昆布多糖硫酸酯对H2O2诱导乳鼠心肌细胞损伤的保护作用Δ

目的研究昆布多糖硫酸酯(LAPS)对过氧化氢(H2O2)诱导乳鼠心肌细胞氧化损伤的保护作用。方法分次消化法分离原代培养的乳鼠心肌细胞,建立H2O2诱导心肌细胞损伤模型;四甲基偶氮唑盐(MTT)法检测不同浓度LAPS对心肌细胞的保护作用;试剂盒检测LAPS对细胞乳酸脱氢酶(LDH)、肌酸磷酸激酶(CK)、丙二醛(MDA)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)含量的影响;利用荧光探针二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)检测细胞内活性氧(ROS)含量。结果100μmol/L的H2O2能明显造成心肌细胞损伤,LAPS能改善H2O2所造成的心肌细胞损伤;LAPS能够明显降低的过氧化损伤心肌细胞外液中LDH和CK含量、明显降低过氧化损伤心肌细胞内MDA和活性氧(ROS)的含量、明显提高过氧化损伤心肌细胞内SOD、GSH-Px和CAT的活力。结论 LAPS对心肌细胞的过氧化损伤有保护作用,与其降低或阻断脂质过氧化反应,降低细胞内ROS含量,进而阻断因ROS对细胞造成的损害,及时修复受损细胞有着密切关系。     

Lipid domain formation modulates activities of snake venom phospholipase A2 enzymes

The goal of the present study is to elucidate the effect of lipid domain formation on activities of Naja naja atra and Bungarus multicinctus phospholipase A₂ (PLA₂) enzymes. Sphingomyelin inhibited enzymatic activity and membrane-damaging activity of PLA₂ against egg yolk phosphatidylcholine (EYPC), while cholesterol and cholesterol sulfate abrogated the inhibitory effect of sphingomyelin. The ability of cholesterol and cholesterol sulfate to abolish the inhibitory effect of sphingomyelin was closely related to their capacity to induce domain formation in EYPC/sphingomyelin vesicles. Laurdan fluorescence measurement revealed that membrane packing of EYPC/sphingomyelin vesicles was differently affected by cholesterol and cholesterol sulfate. Unlike cholesterol, cholesterol sulfate was unable to promote domain formation in dipalmitoylphosphatidylcholine (DPPC) vesicles. Cholesterol increased but cholesterol sulfate reduced PLA₂ activity against DPPC. Self-quenching studies and trinitrophenylation of Lys residues revealed that PLA₂ enzymes adopted different membrane-bound mode upon absorption onto the membrane bilayers comprised of different lipid compositions. Collectively, our data indicate that lipid domain formation regulates PLA₂ activity, and suggest that the physical state of membrane bilayers changes the interactive mode of PLA₂ with phospholipids.     

Effects of Some Antibiotics on Activity of Glucose-6-Phosphate Dehydrogenase from Human Erythrocytes In Vitro and Effect of Isepamicin Sulfate on Activities of Antioxidant Enzymes in Rat Erythrocytes

The purpose of this study was to investigate effects of some antibiotics on glucose-6-phosphate dehydrogenase (G6PD), antioxidant enzymes, and malondialdehyde (MDA). Initially, for in vitro studies, G6PD was purified from human erythrocyte, 9811-fold in a yield of 42.4% by using ammonium sulfate precipitation and 2′,5′ ADP-Sepharose 4B affinity gel. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of four different antibiotics (isepamicin sulfate, meropenem, chloramphenicol, and thiamphenicol glisinat hydrochloride) were investigated on the purified enzyme. K_i value and type of inhibition were determined by means of Lineweaver–Burk graphs and regression analysis graphs. Isepamicin sulfate inhibited the enzyme activity (I₅₀ value, 2.1 mM; K_i value, 1.7 mM), whereas thiamphenicol glisinat hydrochloride activated the G6PD dose dependently. Other drugs showed no inhibition and activation effect. In addition, the effects of isepamicin sulfate on the activities of G6PD, glutathione reductase (GR), superoxide dismutases (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione S-transferase (GST) and MDA contentrations were examined in Sprague-Dawley rat erythrocytes in vivo. A marked alteration in the activities of these enzymes and MDA levels may be the result of oxidative stress in the rats receiving isepamicin sulfate.     

Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora

Two distinct phospholipase A₂ (PLA₂) inhibitory proteins (PLIs) were purified from the serum of the Japanese rat snake, Elaphe climacophora. The 150-kDa inhibitor, a trimer of a 50-kDa subunit, specifically inhibited the basic PLA₂ purified from the venom of Gloydius brevicaudus, whereas the 120-kDa one composed of two distinct 25-kDa subunits, A and B, inhibited both the acidic and basic PLA₂s of G. brevicaudus. On the basis of their amino acid sequences, these inhibitors were assigned as PLIβ and PLIγ, respectively. A PLIα homolog (PLIα-like protein; PLIα-LP) having an apparent molecular weight of 50-kDa and composed of 15-kDa subunits was also purified from the E. climacophora serum. This homolog was immunoreactive with antibody raised against the G. brevicaudus PLIα, but lacked in the inhibitory activity toward the acidic and basic PLA₂s. The cDNAs encoding PLIα-LP, PLIβ, PLIγ-A, and PLIγ-B were cloned from liver RNA, and their nucleotide sequences were compared with those of other venomous and non-venomous snakes.     

A phospholipase A2 from Bothrops asper snake venom activates neutrophils in culture: Expression of cyclooxygenase-2 and PGE2 biosynthesis

In this study, the production of prostaglandin E₂ (PGE₂) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A₂ (PLA₂), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA₂s (cytosolic PLA₂ and Ca²⁺-independent PLA₂) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE₂ levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE₂ production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF₃), an intracellular PLA₂ inhibitor, but not bromoenol lactone (BEL), an iPLA₂ inhibitor, suppressed the MT-III-induced AA and PGE₂ release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE₂. COX-2 isoform is preeminent over COX-1 for production of PGE₂ stimulated by MT-III. PGE₂ and AA release by MT-III probably is related to cPLA₂ activation.     

Molecular Cloning and Pharmacological Properties of an Acidic PLA2 from Bothrops pauloensis Snake Venom

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A₂ (PLA₂) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA₂-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA₂-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA₂-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA₂-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA₂s. The phylogenetic analyses showed that BpPLA₂-TXI forms a group with other acidic D49 PLA₂s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.     

Contribution of oxidative stress in acute intestinal mucositis induced by 5 fluorouracil (5-FU) and its pro-drug capecitabine in rats

This study was designed to examine the contribution of oxidative stress in gastrointestinal disorders after an intraperitoneal administration of 5 fluorouracil (5-FU; 100?mg/kg of body weight (b.w.)) and capecitabine oral administration (500?mg/kg b.w.). The animals were divided into three groups: Group A (NaCl,10?ml/kg of b.w.) considered as control group, group B was intoxicated by 5-FU and group C was the group of animals treated with capecitabine (CAP). To evaluate the secretory and enteropooling effects, we used magnesium sulfate (MgSO₄), 1?ml/100?g of b.w. as a hypersecretion agent . The mucosal gastro-intestinal specimens were scraped and examined for biological markers of oxidative stress and intracellular mediators. These anticancer drugs caused many intestinal damages manifested by an elevation of fluid accumulation and imbalance in electrolytes secretion. The intestinal tissues from treated rats not only showed a significant increase in malondialdehyde (MDA), protein carbonylation and hydrogen peroxide (H₂O₂) production. but also showed a significant depletion of enzymatic and non-enzymatic antioxidant, such as, glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and sulfhydryl groups (-SH). These effects were related with histopathological damage and a perturbation of intracellular mediators. As expected, these disturbances were observed in the group of rats poisoned by the MgSO₄. Data suggest the contribution of oxidative stress in chemotherapy-induced many disorders in intestinal tract.     

