In vitro assessment of cytotoxic,genotoxic and mutagenic effects of antimalarial drugs artemisinin and artemether in human lymphocytes

Artemisinin is a substance extracted from the Chinese plant Artemisia annua L. widely used in natural medicine for the treatment of various diseases. Artemether is a substance synthesized from artemisinin, and both drugs are commonly administered in the treatment of malaria. Although considered effective antimalarial drugs, very little is known about the genotoxic, cytotoxic and mutagenic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin (12.5, 25 and 50?µg/mL) and artemether (7.46; 14.92 and 29.84?µg/mL) in cultured human lymphocytes using the comet assay, the micronucleus test and the cytotoxicity assay for detection of necrosis and apoptosis by acridine orange/ethidium bromide staining. Our results showed a significant increase (p?<?0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p?<?0.05) in the number of lymphocytes with death by necrosis 48?h after treatment. The results demonstrated that these two drugs induce mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.     

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.     

Comparison of in vitro cytotoxicity,estrogenicity and anti‐estrogenicity of triclosan,perfluorooctane sulfonate and perfluorooctanoic acid

Concern with increasing levels of emerging contaminants exists on a global scale. Three commonly observed emerging environmental contaminants: triclosan (2,4,4‐trichloro‐2′‐hydroxydiphenyl ether), a synthetic, broad‐spectrum antibacterial agent, and perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), used in stain‐ and water‐resistant treatments, have become distributed ubiquitously across ecosystems and have been detected in wildlife and humans. MCF‐7 BOS human breast cancer cells were used to investigate the potential for cytotoxicity, estrogenicity and anti‐estrogenicity of these three compounds at environmentally relevant concentrations using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt assay (MTS) and the E‐SCREEN bioassay. The doses used were 0.002–200 µg ml^?1 for triclosan and 0.03–30 µg ml^?1 for PFOS and PFOA. Quantitative results from the MTS assay revealed no significant cytotoxicity at lower concentrations for any of the test compounds; however, both triclosan and PFOA were cytotoxic at the highest concentrations examined (100–200 and 30 µg ml^?1, respectively), while PFOS showed no significant cytotoxicity at any of the concentrations tested. Positive estrogenic responses (P < 0.05) were elicited from the E‐SCREEN at all concentrations examined for triclosan and PFOA and at 30 µg ml^?1 for PFOS. Further, significant anti‐estrogenic activity (P < 0.05) was detected for all compounds tested at all concentrations when cells were co‐exposed with 10^?9 m 17‐β estradiol (E₂). The overall results demonstrated that triclosan, PFOS and PFOA have estrogenic activities and that co‐exposure to contaminants and E₂ produced anti‐estrogenic effects. Each of these compounds could provide a source of xenoestrogens to humans and wildlife in the environment. Published 2011. This article is a US Government work and is in the public domain in the USA.     

Evaluation of cytotoxic concentration–time response in A549 cells exposed to respirable α‐quartz

A causal pathway between quartz, silicosis and lung cancer has been postulated. The aim of our study was to assess cytotoxic effects induced in a human lung epithelial cell line (A549) by exposure to α‐quartz. Cells were exposed to respirable α‐quartz (SRM1878a, NIST) at 25, 50 or 100 µg ml^?1 for 24 h and at 50 or 100 µg ml^?1 for 48 h. Cytotoxic effects were analyzed by scanning electron microscopy (SEM), apoptotic morphology analysis with Hoechst staining and lactate dehydrogenase (LDH) release assay. In cells exposed to α‐quartz for 24 h, a concentration‐dependent bleb development and in particular the localization of blebs at the cell edge at higher concentrations were observed. The blebbing phenomenon was more evident after 48 h of exposure to 50 or to 100 µg ml^?1 of α‐quartz and large blebs were localized at the cell edge. At the same concentrations surface smoothing was also observed. Moreover the presence of holes and tears was detected at the highest concentration both at 24 and 48 h. Results of morphological analysis with Hoechst stain evidenced an increase concentration–time dependent of apoptotic cell percentage that was more marked after 48 h exposure to 100 µg ml^?1 and a prevalence of late apoptosis stage with the increase of exposure time and concentration. Cells exposed to 50 or 100 µg ml^?1 of α‐quartz for 24 and 48 h produced a significant increase in LDH release. The concentration–time‐dependent bleb induction evidenced by SEM correlates with the increase of apoptotic cells and LDH activity release, demonstrating the onset of cytotoxic effects in human lung cells exposed to α‐quartz. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation on cobalt‐oxide nanoparticles cyto‐genotoxicity and inflammatory response in two types of respiratory cells

