ROS-mediated genotoxicity of asbestos-cement in mammalian lung cells <Emphasis Type="Italic">in vitro</Emphasis>

Asbestos is a known carcinogen and co-carcinogen. It is a persisting risk in our daily life due to its use in building material as asbestos-cement powder. The present study done on V79-cells (Chinese hamster lung cells) demonstrates the cytotoxic and genotoxic potential of asbestos-cement powder (ACP) in comparison with chrysotile asbestos. A co-exposure of chrysotile and ACP was tested using the cell viability test and the micronucleus assay. The kinetochore analysis had been used to analyse the pathway causing such genotoxic effects. Thiobarbituric acid-reactive substances were determined as evidence for the production of reactive oxygen species. Both, asbestos cement as well as chrysotile formed micronuclei and induced loss of cell viability in a concentration- and time- dependent way. Results of TBARS analysis and iron chelator experiments showed induction of free radicals in ACP- and chrysotile exposed cultures. CaSO₄ appeared to be a negligible entity in enhancing the toxic potential of ACP. The co-exposure of both, ACP and chrysotile, showed an additive effect in enhancing the toxicity. The overall study suggests that asbestos-cement is cytotoxic as well as genotoxic in vitro. In comparison to chrysotile the magnitude of the toxicity was less, but co-exposure increased the toxicity of both.     

CHEMICAL AND TOXICOLOGICAL CHARACTERIZATION OF AIRBORNE TOTAL SUSPENDED PARTICULATE (TSP) AND PM 10 ORGANIC EXTRACTS

The relationship between chemical composition of airborne particulates and the genotoxicity has been investigated in the atmosphere of Rome, Italy. For this purpose, both total suspended particulate (TSP) and the PM ₁₀ fractions were collected daily inside a green park located in downtown, grouped on a weekly basis and speciated for their burdens of polycyclic aromatic hydrocarbons (PAH) and nitro-PAH. Concurrently, the genotoxicity of the organic extracts was evaluated by the Comet assay (SCGE: single cell gel electrophoresis) on human peripheral blood mononuclear cells (PBMC). The results indicate that organic extracts were able to induce DNA damage and a cytotoxic effect on PBMC. The TSP fraction was more cytotoxic than PM ₁₀ while the genotoxicity of both fractions was comparable. The genotoxic potential of the different samples was highly correlated to the amount of total PAH (correlation coefficient = 0.87), carcinogenic PAH (correlation coefficient = 0.88), B(a)P (correlation coefficient = 0.87) and to a less extend to the concentration of 1-nitropyrene (correlation coefficient = 0.66). The seasonal modulation suggests that in Rome the air is more toxic during winter, however in the warm season oxidized species including nitro-PAHs (evolving from secondary pollution) seem to balance the decrease of PAH concentration rates.     

In Vivo Assessment of the Genotoxicity of PAH-Contaminated Soils in Rats Exposed by Natural Routes

In order to assess the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in vivo, male laboratory rats were exposed during 4 days to a soil highly polluted with PAHs (1200 mg.kg^?1) obtained from a former coke-plant. A range of dilution, from 1 to 1/8 was realized with sand.

Lymphocytes were sampled every day during the exposure. Injured nuclei were quantified by the technique of Comet assay after inclusion in an agarose gel, cells lysis and electrophoresis migration (modified from [1]).

This study showed an optimum response after three days in fact whatever the dilution degree 95% of the nuclei were injured. The dilution range showed a dose-response relationship. Moreover, the quantity of injured nuclei depended on the duration of exposure. In control animals, less than 5% of the nuclei were damaged at the end of the exposure period.

So the mode of exposure has shown in vivo both the bioavailability and the clastogenicity of PAHs from polluted soils. Therefore, Comet assay can be utilized as a biomarker to evaluate the genotoxic effects of a polluted soil.     

Evaluation of the Toxicity of Two Electron-Deficient Half-Sandwich Complexes against Human Lymphocytes from Healthy Individuals

Electron-deficient half-sandwich complexes are a class of under-studied organometallics with demonstrated potential as metallodrug candidates. This study investigates the effect of two 16-electron organoruthenium complexes ([(p-cym)Ru(benzene-1,2-dithiolato)] ( 1 ) and [(p-cym)Ru(maleonitriledithiolate)] ( 2 )) on the cell viability of non-immortalised human lymphocytes from healthy individuals. The genotoxic effects of 1 and 2 in lymphocytes are also investigated by using the Comet and cytokinesis-block micronucleus assays. Gene expression studies were carried out on a panel of genes involved in apoptosis and the DNA damage-repair response. Results show that the two 16-electron complexes do not have significant effect on the cell viability of human lymphocytes from healthy individuals. However, an increase in DNA damage is induced by both compounds, presumably through oxidative stress production.     

