Lead-Induced Genotoxicity in Lymphocytes from Peripheral Blood Samples of Humans: In Vitro Studies

Lead is a known toxicant that has been implicated in encephalopathy in children and may affect the gastrointestinal and hematopoietic and other systems in adults. In fact, lead has been shown to compete with calcium for entry into the synaptosome and induce toxic effects. The aim of the current study was to evaluate the cytotoxic and genotoxic effects of lead by using lymphocytes from human peripheral blood in vitro. The LC₅₀ for lead nitrate as determined by Trypan blue dye exclusion technique was found to be 3.14 mM. Chromosomal aberration frequency at sublethal doses (1/10 of LC₅₀) as determined by examining the metaphase chromosomes (karyotyping) did not show significant aberrations except for some aneuploidy and about 2–4% gaps, breaks (3–4%), and about 5% satellite associations. However, significant DNA damage was determined by SCGE (Comet assay). The comet tail length proportionately increased with increasing lead nitrate concentration. Thus, Pb can induce single-strand DNA breaks, possibly by competing with metal binding sites.     

Cytogenetic Biomarkers of Carbofuran Toxicity Utilizing Human Lymphocyte Cultures In Vitro

Short-term lymphocyte cultures from human peripheral blood samples were incubated with various aliquots of the carbofuran pesticide. After 48 h of initiation and 24 h of exposure to the carbofuran pesticide aliquots, it was seen that carbofuran caused an increase in the frequency of chromosomal aberrations, and the increase was significant (p < 0.05) in treated samples compared to controls. Karyotype analysis revealed more satellite associations, gaps, and breaks in treated samples. Single-strand breaks in the DNA assessed by comet assay revealed that the pesticide caused increase in the comet tail length implicating genotoxicity in somatic cells. The LD₅₀ of carbofuran was found to be 18 µM as calculated by probit analysis and determined by trypan blue dye exclusion method. The results presented here indicate that in vitro assays could be used as indicators of cyto- and genotoxicity of the pesticide, and their end points could be used as biomarkers.     

Effect of titanium surface modified by plasma energy source on genotoxic response in vitro

Titanium (Ti) is currently the most widely used material for the manufacture of orthopedic and dental implants. Changes in the surface of commercial pure Ti (cp Ti) can determine the functional response of cells, and is therefore a critical factor for the success of the implant. However, the genotoxicity of titanium surfaces has been poorly studied. Thus, the purpose of this study was to evaluate the genotoxic potential of a new titanium surface developed by plasma treatment using argon-ion bombardment and compare it with an untreated titanium surface. Accordingly, comet assay, analysis of chromosomal aberrations (CAs), and Cytokinesis Block Micronucleus (CBMN) assay were carried out, using CHO-K1 (Chinese hamster ovary) cells grown on both titanium surfaces. Our results show that the untreated titanium surface caused a significant increase in % tail moment, in the number of cells with CAs, tetraploidy, micronucleus frequency, and other nuclear alterations when compared with the negative control and with the plasma-treated titanium surface. This difference may be attributed to increased surface roughness and changes in titanium oxide layer thickness.     

The Assesment of Genotoxicity of Carbamazepine Using Cytokinesis-Block (CB) Micronucleus Assay in Cultured Human Blood Lymphocytes

The genotoxic effect of CBZ has been investigated in few studies. There is little evidence linking carbamazepine (CBZ) with any genotoxic effects, particularly in vitro micronucleus test using cytogenesis-block technique. In this study, the genotoxicity of the antiepileptic drug, carbamazepine, was tested using cytokinesis-block (CB) micronucleus assay. In vitro analysis was performed in human blood lymphocytes from four healthy persons at five different concentrations of carbamazepine (6, 8, 10, 12, 14 μg/mL). Genotoxic potential and cytotoxic effects of carbamazepine were evaluated by using micronucleus assay and cytokinesis-block proliferation index (CBPI), called the parameter of cytotoxicity in human peripheral blood lymphocyte cultures, respectively. The results of this study indicate that CBZ caused the genotoxic effect under in vitro conditions, except at the dose of 6 μg/mL, and cytotoxic effects of carbamazepine were revealed by a decrease in the cytokinesis-block proliferation index at all the concentrations.     