Guanidination of notexin promotes its phospholipase A2 activity-independent fusogenicity on vesicles with lipid-supplied negative curvature

To address the requirement of phospholipase A₂ (PLA₂) activity in membrane fusion events and membrane perturbation activity of notexin and guanidinated notexin (Gu-notexin), the present study was conducted. Notexin and Gu-notexin did not show PLA₂ activity after the removal of Ca²⁺ with EDTA. Metal-free notexin and Gu-notexin were found to induce membrane leakage and fusion of phospholipid vesicles. Fusogenic activity of native and modified notexin correlated positively with their membrane-damaging activity underlying the deprivation of PLA₂ activity. Compared with Ca²⁺-bound Gu-notexin, fusogenicity of metal-free Gu-notexin was notably increased by incorporation of cholesterol, cholesterol sulfate, phosphatidylethanolamine, α-tocopherol and phosphatidic acid that supplied negative curvature into phospholipid bilayer. The ability of Gu-notexin to induce membrane fusion of vesicles with lipid-supplied negative curvature was higher than that of notexin regardless of the absence or presence of Ca²⁺. Consistently, metal-free Gu-notexin markedly induced membrane fusion of red blood cells (RBCs) compared with metal-free notexin, and fusion activity of metal-free Gu-notexin on cholesterol-depleted RBCs notably reduced. Compared with notexin, Gu-notexin highly induced uptake of calcein-loaded phosphatidylcholine (PC)/cholesterol and PC/cholesterol sulfate vesicles by K562 cells in the presence of EDTA. Taken together, our data suggest that notexin and Gu-notexin could induce vesicle leakage and fusion via a PLA₂ activity-independent mechanism, and guanidination promotes PLA₂ activity-independent fusogenicity of notexin on vesicles with lipid-supplied negative curvature.     

Cicletanine sulfate: inhibition of anion transport systems and natriuretic activity

Summary In contrast with cicletanine, its urinary sulfoconjugate metabolite (cicletanine sulfate) was active on membrane ion transport in human red blood cells. Cicletanine sulfate was a more potent inhibitor of the Na⁺ dependent [Cl^–/HC0₃ ^–] exchanger (IC₅₀ = 9±3 × 10^–5 mol/l; mean±SD of 4 experiments) than cicletanine (IC₅₀ = 10^–3 mol/l). This inhibitory potency was intermediate between that of xipamide (IC₅₀ = 2 × 10^–5 mol/l) and that of furosemide (IC₅₀ = 2 × 10^–4 mol/l). Moreover, cicletanine sulfate exhibited modest inhibitory potency against the [Na⁺,K⁺,Cl^–]-cotransport system (IC₅₀ = 1±0.3 × 10^–3 mol/I; mean±SD of 4 experiments) and poor inhibitory activity against the [K⁺,CI^–]-cotransport system. Cicletanine sulfate was unable to modify the activity of Cl^–-independent membrane carriers(Na⁺ : H⁺exchanger,Ca ²⁺pump, Na⁺ : Li⁺ countertransport system and Na⁺,K⁺ pump). Following renal intraarterial administration in rats, cicletanine sulfate and not cicletanine, exhibited salidiuretic activity. In conclusion, the urinary sulfo-conjugate of cicletanine is an active anion transport inhibitor and natriuretic metabolite. In fact, this metabolite may be responsible for the salidiuretic action of cicletanine. Send offprint requests to R. Garay at the above address     

Different mechanisms of the inhibition of the transient outward current in rat ventricular myocytes