The increasing use of cobalt oxide (Co₃O₄) nanoparticles (NPs) in several applications and the suggested genotoxic potential of Co‐oxide highlight the importance of evaluating Co₃O₄ NPs toxicity. Cyto‐genotoxic and inflammatory effects induced by Co₃O₄ NPs were investigated in human alveolar (A549), and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1. The physicochemical properties of tested NPs were analysed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cytotoxicity was studied to analyze cell viability (WST1 test) and membrane damage (LDH assay), direct/oxidative DNA damage was assessed by the Formamido‐pyrimidine glycosylase (Fpg)‐modified comet assay and inflammation by interleukin (IL)‐6, IL‐8 and tumor necrosis factor‐alpha (TNF‐α) release (ELISA). In A549 cells, no cytotoxicity was found, whereas BEAS‐2B cells showed a viability reduction at 40 µg ml^–1 and early membrane damage at 1, 5 and 40 µg ml–1. In A549 cells, direct and oxidative DNA damage at 20 and 40 µg ml^–1 were detected without any effects on cytokine release. In BEAS‐2B cells, significant direct DNA damage at 40 µg ml^–1 and significant oxidative DNA damage with a peak at 5 µg ml^–1, that was associated with increased TNF‐α release at 1 µg ml^–1 after 2 h and increased IL‐8 release at 20 µg ml^–1 after 24 h, were detected. The findings show in the transformed alveolar cells no cytotoxicity and genotoxic/oxidative effects at 20 and 40 µg ml^–1. In normal bronchial cells, moderate cytotoxicity, direct DNA damage only at the highest concentration and significant oxidative‐inflammatory effects at lower concentrations were detected. The findings confirm the genotoxic‐oxidative potential of Co₃O₄ NPs and show greater sensitivity of BEAS‐2B cells to cytotoxic and oxidative‐inflammatory effects suggesting the use of different cell lines and multiple end‐points to elucidate Co₃O₄ NPs toxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

Protective effects of Scutellaria lindbergii root extract against oxidative-induced cell and DNA damage in mouse fibroblast-like cells

Context: Scutellaria lindbergii Rech. f. (Lamiaceae) is an Iranian species of Scutellaria which has been shown to exert antimicrobial, antioxidant and cytotoxic effects. Objective: The protective properties of total methanol extract (TME) of S. lindbergii and its fractions (defatted and CH₂Cl₂) were investigated against cytotoxic and genotoxic effects of H₂O₂ in NIH 3T3 cell line as non-malignant cells. Materials and methods: The cells were incubated with different concentrations of S. lindbergii root extracts [TME (15–250?μg ml^?1), defatted fraction (15–500?μg ml^?1) and CH₂Cl₂ fraction (5–40?μg ml^?1)] and toxic concentration of H₂O₂ (200?µM) at 37?°C for 2?h concurrently and Cell viability was quantitated by MTT assay. The antigenotoxic effect of extracts was investigated using comet assay. The cells were incubated with extracts [TME (25–250?μg ml^?1), defatted fraction (25–500?μg ml^?1) and CH₂Cl₂ fraction (5–40?μg ml^?1)] and H₂O₂ (25?µM) at 4?°C for 20?min, then the comet assay was performed. DNA damage was expressed as percentage tail DNA. Results: Total methanol extract of S. lindbergii and its fractions had a significant inhibitory effect on DNA damage. The IC₅₀ values of TME, defatted fraction and CH₂Cl₂ fraction against DNA damage were determined as 48, 138 and 8?μg ml^?1, respectively. Conclusion: S. lindbergii extracts can prevent oxidative DNA damage, which is likely due to its flavonoids and phenolic compounds as antioxidant constituents.     

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.     