Comparative Study of the Cytotoxicity and Genotoxicity of Alpha- and Beta-Asarone

The cytotoxicity of alpha- and beta-asarone was investigated with the BrdU assay in HepG2-cells. Alpha-asarone was found to be more toxic than beta-asarone after 24 hours of treatment. Investigation of the genotoxicity using the micronucleus assay in the HepG2-cell system showed that only after metabolic activation by a liver microsomal preparation, beta-asarone was able to induce micronuclei at concentrations higher than 50 μg/mL.     

Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC₅₀: 11 μM; 48 h IC₅₀: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC₅₀: 47 μM; 48 h IC₅₀: 17 μM). IC₅₀ values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC₅₀ values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7–123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.     

Evaluation of the genotoxicity and cytotoxicity of fexofenadine in cultured human peripheral blood lymphocytes

Fexofenadine (FXF) is a new non-sedating antihistamine used in the treatment of seasonal allergic rhinitis and chronic idiopathic urticaria. Studies on FXF genotoxicity and cytotoxicity in cultured human peripheral blood lymphocytes have not been reported so far. Therefore, the present study is the first report investigating the genotoxic and cytotoxic effects of FXF in cultured human peripheral blood lymphocytes in vitro. Cultures were treated with FXF at three concentrations (50, 100 and 150 μg/ml) for 24 and 48 h. Endpoints analyzed included: mitotic index (MI), nuclear division index (NDI), chromosomal aberrations (CA) and micronucleus (MN) assay. Mitomycin C (MMC) was used as a positive control. The results of CA and MN assays showed that FXF was not genotoxic at all the concentrations tested, meanwhile MI and NDI results showed dose-dependent decrease and significant differences were found for at least one concentration. In conclusion, the results of this study suggest that FXF has a cytotoxic effect but not genotoxic effect on human peripheral blood lymphocyte cultures. Further cytogenetic studies, especially about the cell cycle kinetics of FXF are required to elucidate the decreases in dividing cells, and biomonitoring studies should also be conducted with patients receiving therapy with this drug.     

Cytotoxicity of mebendazole against established cell lines from the human,rat, and mouse liver

The direct cytotoxicity of mebendazole (MBZ) was investigated by using cell lines derived from human, mouse and rat liver. It was demonstrated that Chang liver cells (derived from human liver) were more sensitive to the cytotoxic effects of MBZ than the other two cell lines. Longer incubation of the cells with MBZ resulted in stronger toxicity, and the cytotoxicity was dependent on the MBZ concentration above a certain threshold value (0.25–0.50 mg/l in a 42-h culture). Inhibition of the proliferation of Chang liver cells by MBZ was detected at a concentration of 0.008 mg/l, a lower concentration than that having a cytotoxic effect. The other two cell lines were less sensitive to the inhibitory effect of MBZ. Proliferation of human mononuclear cells following stimulation by phytohemagglutinin (PHA) was inhibited by MBZ, and this inhibition was more extensive than that of cells stimulated with whole formalin-treatedPseudomonas aeruginosa. It is suggested that dividing cells may be more sensitive to MBZ cytotoxicity. This anti-proliferative effect may be related to its clinically known side effects, such as hepatotoxicity and bone marrow suppression.     