An in vitro liver model - assessing oxidative stress and genotoxicity following exposure of hepatocytes to a panel of engineered nanomaterials

ABSTRACT: BACKGROUND: Following exposure via inhalation, intratracheal instillation or ingestion some nanomaterials(NM) have been shown to translocate to the liver. Since oxidative stress has been implicatedas a possible mechanism for NM toxicity this study aimed to investigate the effects of variousmaterials (five titanium dioxide (TiO2), two zinc oxide (ZnO), two multi-walled carbonnanotubes (MWCNT) and one silver (Ag) NM) on oxidative responses of C3A cell line as amodel for potential detrimental properties of nanomaterials on the liver. RESULTS: We noted a dose dependant decrease in the cellular glutathione content following exposure ofthe C3A cells to Ag, the ZnO and the MWCNTs. Intracellular ROS levels were alsomeasured and shown to increase significantly following exposure of the C3A to the lowtoxicity NMs (MWCNT and TiO2). The antioxidant Trolox in part prevented the detrimentaleffect of NMs on cell viability, and decreased the NM induced IL8 production after exposureto all but the Ag particulate. Following 4 hr exposure of the C3A cells to sub-lethal levels ofthe NMs, the largest amount of DNA damage was induced by two of the TiO2 samples (7 nmand the positively charged 10 nm particles). CONCLUSIONS: All ten NMs exhibited effects on the hepatocyte cell line that were at least in partROS/oxidative stress mediated. These effects included mild genotoxicity and IL8 productionfor all NM except the Ag possibly due to its highly cytotoxic nature.     

Comparison of Cytotoxic,Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells

Secondary metabolites as a potential source of anticancer therapeutics have been the subject of many studies. Since hypericin, a metabolite isolated from Hypericum perforatum L., shows several biomedical properties applicable in oncology, the aim of our study was to investigate its potential precursor skyrin in terms of genotoxic and DNA-protective effects. These skyrin effects were analyzed by cell-free methods, and cytotoxicity was estimated by an MTT assay and by a trypan blue exclusion test, while the genotoxic/antigenotoxic potential was examined by comet assay using non-cancerous human lymphocytes and the HepG2 cancer cell line. Skyrin did not show DNA-damaging effects but rather exhibited DNA-protectivity using a DNA-topology assay. However, we observed only weak antioxidant and chelating skyrin properties in other cell-free methods. Regarding the cytotoxic activity of skyrin, HepG2 cells were more prone to skyrin-induced death in comparison to human lymphocytes. Skyrin in non-cytotoxic concentrations did not exhibit elevated genotoxicity in both cell types. On the other hand, skyrin displayed moderate DNA-protective effects that were more noticeable in the case of non-cancerous human lymphocytes. The potential genotoxic effects of skyrin were not observed, and its DNA-protective capacity was more prominent in non-cancerous cells. Therefore, skyrin might be a promising agent used in anticancer therapy.     

Hemocompatibility,cytotoxicity, and genotoxicity of poly(methylmethacrylate)/nanohydroxyapatite nanocomposites synthesized by melt blending method

The aim of this study was to show the hemocompatibility, cytotoxicity, and genotoxicity of nanocomposites that were synthesized with different molecular weights of poly(methyl methacrylate) (PMMA) and different concentrations of nanohydroxyapatite (nHAp). Different techniques to characterize the nanocomposites were used. The cytotoxicity and genotoxic effects of the polymers and nanocomposites on human lymphocytes were determined by acid phosphatase assay, viability test, and comet assay. Moreover, hemocompatibility test was performed. It was found that all of the PMMA/nHAp nanocomposites are highly hemocompatible and biocompatible, none of the nanocomposites showed a cytotoxic effect, and nHAp addition decreased the genotoxicity.     