Pharmacokinetic study of two acetylcholinesterase reactivators, trimedoxime and newly synthesized oxime K027, in rat plasma

K027 [1‐(4‐hydroxyiminomethylpyridinium)‐3‐(4‐carbamoylpyridinium)–propane dibromide] is a promising new reactivator of organophosphate‐ or organophosphonate‐inhibited acetylcholinesterase (AChE) with low acute toxicity and broad spectrum efficacy. The aim of the present study was to compare the pharmacokinetics of both compounds. Male Wistar rats (body weight = 320 ± 10 g) were administered a single intramuscular dose of K027 (22.07 mg kg^?1) and an equimolar dose of trimedoxime. Blood was collected at various time intervals until 180 min. Plasma samples were analyzed by reversed‐phase HPLC with ultraviolet (UV) detection. The recovery of both oximes from the plasma was approximately 90% and a linear relationship (R² > 0.998) was observed between the peak areas and concentrations of calibrated standards in the range 1–100 µg ml^?1. Near‐identical plasma profiles were obtained for both compounds. No differences were found in the mean ± SD values of C_max (18.6 ± 2.5 vs 20.0 ± 6.3 µg ml^?1, P = 0.72) and AUC_0–180min (2290 ± 304 vs 2269 ± 197 min µg ml^?1, P = 0.84). However, the percentage coefficient of variation of the first‐order rate constant of absorption (k_a) was 3‐fold higher (P < 0.01) providing evidence for more erratic absorption of intramuscular trimedoxime as compared with K027. In conclusion, oxime K027 might have superior pK properties that may be translated in its faster absorption and subsequent tissue distribution. Copyright © 2011 John Wiley & Sons, Ltd.     

Inhibitory effect of pisosterol on human glioblastoma cell lines with C‐MYC amplification

Despite the remarkable progress in the characterization of the molecular pathogenesis of glioblastoma multiforme (GBM), these tumors remain incurable and, in most cases, refractory to aggressive cytotoxic treatments. We conducted a morphological and cytogenetic study in two GBM cell lines (U343 and AHOL1), before and after treatment with pisosterol (at 0.5, 1.0 and 1.8 µg ml^?1), a triterpene isolated from the fungus Pisolithus tinctorius. No significant alteration was observed in the morphology and frequency of chromosomal abnormalities in the cell lines analyzed after treatment with pisosterol. Using fluorescence in situ hybridization analysis with a locus‐specific probe for C‐MYC showed that 72% of U343 and 65% of AHOL1 cells contained more than two alleles of C‐MYC before treatment. After treatment, no effects were detected at lower concentrations of pisosterol (0.5 and 1.0 µg ml^?1). However, at 1.8 µg ml^?1 of pisosterol, only 33% of U343 cells and 15% of AHOL1 cells presented more than two fluorescent signals, suggesting that pisosterol blocks the cells with gene amplification. Cells that do not show a high degree of C‐MYC gene amplification have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti‐cancer therapy. Copyright © 2010 John Wiley & Sons, Ltd.     

In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL‐2 production in human T‐cells

Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)‐2 production in the human Jurkat T‐cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD‐3/anti CD‐28, or anti CD‐3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml^?1 PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml^?1 PFOA. Jurkat cells stimulated with PHA/PMA or anti CD‐3 exhibited decreased IL‐2 production beginning at 50 µg PFOS ml^?1 and 5 µg PFOS ml^?1 respectively, but stimulation with anti‐CD3/anti‐CD28 resulted in no changes compared with the control. Addition of the PPAR‐alpha antagonist GW6471 to PFOS‐dosed cells stimulated with PHA/PMA resulted in decreases in IL‐2 production starting at 50 µg PFOS ml^?1, which suggests PFOS affected T‐cell IL‐2 production via PPAR‐alpha‐independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL‐2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL‐2 production, but PFOS suppressed IL‐2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL‐2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

Kauren‐19‐oic acid induces DNA damage followed by apoptosis in human leukemia cells