Comparison Between the Genotoxicity of cis-Pt(II) Complex of 3-Aminoflavone and cis-DDP in Lymphocytes Evaluated by the Comet Assay

cis-bis(3-aminoflavone)dichloroplatinum(II) [cis-Pt(II) complex of 3-aminoflavone] is an analog of cis-DDP characterized by low cytotoxicity and anticancer properties in vivo. In order to evaluate genotoxic properties of this chemical compound, the comet assay in human lymphocytes was used. The analysis of DNA damage after 1-h cell incubation with cis-Pt(II) complex of 3-aminoflavone and cis-DDP was carried out, and DNA repair kinetics were evaluated after 0.5-h, 1-h, and 1.5-h postexposure incubation. It has been shown that cis-Pt(II) complex of 3-aminoflavone causes the increase in tail moments in comparison with cis-DDP. On the other hand, the decrease in these values caused by cis-DDP was connected mainly with the presence of DNA and DNA–protein cross-links. The decrease in tail moments after cis-Pt(II) complex of 3-aminoflavone lymphocyte treatment was already observed after 0.5-h postexposure incubation, whereas in the variant with hydrogen peroxide application these values after cis-Pt(II) complex of 3-aminoflavone addition were higher in comparison with cis-DDP. Results obtained on the basis of the comet assay could confirm the occurrence of DNA breaks and cross-links induced by cis-Pt(II) complex of 3-aminoflavone.     

A Rapid Colorimetric Assay for Sulfur Mustard Cytotoxicity Using Isolated Human Peripheral Blood Lymphocytes and Keratinocytes

Sulfur mustard (SM) is a potent vesicating agent that has pronounced cytotoxic effects as well as mutagenic, carcinogenic, and radiomimetic properties. Isolated human peripheral blood lymphocytes (PBLs) and human epidermal keratinocytes (HEKs) have been used as in vitro models for determining SM-induced cytotoxicity. A recently developed colorimetric assay (the CellTiter 96 AQ ueous Non-radioactive Cell Proliferation Assay) was assessed using both of the in vitro models described above. Using 24- or 96-well microplates, reproducible (± 10%) SM dose/response curves for both types of human cells were obtained using a spectrophotometric microplate reader set at 490 nm. After a 4-h incubation time, as many as 96 sample wells could be measured within 45 s using this commonly available equipment. Multiple plates of samples can be run immediately. This technique may facilitate cytotoxicity investigations of new candidate compounds for both prophylaxis of and therapy for SM intoxication.     

Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on fish cells in vitro

Intrinsic genotoxic and cytotoxic potential of titanium dioxide (TiO2) engineered nanoparticles (ENPs) were evaluated in a metabolically competent, established fish cell line derived from rainbow trout (Oncorhyncus mykiss) gonadal tissue (i.e. RTG-2 cells). Prior to evaluation of the toxic potential, mean size of the ENPs was determined using transmission electron microscopy (TEM). As a prerequisite, an extensive characterisation of the ENPs was carried out following sonication which enabled the synthesis of an efficient dosing strategy for the cells in which exposure in phosphate buffered saline (PBS) gave an optimal agglomeration effects compared to distilled water (H2O) and minimal essential media (MEM). Interaction of the ENPs with cells under scanning electron microscope (SEM) was also studied. The genotoxic and cytotoxic potential of the ENPs were determined either alone or in combination with ultraviolet radiation (i.e. UVA). Whilst genotoxic potential was determined by evaluating DNA strand breaks using single cell gel electrophoresis (SCGE) or the comet assay and induction of cytogenetic damage using cytokinesis-blocked micronucleus (MN) assay, cytotoxicity was determined by measuring the retention of supra vital stain, neutral red, by the lysosomes using the neutral red retention (NRR) assay. In addition, while performing the comet assay, lesion specific bacterial endonuclease, formamidopyrimidine DNA glycosylase (Fpg), which recognises oxidised purine bases, was used to determine oxidative DNA damage. The results suggested that the highest concentration of the ENPs (i.e. 50 microg ml(-1)) did not produce elevations in DNA damage over 4 h (comet assay), 24 h (modified comet assay) or 48 h (MN assay) exposures in the absence of UVA irradiation, although there was a significant reduction in lysosomal integrity over 24 h exposure (NRR assay). The induction of MN did not show any enhanced levels as a function of ENP concentration. A significantly increased level of strand breaks was observed in combination with UVA (3 kJ m(-2)). In general, the NRR assay suggested elevated levels of cytotoxicity when the UVA exposure was carried out with MEM compared to PBS, although both showed an increase when in combination with the highest concentration of ENPs (i.e. 50 microg ml(-1)). Overall, the study emphasises the need for adoption of an holistic approach while evaluating the potential toxic effects of ENPs in which appropriate measures should be taken to avoid agglomeration or aggregation to facilitate efficient cellular uptake to evaluate potential biological responses.     