Antioxidant,Cytotoxic, Genotoxic,and DNA-Protective Potential of 2,3-Substituted Quinazolinones: Structure—Activity Relationship Study

The evaluation of antioxidant compounds that counteract the mutagenic effects caused by the direct action of reactive oxygen species on DNA molecule is of considerable interest. Therefore, a series of 2,3-substituted quinazolinone derivatives (Q1–Q8) were investigated by different assays, and the relationship between their biological properties and chemical structure was examined. Genotoxicity and the potential DNA-protective effects of Q1–Q8 were evaluated by comet assay and DNA topology assay. Antioxidant activity was examined by DPPH-radical-scavenging, reducing-power, and total antioxidant status (TAS) assays. The cytotoxic effect of compounds was assessed in human renal epithelial cells (TH-1) and renal carcinoma cells (Caki-1) by MTT assay. Analysis of the structure–activity relationship disclosed significant differences in the activity depending on the substitution pattern. Derivatives Q5–Q8, bearing electron-donating moieties, were the most potent members of this series. Compounds were not genotoxic and considerably decreased the levels of DNA lesions induced by oxidants (H₂O₂, Fe²⁺ ions). Furthermore, compounds exhibited higher cytotoxicity in Caki-1 compared to that in TH-1 cells. Substantial antioxidant effect and DNA-protectivity along with the absence of genotoxicity suggested that the studied quinazolinones might represent potential model structures for the development of pharmacologically active agents.     

Examination of Surfactant Protein D as a Biomarker for Evaluating Pulmonary Toxicity of Nanomaterials in Rat

This work studies the relationship between lung inflammation caused by nanomaterials and surfactant protein D (SP-D) kinetics and investigates whether SP-D can be a biomarker of the pulmonary toxicity of nanomaterials. Nanomaterials of nickel oxide and cerium dioxide were classified as having high toxicity, nanomaterials of two types of titanium dioxides and zinc oxide were classified as having low toxicity, and rat biological samples obtained from 3 days to 6 months after intratracheal instillation of those nanomaterials and micron-particles of crystalline silica were used. There were different tendencies of increase between the high- and low-toxicity materials in the concentration of SP-D in bronchoalveolar-lavage fluid (BALF) and serum and in the expression of the SP-D gene in the lung tissue. An analysis of the receiver operating characteristics for the toxicity of the nanomaterials by SP-D in BALF and serum showed a high accuracy of discrimination from 1 week to 3 or 6 months after exposure. These data suggest that the differences in the expression of SP-D in BALF and serum depended on the level of lung inflammation caused by the nanomaterials and that SP-D can be biomarkers for evaluating the pulmonary toxicity of nanomaterials.     

Insight into Factors Influencing Wound Healing Using Phosphorylated Cellulose-Filled-Chitosan Nanocomposite Films

Marine polysaccharides are believed to be promising wound-dressing nanomaterials because of their biocompatibility, antibacterial and hemostatic activity, and ability to easily shape into transparent films, hydrogels, and porous foams that can provide a moist micro-environment and adsorb exudates. Current efforts are firmly focused on the preparation of novel polysaccharide-derived nanomaterials functionalized with chemical objects to meet the mechanical and biological requirements of ideal wound healing systems. In this contribution, we investigated the characteristics of six different cellulose-filled chitosan transparent films as potential factors that could help to accelerate wound healing. Both microcrystalline and nano-sized cellulose, as well as native and phosphorylated cellulose, were used as fillers to simultaneously elucidate the roles of size and functionalization. The assessment of their influences on hemostatic properties indicated that the tested nanocomposites shorten clotting times by affecting both the extrinsic and intrinsic pathways of the blood coagulation system. We also showed that all biocomposites have antioxidant capacity. Moreover, the cytotoxicity and genotoxicity of the materials against two cell lines, human BJ fibroblasts and human KERTr keratinocytes, was investigated. The nature of the cellulose used as a filler was found to influence their cytotoxicity at a relatively low level. Potential mechanisms of cytotoxicity were also investigated; only one (phosphorylated microcellulose-filled chitosan films) of the compounds tested produced reactive oxygen species (ROS) to a small extent, and some films reduced the level of ROS, probably due to their antioxidant properties. The transmembrane mitochondrial potential was very slightly lowered. These biocompatible films showed no genotoxicity, and very importantly for wound healing, most of them significantly accelerated migration of both fibroblasts and keratinocytes.     