This study evaluated the potential cytotoxicity of the natural diterpenoids kauren‐19‐oic acid (KA), 14‐hydroxy‐kaurane (1) and xylopic acid (2), and semi‐synthetic derivatives of KA (3–5) towards human cancer cell lines (K562, HL60, MDA‐MB435 and SF295) and lymphocytes. Mouse erythrocytes were used to verify a possible hemolytic activity Cytotoxicity mechanisms were investigated in HL60 cells. KA showed a moderate antiproliferative effect in MTT assay towards all cancer cells (IC₅₀, 9.1–14.3 µg ml^?1). However, KA appeared not selective to cancer cells, since it also inhibited the lymphocytes proliferation (IC₅₀, 12.6 µg ml^?1). Unlike KA, compounds 1–5 displayed no cytotoxicity and were also free from antiproliferative and hemolytic effects, suggesting that the exocyclic double bond (C16) unit may be the active pharmacophore of KA cytotoxicity. KA‐treated HL60 cells displayed decreased proliferation (5‐bromo‐2';‐deoxyuridine incorporation assay) and topoisomerase I activity (DNA relaxation assay). These assays revealed that KA primarily intercalates with DNA and not with topoisomerase I. Fluorescence microscopy using AO/EB (acridine orange/ethidium bromide) staining indicated that KA can induce both apoptosis and necrosis in HL‐60 cell cultures, which corroborate the findings with MTT. From these findings, we conclude that KA, although demonstrating cytotoxic potential, may have a limited or poor therapeutic potential due to lack of selectivity to tumor cells. Further studies on the structure modification of KA and the mechanism of the new derivatives are currently in progress. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo genotoxicity and cytotoxicity assessment of a novel quinoxalinone with trichomonacide activity

The compound VAM2‐6 (1‐methyl‐7‐nitro‐4‐(5‐(piperidin‐1‐yl)pentyl)‐3,4‐dihydroquinoxalin‐2(1H)‐one) has previously been shown to have an in vitro efficacy of 100% at a concentration of 100 µg ml^–1 against Trichomonas vaginalis, a protozoon parasite that causes the sexually transmitted disease trichomoniasis. Because VAM2‐6 is a quinoxaline derivative and given the lack of studies on the genotoxic activity of this compound, the present study was undertaken to evaluate its ability to induce DNA damage in the peripheral blood of mice using single‐cell gel electrophoresis (SCGE or comet assay) and the micronucleus (MN) assay. Cell viability was assessed using a fluorochrome‐mediated viability test. The compound was tested on CD1 mice at 60, 40 and 10 mg kg^–1 body weight administrated intraperitoneal (i.p.) in a single dose. Peripheral blood samples were collected 24 and 48 h after treatment. N‐Ethyl‐N‐nitrosourea (ENU) was used as a positive control for the comet and micronucleus assays. The results showed that i.p. VAM2‐6 induced single‐strand DNA breaks and increased the average number of micronuclei in the treated mice in a dose‐dependent manner at 60, 40 and 10 mg kg^–1. Cell viability decreased at 24 h but recovered at 48 h for all three evaluated doses. Therefore, the chemical structure of VAM2‐6 should be modified to reduce its genotoxic potential. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro toxicological assessment of iron oxide,aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types

Metallic nanoparticles (NPs) have a variety of applications in different industries including pharmaceutical industry where these NPs are used mainly for image analysis and drug delivery. The increasing interest in nanotechnology is largely associated with undefined risks to the human health and to the environment. Therefore, in the present study cytotoxic and genotoxic effects of iron oxide, aluminium oxide and copper nanoparticles were evaluated using most commonly used assays i.e. Ames assay, in vitro cytotoxicity assay, micronucleus assay and comet assay. Cytotoxicity to bacterial cells was assessed in terms of colony forming units by using Escherichia coli (gram negative) and Bacillus subtilis (gram positive). Ames assay was carried out using two bacterial strains of Salmonella typhimurium TA98 and TA100. Genotoxicity of these NPs was evaluated following exposure to monkey kidney cell line, CHS-20. No cytotoxic and genotoxic effects were observed for iron oxide, and aluminium oxide NPs. Copper NPs were found mutagenic in TA98 and in TA100 and also found cytotoxic in dose dependent manner. Copper NPs induced significant (p?相似文献   

The potential of synthetic thiourea compound to reduce the cytotoxic and genotoxic effects of paraquat in Hordeum vulgare and cultured human lymphocytes