The mechanism of drug-induced inhibition of the transient outward current, I_to, has been investigated in rat ventricular myocytes using the whole cell patch clamp technique. I_to was activated by 300 ms depolarizing voltage clamp steps in 10 mV increments from –50 mV up to +40 mV. At +40 mV, I_to peaked after about 3 ms, and the time course of inactivation was appropriately described by two time constants, _fast = 17 ms and _slow = 203 ms. Verapamil, quinidine sulfate and nifedipine preferentially depressed I_to at the end of the 300 ms depolarizing voltage clamp step; the inactivation of I_to was accelerated by all drugs, whereas peak I_to was less affected. The time course of drug action at +40 mV was calculated by the fractional changes of I_to. Verapamil, quinidine sulfate and nifedipine exerted a block of I_to. increasing during the depolarizing voltage clamp step. The onset of block in response to verapamil, quinidine sulfate and nifedipine (30 mol/each) was appropriately described by monoexponential functions with time constants _on = 9.3, 1.7 and 1.1 ms, respectively. Relief from block by verapamil, quinidine sulfate and nifedipine at –50 mV was assessed by comparison of the recovery process of peak I_to from inactivation with or without drugs. _off amounted to 695 ms in the case of quinidine sulfate; verapamil and nifedipine did not significantly affect the recovery process so that the determination of the time course of relief from block was not possible. 4-Aminopyridine preferentially depressed peak I_to in a concentration-dependent manner, whereas I_to at the end of the 300 ms depolarizing voltage step remained unaffected. The block of I_to by 4-aminopyridine (3 mmol/l) decreased during the voltage step from –50 mV to +40 mV. Relief from block was described by _off = 30.4 ms. The efficacy of 4-aminopyridine was diminished at short and enhanced at long pulse intervals (reverse use-dependence). The time course of 4-aminopyridine-induced block of Ito was described by _on = 1561 ms. Phenylephrine (30 mol/l),papaverine (30 mol/I) and tetraethylammonium chloride (5 mmol/l) reduced I_to at the peak and at the end of the 300 ms depolarizing voltage step in a time-independent manner. It is concluded that verapamil, quinidine sulfate and nifedipine bind to the I_to channel in the open state at positive membrane potentials. In contrast, 4-aminopyridine obviously binds to the channel in the closed state at negative membrane potentials. Phenylephrine, papaverine and tetraethylammonium chloride seem to block I_to independent of the channel state. Correspondence to: H. Nawrath at the above address     

Purification and characterization of GTP cyclohydrolase I fromStreptomyces tubercidicus, a producer of tubercidin

GTP cyclohydrolase I catalyzing the first reaction in the biosynthesis of pterin moiety of folic acid in bacteria, was purified fromStreptomyces tubercidicus by at least 203-fold with a yield of 32% to apparent homogeneity, using ammonium sulfate fractionation, DEAE-cellulose, Sepharose CL-6B, and hydroxylapatite column chromatography. The molecular weight of the native enzyme was estimated to be 230,000 daltons by gel permeation chromatography. The purified enzyme gave a single band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was apparently 58,000 daltons. These results indicate that the enzyme consists of four subunits with the same molecular weight. TheK _m andV _max values for GTP of the purified enzyme were determined to be 80 μM and 90 nmol/min (mg protein), respectively. The optimum pH and temperature for the enzyme reaction were pH 7. 5∼8.5 and 40∼42°C, respectively. Coenzyme or metal ion was not required for the enzyme activity. The enzyme activity was inhibited by most divalent cations, while it was slightly activated by potassium ion. In case of nucleotides, CTP, GMP, GDP, and UTP inhibited enzyme activity, among which GDP exhibited the strongest inhibitory effect.     

Selective Toxicity of the Antimalarial Primaquine—Evidence for Both Uncoupling and Inhibitory Effects of a Metabolite on the Energetics of Mitochondria and Its ATP Synthase Complex

A methylene-linked dimeric metabolite of primaquine can exist as an oxidized form (PD⁺) or a reduced form (PDH). Using a rat liver mitochondrial preparation, low concentrations (E _50% = 12 µM) of PD⁺ were found to act as a classical uncoupler of oxidative phosphorylation leading to a stimulation of mitochondrial respiration. At higher concentrations, PD⁺ was found to inhibit a purified F₁-ATPase preparation, while its effects on the complete ATP synthase complex (F₀F₁-ATPase) was bimodal. A working hypothesis relating F₀F₁-ATPase and glucose-6-phosphate dehydrogenase activity to the selective toxicity of primaquine on the malaria parasite and host is presented.     