氨水为沉淀剂从水晶石废料中提纯 二氧化铈的实验研究

以工业固体废弃物水晶石为原料,采取一定方法将二氧化硅与二氧化铈分离后进行二氧化铈的提取,有效解决废弃物堆放并实现其资源化。以氨水为沉淀剂,将分离后的二氧化铈进行提纯,研究了氨水浓度、静置时间、pH、焙烧温度和时间对二氧化铈纯度和质量的影响。实验结果表明：在氨水浓度为6 mol/L、pH=8、静置30 min、焙烧温度为450 ℃、焙烧时间为2 h条件下,得到二氧化铈的含量接近原料的实际值。     

Novel Psychoactive Phenethylamines: Impact on Genetic Material

Psychedelic and stimulating phenethylamines belong to the family of new psychoactive substances (NPS). The acute toxicity framework has begun to be investigated, while studies showing genotoxic potential are very limited or not available. Therefore, in order to fill this gap, the aim of the present work was to evaluate the genotoxicity by treating TK6 cells with 2C-H, 2C-I, 2C-B, 25B-NBOMe, and the popular 3,4-Methylenedioxymethylamphetamine (MDMA). On the basis of cytotoxicity and cytostasis results, we selected the concentrations (6.25–35 µM) to be used in genotoxicity analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by an automated flow cytometric protocol. All substances, except MDMA, resulted genotoxic; therefore, we evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the demonstrated genotoxicity. The obtained results showed a statistically significant increase in ROS levels for all genotoxic phenethylamines confirming this hypothesis. Our results highlight the importance of genotoxicity evaluation for a complete assessment of the risk associated also with NPS exposure. Indeed, the subjects who do not have hazardous behaviors or require hospitalization by using active but still “safe” doses could run into genotoxicity and in the well-known long-term effects associated.     

Chlorinated Polycyclic Aromatic Hydrocarbons Associated with Drinking Water Disinfection: Synthesis,Formation under Aqueous Chlorination Conditions and Genotoxic Effects

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent and toxic organic micropollutants present in water and several of them are mutagenic and carcinogenic. Although it has been shown that chlorinated derivatives of PAHs (Cl-PAHs) may be formed during the water chlorination procedure, little is known about their potential genotoxic and carcinogenic effects. The objectives of the present work were to prepare and characterize the major chlorinated derivatives of benzo[a]pyrene (BaP) and fluoranthene (Fluo), to develop an analytical methodology for their quantification in water samples and to analyse their potential genotoxicity. Chlorinated standards were prepared by a newly developed two phase method (water/n-hexane) using sodium hypochlorite. 6-Chloro-benzo[a]pyrene was selectively obtained from BaP, while 1,3-dichloro-fluoranthene and 3-chloro-fluoranthene were obtained from Fluo. All products were isolated and characterized by nuclear magnetic resonance and mass spectrometry. The formation of BaP- and Fluo-chlorinated derivatives under aqueous chlorination conditions was observed using a SPE-HPLC-FLD methodology. In addition, the cytotoxic and genotoxic activities of the three chlorinated derivatives were analyzed in comparison to their parent compounds, in a human-derived hepatoma cell line using the neutral red uptake and comet assays, respectively. The results showed that, at the equimolar doses of 100 and 125 μM, 6-Cl-BaP was able to induce a significantly higher level of DNA damage than BaP, suggesting a more potent genotoxic effect. In contrast, neither Fluo nor its chlorinated derivatives were genotoxic in the same cell line. The identification of new and possibly hazardous water chlorination by-product from PAHs emphasizes the need to minimize total organic carbon content of raw water and the implementation of safer water disinfection methods.     

彗星试验在新农药毒性检测中的应用

综述了彗星试验在农药遗传毒性检测中的应用现状,测试了新型嘧啶苄胺除草剂一丙酯草醚等5种新农药对小鼠骨髓细胞和生殖细胞的DNA损伤情况,摸索了最适的彗星试验方法条件,比较不同细胞的敏感性,建立彗星试验标准操作规程(SOP),最终达到彗星试验在新农药遗传毒性检测中的应用和推广。     

Benzo[a]pyrene-Induced Genotoxicity in Rats Is Affected by Co-Exposure to Sudan I by Altering the Expression of Biotransformation Enzymes

The environmental pollutant benzo[a]pyrene (BaP) is a human carcinogen that reacts with DNA after metabolic activation catalysed by cytochromes P450 (CYP) 1A1 and 1B1 together with microsomal epoxide hydrolase. The azo dye Sudan I is a potent inducer of CYP1A1/2. Here, Wistar rats were either treated with single doses of BaP (150 mg/kg bw) or Sudan I (50 mg/kg bw) alone or with both compounds in combination to explore BaP-derived DNA adduct formation in vivo. Using ³²P-postlabelling, DNA adducts generated by BaP-7,8-dihydrodiol-9,10-epoxide were found in livers of rats treated with BaP alone or co-exposed to Sudan I. During co-exposure to Sudan I prior to BaP treatment, BaP-DNA adduct levels increased 2.1-fold in comparison to BaP treatment alone. Similarly, hepatic microsomes isolated from rats exposed to Sudan I prior to BaP treatment were also the most effective in generating DNA adducts in vitro with the activated metabolites BaP-7,8-dihydrodiol or BaP-9-ol as intermediates. DNA adduct formation correlated with changes in the expression and/or enzyme activities of CYP1A1, 1A2 and 1B1 in hepatic microsomes. Thus, BaP genotoxicity in rats in vivo appears to be related to the enhanced expression and/or activity of hepatic CYP1A1/2 and 1B1 caused by exposure of rats to the studied compounds. Our results indicate that the industrially employed azo dye Sudan I potentiates the genotoxicity of the human carcinogen BaP, and exposure to both substances at the same time seems to be hazardous to humans.     