Cumulative genotoxic and apoptotic effects of xenobiotics in a mini organ culture model of human nasal mucosa as detected by the alkaline single cell microgel electrophoresis assay and the annexin V-affinity assay

Three-dimensional mini organ cultures of human inferior nasal turbinate epithelia have proved to be a useful tool in genotoxicology studies. They allow repetitive or chronic exposure of cells to xenobiotics in a well-preserved organ-specific mucosal architecture for an extended period of time. It is the aim of the present study to concurrently monitor cumulative genotoxic and apoptotic effects of sodium dichromate, N-nitrosodiethylamine (NDEA) and N-methyl-N-nitro-N-nitroso-guanidine (MNNG). Mini organs were raised by separating fresh specimens of human inferior nasal turbinates (n=11) into 1 mm3 sized pieces and culturing them on multiwell plates with bronchial epithelial basal medium for 6 days. Aliquots of the mini organs were subsequently exposed to sodium dichromate (1.0 mM, 1h), NDEA (50 mM, 1h) or MNNG (0.07 mM, 1h) on days 7, 9 and 11 versus a single exposure on day 11 only. DNA fragmentation and apoptotic events were assessed on day 11 using the alkaline single cell microgel electrophoresis assay (comet assay) and the annexin V-affinity assay. Significant DNA fragmentation could be demonstrated after a single exposure of the mini organs to sodium dichromate. Following three subsequent incubations, there was a further increase in the genetic damage observed, accompanied by an increase in the rate of apoptotic cells. In contrast, after single and triple incubation with NDEA there was neither an increase in genetic damage nor in the fraction of apoptotic cells detectable. Repetitive exposure to MNNG resulted in an accumulation of DNA damage without an observable increase in apoptosis. The results verify the need to assess apoptosis in genotoxicology research and to investigate cumulative effects of xenobiotics. Three-dimensional mini organ cultures of human upper aerodigestive tract epithelia have shown to be well-suited for improving the ability to distinguish between cumulative genotoxic and apoptotic effects.     

In vitro study on the genotoxicity of dichloromethane extracts of valerian (DEV) in human endothelial ECV304 cells and the effect of vitamins E and C in attenuating the DEV-induced DNA damages

To study the genotoxicity of valepotriates in vitro, the degree of DNA damage in human endothelial cell line ECV304 treated with 5-60 microg/mL of dichloromethane extracts of valerian (DEV) was analyzed by the Comet assay. No DNA damage was observed in ECV304 cells after culture for 48 h in the presence of 5,10, and 20 microg/mL of DEV. But a moderate degree of DNA damage was observed in the cells treated with 40 or 60 microg/mL of DEV. Quantitative analyses of DNA damage in the presence of antioxidants vitamin E (VE) and vitamin C (VC) were also carried out. The study revealed that both VE and VC exhibited a biphasic effect, reducing DEV-induced DNA damages at low concentrations but increasing them at high concentrations. We concluded that (1). the observed DNA damage in ECV304 cells induced by high concentrations of DEV was mainly through epigenetic mechanisms, i.e., reactive oxygen species mediated oxidative DNA damage (2). at the low doses, DEV did not appear to have any significant genotoxicity in ECV304 cells, and (3). VE and VC, at proper concentrations, can reduce or eliminate the adverse effects derived from high doses of DEV. This study should serve as scientific guidance for clinical therapy of valerian preparation.     