In praise of H2O2, the versatile ROS,and its vanadium complexes

Hydrogen peroxide (H₂O₂) is generated in mitochondria in aerobic cells as a minor product of electron transport, is inhibited selectively by phenolic acids (in animals) or salicylhydroxamate (in plants) and is regulated by hormones and environmental conditions. Failure to detect this activity is due to presence of H₂O₂-consuming reactions or inhibitors present in the reaction mixture. H₂O₂ has a role in metabolic regulation and signal transduction reactions. A number of enzymes and cellular activities are modified, mostly by oxidizing the protein-thiol groups, on adding H₂O₂ in mM concentrations. On complexing with vanadate, also occurring in traces, H₂O₂ forms diperoxovanadate (DPV), stable at physiological pH and resistant to degradation by catalase. DPV was found to substitute for H₂O₂ at concentrations orders of magnitude lower, and in presence of catalase, as a substrate for user reaction, horseradish peroxidase (HRP), and in inactivating glyceraldehyde-3-phosphate dehydrogenase. superoxide dismutase (SOD) -sensitive oxidation of NADH was found to operate as peroxovanadate cycle using traces of DPV and decameric vanadate (V₁₀) and reduces O₂ to peroxide (DPV in presence of free vanadate). This offers a model for respiratory burst. Diperoxovanadate reproduces several actions of H₂O₂ at low concentrations: enhances protein tyrosine phosphorylation, activates phospholipase D, produces smooth muscle contraction, and accelerates stress induced premature senescence (SIPS) and rounding in fibroblasts. Peroxovanadates can be useful tools in the studies on H₂O₂ in cellular activities and regulation.     

Purification and characterization of abelesculin,a novel ribosome-inactivating protein from the mature seeds of <Emphasis Type="Italic">Abelmoschus esculentus</Emphasis>

A novel, type-1 ribosome-inactivating protein (RIP), abelesculin, from the mature seeds of Abelmoschus esculentus was successively purified to homogeneity using ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography. Abelesculin was found to have a molecular mass of 30 kDa and a pI of more than 10.1. The protein depurinates 28S rRNA in the ribosomes of rat liver with an EC₅₀ (concentration causing 50% cleavage) of 62.8 pM. Study of the sequence of the 26 N-terminal amino acids of the protein revealed a close relationship with other RIPs. This study reports for the first time that RIPs exist in Malvaceae plants.     

Role of urinary alpha1-antitrypsin in padutin® (kallikrein) inactivation

Summary An inverse relationship between proteolytic activity in the presence of kallikrein 0.4 IU and urinary alpha₁-antitrypsin concentration has been demonstrated. This protease inhibitor can directly inactivate kallikrein activity. The inhibition was abolished by removal of urinary alpha₁-antitrypsin by trypsinsepharose treatment. Inhibition could be reversed by addition of purified alpha₁-antitrypsin. These effects could not be demonstrated with inter-alpha-trypsin inhibitor or alpha₂-macroglobulin. The inhibitory effect of alpha₁-antitrypsin on kallikrein activity should be taken into account in studies in which kallikrein activity is estimated.     

Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier

Summary The neuronal noradrenaline uptake mechanism (uptake₁) has been further characterized. For a number of substrates of uptake, the half-saturating concentration (K _m) and the maximal initial transport rate (V _max) were determined. Furthermore, the dissociation constants (K _D) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of V _max, K _m, and K _D. Neither K _m and V _max nor K _D and V _max were found to be correlated. However, the discrepancy between K _m and K _D expressed as the ratio, K_m/K_D, was negatively correlated with V _max (r = – 0.9315, n = 7, p < 0.01).For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake₁. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (K _m) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (K _D). (2) The discrepancy between K _m and K _D is expected to be negatively correlated with the maximal initial transport rate of the substrate (V _max).The experimental results are in good agreement with the proposed model for uptake,. Especially the negative correlation between K _m/K _D and V_max supports the hypothesis that desipramine inhibits uptake, via binding to the substrate recognition site of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to E. Schömig at the above address     