This work evaluated the ability of one synthetic compound 1‐(4‐fluorophenylthiocarbamoyl)‐4‐methyl‐piperazine (FTMP), thiourea derivative to reduce cytoxic and genotoxic effects of free radical inducer paraquat (PQ) in two different test systems Hordeum vulgare and human lymphocytes in vitro. The mitotic index was used as a marker for cytotoxicity. To indicate genotoxicity, chromosome aberrations test and micronucleus induction test were used. FTMP manifested a weak genotoxic effect in both test systems. Clear evidence was obtained that conditioning treatment with FTMP (10^?6, 5 × 10^?6, and 10^?5 mol/l) could decrease chromosome aberrations and micronuclei induced by PQ in both test systems. “Aberration hot spots” in heterochromatin containing segments were reduced. The present data show that the thiourea synthetic compound FTMP provides genome protection against the harmful action of oxidative stress inductor PQ. Human lymphocytes were found to be more sensitive to the cytotoxic and clastogenic effects of FTMP conditioning treatment than Hordeum vulgare. Revealing the protective action of newly synthesized compounds could contribute to the improvement of our present knowledge of the mechanisms of mutagenesis and antimutagenesis. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Toxicokinetic study of melamine in the presence and absence of cyanuric acid in rats

Several lines of evidence show that the nephrotoxic effect of melamine (MEL) in animals is consistent with combined ingestion of MEL and cyanuric acid (CYA). The aim of the present study was to compare the toxicokinetics of MEL in the presence and absence of CYA, and to elucidate the correlation between toxicity and kinetic properties of MEL. Sprague–Dawley rats were administered a single oral dose of MEL (100 mg kg^?1) with or without CYA (100 mg kg^?1). Plasma and tissue samples were analyzed by liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay. Significant changes in toxicokinetic parameters of MEL such as lower maximum concentration (7.4 ± 3.5 vs 78.0 ± 11.0 µg ml^?1) and area under curve (94.9 ± 53.5 vs 295.1 ± 93.7 µg h ml^?1), higher plasma elimination half‐life (7.0 ± 3.3 vs 2.5 ± 0.3 h) and volume of distribution (11 505.5 ± 5030.3 vs 1312.7 ± 337.7 ml kg^?1), as well as significantly higher concentration of MEL in rat kidney (2.96–274.15 vs < 1 µg g^?1) were detected in the CYA co‐administration group when compared with MEL alone group (P < 0.05). The differences in kinetic parameters between the two groups meant that CYA co‐administration could lower absorption, slow excretion and induce tissue accumulation of MEL, which correlated well with the generation and development of renal toxicity. In conclusion, co‐administration with CYA leads to the alteration of the kinetic characteristics of MEL, which provides an additional explanation for renal toxicity. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of cobalt ferrite nanoparticles induced genotoxicity on human peripheral lymphocytes: comparison of size and organic grafting-dependent effects

We aimed to detect the potential genotoxic effects of CoFe₂O₄ particles (CoFe), of different size, by means of the micronucleus test (MN) in human lymphocytes for the possible use in hyperthermic treatments. CoFe of 5.6 nm (CoFe06) showed a significant decrease of the cytokinesis blocked proliferation index (CBPI) (p<0.05), in parallel to a significant increase in the frequency of micronucleated binucleated lymphocytes (BNMN) (p<0.05). For CoFe of 10 µm (CoFe10) a significant increase in BNMN was found, but any cytotoxic effect. For larger CoFe particles of 120 µm (CoFe120) any significant effect in both indices was detected. To verify if these effects could be triggered by the release in the medium of Co²⁺ source, we organically grafted CoFe06 (CoFe06Lig) to analyze if there was a reduction of the Co⁺²-induced toxic effect. CoFe06Lig showed still a statistically significant genotoxic effect (p<0.05), but 4-fold less than that observed for the same NP, but ‘naked’.     

Analysis of cytotoxic effects of silver nanoclusters on human peripheral blood mononuclear cells ‘in vitro’

The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most used nanomaterials in consumer products. Therefore, an understanding of the interactions (unwanted toxicity) between nanoparticles and human cells is of significant interest. The aim of this study was to assess the in vitro cytotoxicity effects of silver nanoclusters (AgNC, < 2 nm diameter) on peripheral blood mononuclear cells (PBMC). Using flow cytometry and comet assay methods, we demonstrate that exposure of PBMC to AgNC induced intracellular reactive oxygen species (ROS) generation, DNA damage and apoptosis at 3, 6 and 12 h, with a dose‐dependent response (0.1, 1, 3, 5 and 30 µg ml^–1). Advanced electron microscopy imaging of complete and ultrathin‐sections of PBMC confirmed the cytotoxic effects and cell damage caused by AgNC. The present study showed that AgNC produced without coating agents induced significant cytotoxic effects on PBMC owing to their high aspect ratio and active surface area, even at much lower concentrations (<1 µg ml^–1) than those applied in previous studies, resembling what would occur under real exposure conditions to nanosilver‐functionalized consumer products. Copyright © 2015 John Wiley & Sons, Ltd.     