The effect of 3-methylfuran inhalation exposure on the rat nasal cavity

The acute inhalation toxicity of 3-methylfuran (3MF) was investigated in female CD/CR rats. Animals were killed 1, 3, 6, 9, 12, 15 and 30 days following a 1-h exposure to 148 mumol 3MF/1. Relatively selective 3MF-induced necrosis of the olfactory epithelium was seen at day 1 post exposure. Subsequent resolution of the acute olfactory necrosis was not complete and resulted in partial occlusive fibrosis of the nasal cavity as seen at 30 days. Pretreatment of the animals with piperonyl butoxide (PB) did not block 3MF-induced olfactory epithelial necrosis although it prevented Clara cell necrosis when given at a dose of 800 mg/kg intraperitoneally 1 h before exposure to 3MF.     

The effect of smoke generation and manipulation variables on the cytotoxicity of mainstream and sidestream cigarette smoke to monolayer cultures of L-929 cells

A system for the simultaneous exposure of monolayer cell cultures to mainstream (MS) and sidestream (SS) smoke from the same cigarette was utilized to study the effects of smoke generation and manipulation variables on the cytotoxicity of smoke to monolayer cultures of mouse fibroblast-like L-929 cells. The cytotoxicity of MS smoke was decreased with increasing smoke age (up to 8.7 s), smoke dilution, and the quantity of activated charcoal in filters. Acetate filters had little effect on cell mortality, and the age-of-smoke effect was not evident for MS smoke generated with a low puff volume and rapid dilution. The cytotoxicity of SS smoke also decreased rapidly with increasing smoke age and dilution. The results indicate that the gas phase of smoke may be of major importance in generating the observed toxic effects. These results may be of potential future significance in defining the requirements for a less toxic cigarette, in considering the hazards of SS smoke, and in evaluating in vivo inhalation studies.     

免疫磁珠分选人外周血T淋巴细胞的方法探讨

目的：探索一种用免疫磁珠分选人外周血T淋巴细胞更高效的方法。方法：用Ficoll密度梯度离心法先分离出人单核细胞（PBMC）,选用EasySep正选人CD3试剂盒,用两种不同的操作方法分离T淋巴细胞并进行比较。结果：两种方法有显著差异。结论：用免疫磁珠分选人外周血T淋巴细胞的方法值得推广。     

Formaldehyde and Glutaraldehyde and Nasal Cytotoxicity: Case Study Within the Context of the 2006 IPCS Human Framework for the Analysis of a Cancer Mode of Action for Humans

Formaldehyde and glutaraldehyde cause toxicity to the nasal epithelium of rats and mice upon inhalation. In addition, formaldehyde above certain concentrations induces dose-related increases in nasal tumors in rats and mice, but glutaraldehyde does not. Using the 2006 IPCS human framework for the analysis of cancer mode of action (MOA), an MOA for formaldehyde was formulated and its relevance was tested against the properties of the noncarcinogenic glutaraldehyde. These compounds produce similar patterns of response in histopathology and in genotoxicity tests (although formaldehyde has been much more extensively tested studied). The MOA is based on the induction of sustained cytotoxicity and reparative cell proliferation induced by formaldehyde at concentrations that also induce nasal tumors upon long-term exposure. Data on dose dependency and temporal relationships of key events are consistent with this MOA. While a genotoxic MOA can never be ruled out for a compound that is clearly genotoxic, at least in vitro, the nongenotoxic properties fundamental to the proposed MOA can explain the neoplastic response in the nose and may be more informative than genotoxicity in risk assessment. It is not yet fully explained why glutaraldehyde remains noncarcinogenic upon inhalation, but its greater inherent toxicity may be a key factor. The dual aldehyde functions in glutaraldehyde are likely to produce damage resulting in fewer kinetic possibilities (particularly for proteins involved in differentiation control) and lower potential for repair (nucleic acids) than would be the case for formaldehyde. While there have been few studies of possible glutaraldehyde-associated cancer, the evidence that formaldehyde is a human carcinogen is strong for nasopharyngeal cancers, although less so for sinonasal cancers. This apparent discrepancy could be due in part to the classification of human nasal tumors with tumors of the sinuses, which would receive much less exposure to inhaled formaldehyde. Evaluation of the human relevance of the proposed MOA of formaldehyde in rodents is restricted by human data limitations, although the key events are plausible. It is clear that the human relevance of the formaldehyde MOA in rodents cannot be excluded on either kinetic or dynamic grounds.     