Evaluation of Interactions of Selected Olivacine Derivatives with DNA and Topoisomerase II

Olivacine and ellipticine are model anticancer drugs acting as topoisomerase II inhibitors. Here, we present investigations performed on four olivacine derivatives in light of their antitumor activity. The aim of this study was to identify the best antitumor compound among the four tested olivacine derivatives. The study was performed using CCRF/CEM and MCF-7 cell lines. Comet assay, polarography, inhibition of topoisomerase II activity, histone acetylation, and molecular docking studies were performed. Each tested compound displayed interaction with DNA and topoisomerase II, but did not cause histone acetylation. Compound 2 (9-methoxy-5,6-dimethyl-1-({[1-hydroxy-2-(hydroxymethyl)butan-2-yl]amino}methyl)-6H-pyrido[4,3-b]carbazole) was found to be the best candidate as an anticancer drug because it had the highest affinity for topoisomerase II and caused the least genotoxic damage in cells.     

从废玻璃渣中提取氧化铈的研究

为了实现玻璃生产过程中废玻璃的综合利用,得到经济价值高、加工成本低的氧化铈。以某企业的废玻璃为原料,经稀盐酸预处理后溶解于浓盐酸中提取氧化铈。分别研究了稀盐酸的浓度和加入量、浓盐酸的加入量、水浴时间和温度等因素对氧化铈产率的影响,并得出了最佳提取条件:稀盐酸浓度为2 mol/L、稀盐酸加入量为100 mL、浓盐酸加入量为250 mL、水浴时间为2 h、水浴温度为95℃。     

Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver

Background

Widespread occupational exposure to carbon black nanoparticles (CBNPs) raises concerns over their safety. CBNPs are genotoxic in vitro but less is known about their genotoxicity in various organs in vivo.

Methods

We investigated inflammatory and acute phase responses, DNA strand breaks (SB) and oxidatively damaged DNA in C57BL/6 mice 1, 3 and 28 days after a single instillation of 0.018, 0.054 or 0.162 mg Printex 90 CBNPs, alongside sham controls. Bronchoalveolar lavage (BAL) fluid was analyzed for cellular composition. SB in BAL cells, whole lung and liver were assessed using the alkaline comet assay. Formamidopyrimidine DNA glycosylase (FPG) sensitive sites were assessed as an indicator of oxidatively damaged DNA. Pulmonary and hepatic acute phase response was evaluated by Saa3 mRNA real-time quantitative PCR.

Results

Inflammation was strongest 1 and 3 days post-exposure, and remained elevated for the two highest doses (i.e., 0.054 and 0.162 mg) 28 days post-exposure (P < 0.001). SB were detected in lung at all doses on post-exposure day 1 (P < 0.001) and remained elevated at the two highest doses until day 28 (P < 0.05). BAL cell DNA SB were elevated relative to controls at least at the highest dose on all post-exposure days (P < 0.05). The level of FPG sensitive sites in lung was increased throughout with significant increases occurring on post-exposure days 1 and 3, in comparison to controls (P < 0.001-0.05). SB in liver were detected on post-exposure days 1 (P < 0.001) and 28 (P < 0.001). Polymorphonuclear (PMN) cell counts in BAL correlated strongly with FPG sensitive sites in lung (r = 0.88, P < 0.001), whereas no such correlation was observed with SB (r = 0.52, P = 0.08). CBNP increased the expression of Saa3 mRNA in lung tissue on day 1 (all doses), 3 (all doses) and 28 (0.054 and 0.162 mg), but not in liver.

Conclusions

Deposition of CBNPs in lung induces inflammatory and genotoxic effects in mouse lung that persist considerably after the initial exposure. Our results demonstrate that CBNPs may cause genotoxicity both in the primary exposed tissue, lung and BAL cells, and in a secondary tissue, the liver.