Chromatographic determination of drugs of abuse in vitreous humor using solid‐phase extraction

A simple method is presented for the simultaneous determination of morphine, 6‐acetylmorphine, codeine, cocaine, benzoylecgonine, cocaethylene, methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in vitreous humor by high‐performance liquid chromatography with photodiode array detector after solid‐phase extraction with Oasis® HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerra® RP8 column (250 × 4.6 mm i.d., 5 µm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradient mode. A linear response from the detector was obtained within the concentration range of 0.1–4 µg ml^?1, with correlation coefficients higher than 0.99. The limits of detection were lower than 30 ng ml^?1 for all the drugs studied, the coefficients of variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs except for EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming from individuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in five cases and heroin in three cases. Average concentrations of 0.30 µg ml^?1 for morphine, 0.24 µg ml^?1 for 6‐acetylmorphine, 0.10 µg ml^?1 for codeine, 0.81 µg ml^?1 for cocaine, 1.26 µg ml^?1 for benzoylecgonine, 0.15 µg ml^?1 for cocaethylene, 0.11 µg ml^?1 for methadone and 0.68 µg ml^?1 for EDDP were obtained. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigation of cytotoxic and genotoxic effects of the antihistaminic drug,loratadine, on human lymphocytes

Context: Loratadine (LOR) is a new generation antihistamine used in the treatment of allergic disorders. Objective: The aim of this study was to evaluate the cytogenotoxic effect of LOR on human peripheral blood lymphocytes. Materials and methods: We investigated the genotoxic effect of this drug in cultured human peripheral blood lymphocytes using sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronucleus (MN) assay in culture conditions. Proliferation index (PI), mitotic index (MI) and nuclear division index (NDI) were also calculated to determine the cytotoxic/cytostatic effect. Cultures were treated with LOR at three concentrations (5, 15 and 25?µg/ml) for 48?h. Results: Although the MI significantly decreased at the higher concentrations (15 and 25?µg/ml) compared with negative (solvent) control, LOR indicated weaker cytotoxic potential in PI and NDI values at all the tested concentrations. LOR increased the frequencies of SCE, CA and MN in all lymphocyte cultures. However, significant increase was observed in MN at the medium and highest doses (15 and 25?µg/ml) and in CA at the medium dose (15?µg/ml) compared with negative (solvent) control culture. Our results indicate that LOR has cytotoxic and genotoxic effects on human peripheral blood lymphocyte cultures. Discussion: Although most of previously findings have shown that LOR does not reflect genotoxicity, our results indicated that it may be a genotoxic drug. Conclusion: More studies are necessary to elucidate the relationship between cytotoxic, genotoxic and apoptotic effects, and to make a possible risk assessment in patients receiving therapy with this drug.     

The adverse effects of low‐dose exposure to Di(2‐ethylhexyl) phthalate during adolescence on sperm function in adult rats

Di(2‐ethylhexyl) phthalate (DEHP) is the most crucial phthalate derivative added to polyvinyl chloride as a plasticizer. This study examined the effects of low‐dose exposure to DEHP during adolescence on sperm function in adult rats. The male rats were daily gavaged with 30, 100, 300, and 1000 µg kg^?1 of DEHP or corn oil from postnatal day (PND) 42 until PND 105. The selection of DEHP doses ranged from the mean daily intake by the normal‐population exposure levels to no‐observed‐adverse‐effect level of DEHP for the endpoints evaluated until adulthood. Significant increases in the percentage of sperm with tail abnormality, tendency for sperm DNA fragmentation index (DFI) and percentage of sperm with DFI were found in those exposed to 100, 300, and 1000 µg kg^?1 (P < 0.05). We observed a significant increase of hydrogen peroxide (H₂O₂) generation in the sperm of the 1000 µg kg^?1 group compared with the control group (P < 0.05). The excessive production of sperm H₂O₂ coincided with an increase in sperm DFI. In this study, the lowest‐observed‐adverse‐effect level for sperm toxicity was considered to be 100 µg DEHP/kg/day in sperm morphology and chromatin DNA damage. Further research is necessary to clarify the mechanisms of DEHP‐related sperm ROS generation on sperm DNA damage. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 706–712, 2016.