg/mL). The DNA hypomethylation incurred upon MnONP exposure was comparable with that of the DNA methylation blocker 5-azacytidine. This can be ascribed to its clastogenic potential mediated by the formation of H2O2, OH and O2¯. There are no reports on the epigenotoxicity of nanomaterials in plants utilizing the detection of DNA damage and DNA methylation. This can open up new avenues of research on the assessment of the epigenotoxic impact of environmentally relevant nanoparticles using bryophytes as model indicator plant system.

Genotoxicity of engineered nanoparticles in higher plants.

Nanoparticles (NPs) are an emerging environmental threat. However, studies of NPs in different environmental components are limited. In this review, we discuss studies that have evaluated the genotoxicity of NPs in higher plants. Among the 29 studies reviewed, silver NPs were most studied (n = 7 articles), with fewer studies reporting the genotoxicity of carbon nanotubes (n = 3), titanium dioxide NPs (n = 4), and zinc oxide NPs (n = 3). Most of the genotoxicity studies were performed in the model plant systems Allium sp (n = 22), Nicotiana sp (n = 4) and Vicia sp (n = 4) using chromosome aberration (n = 22), micronucleus (n = 15) and comet assays (n = 14). Genotoxicity was observed in most of the studies; however, many studies did consider key determinants of NP toxicity such as particle characterization, dissolution, and uptake. From this review, we propose a set of guidelines that should be considered when reporting results of NP toxicity in plants.