Origin of the different phytotoxicity and biotransformation of cerium and lanthanum oxide nanoparticles in cucumber

Abstract

To investigate how the physicochemical properties of nanoparticles (NPs) affect their biological and toxicological effects, we evaluated the phytotoxicity of CeO₂ and La₂O₃ NPs to cucumber (Cucumis sativus) plants and tried to clarify the relation between physicochemical properties of NPs and their behaviors. CeO₂ NPs had no phytotoxicity to cucumber at all tested concentrations, while La₂O₃ NPs showed significant inhibition on root elongation (?≥?2?mg/L), shoot elongation (at 2000?mg/L), root biomass (?≥?2?mg/L), and shoot biomass (?≥?20?mg/L), as well as induced more reactive oxygen species and cell death in roots (2000?mg/L). The different distribution and speciation of Ce and La in plants were determined by synchrotron-based micro X-ray fluorescence microscopy and X-ray absorption spectroscopy. In the aerial parts, all of La was combined with phosphate or carboxylic group, while a fraction of Ce was changed to Ce(III)–carboxyl complexes, implying that La₂O₃ acted as its ionic form, while CeO₂ displayed the behavior of particles or particle–ion mixtures. The higher dissolution of La₂O₃ than CeO₂ NPs might be the reason for their significant difference in phytotoxicity and transporting behaviors in cucumbers. To our knowledge, this is the first detailed study of the relation between the level of dissolution of NPs and their behaviors in plant systems.     

Effect of NO2 and SO2 inhalation on benzo(a)pyrene metabolism in rat lung

Male Wistar rats were continuously exposed to NO₂ (14.4 ppm), SO₂ (46.5 ppm) and to a mixture of both gases and their effect on lung microsomal aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) activity was determined. The pre-exposed animals were administered methylcholanthrene (MC) to investigate the exposure effect on enzyme inducibility and pattern of B(a)P metabolites. NO₂ significantly increased AHH activity but no marked change was noted with SO₂. Induction of AHH by MC was markedly inhibited by SO₂, only slightly by mixture of NO₂-SO₂ but not with NO₂ alone.     

An in vivo study of nanorod,nanosphere, and nanowire forms of titanium dioxide using Drosophila melanogaster: toxicity,cellular uptake,oxidative stress,and DNA damage

ABSTRACT

The biological impact of nanomaterials (NMs) is determined by several factors such as size and shape, which need to be taken into consideration in any type of analysis. While investigators often prefer to conduct in vitro studies for detection of any possible adverse effects of NMs, in vivo approaches yield more relevant data for risk assessment. For this reason, Drosophila melanogaster was selected as a suitable in vivo model to characterize the potential risks associated with exposure nanorods (NRs), nanospheres (NSs), nanowires (NWs) forms of titanium dioxide (TiO₂), and their microparticulated (or bulk) form, as TiO₂. Third instar larvae (72 hr old larvae) were fed with TiO₂ (NRs, NSs, or NWs) and TiO₂ at concentrations ranging from 0.01 to 10 mM. Viability (toxicity), internalization (cellular uptake), intracellular reactive oxygen species (ROS) production, and genotoxicity (Comet assay) were the end-points evaluated in hemocyte D. melanogaster larvae. Significant intracellular oxidative stress and genotoxicity were noted at the highest exposure concentration (10 mM) of TiO₂ (NRs, NSs, or NWs), as determined by the Comet assay and ROS analysis, respectively. A concentration–effect relationship was observed in hemocytes exposed to the NMs. Data demonstrated that selected forms of TiO₂.-induced genotoxicity in D. melanogaster larvae hemocytes indicating this organism is susceptible for use as a model to examine in vivo NMs-mediated effects.