Estimation of Apoptosis and Necrosis Caused by Pesticides In Vitro on Human Lymphocytes Using DNA Diffusion Assay

Organophosphorus pesticides like monocrotophos, profenofos, chlorpyrifos, and acephate are most commonly used in India for agriculture and public health programs. Previous studies have revealed that at low doses, organophosphorus pesticides not only act as genotoxic agents but also affect several other biochemical pathways. The aim of the current investigation was to assess apoptosis and necrosis caused by these pesticides on human peripheral blood lymphocytes under in vitro conditions using the DNA diffusion assay. Our studies have revealed that all the above pesticides induced apoptosis and necrosis in cultured human peripheral blood lymphocytes in in vitro conditions. The results are statistically significant (p < 0.001). Data on these alterations of immune cells are required for understanding the subchronic effects mediated by pesticides on nontarget organisms.     

Evaluation of the genotoxic and antigenotoxic potential of Baccharis dracunculifolia extract on V79 cells by the comet assay

Baccharis dracunculifolia (Asteraceae), the main botanical source of green propolis, is a shrub of the Brazilian ‘cerrado’. In folk medicine it is used as an anti‐inflammatory agent, mainly for the treatment of gastrointestinal diseases. The aim of the present study was to evaluate the genotoxic and antigenotoxic effects of B. dracunculifolia ethyl acetate extract (Bd‐EAE) on Chinese hamster lung fibroblasts (V79 cells) by the comet assay. Methyl methanesulfonate (MMS; 200 μM ) was used as an inducer of DNA damage. Genotoxicity was evaluated using four different concentrations of Bd‐EAE: 12.5, 25.0, 50.0 and 100.0 μg ml^?1. Antigenotoxicity was assessed before, simultaneously, and after treatment with the mutagen. The results showed a significant increase in the frequency of DNA damage in cultures treated with 50.0 and 100.0 μg ml^?1 Bd‐EAE. Regarding its antigenotoxic potential, Bd‐EAE reduced the frequency of DNA damage induced by MMS. However, this chemopreventive activity depended on the concentrations and treatment regimens used. The antioxidant activity of phenolic components present in Bd‐EAE may contribute to reduce the alkylation damage induced by MMS. In conclusion, our findings confirmed the chemopreventive activity of Bd‐EAE and showed that this effect occurs under different mechanism. Copyright © 2009 John Wiley & Sons, Ltd.     

Mutagenic, cytotoxic, and genotoxic properties of tobacco smoke produced by cigarillos available on the Canadian market

Cigarillos (aka little cigars) have been increasing in popularity unlike cigarettes; but relatively little is known about the toxicology of the mainstream smoke (MSS) from such products. Therefore, the objective of this work was to compare the toxicological properties of the MSS (Health Canada Intensive smoking conditions) from a range of cigarillo products with the toxicological properties of MSS of cigarettes. Three in vitro assays were used to evaluate the toxicities of the MSS total particulate matter (TPM): (1) mutagenicity using Ames assay with Salmonella strains TA98 and TA100 with S9 metabolic activation (+S9); (2) cytotoxicity using the Neutral Red Uptake (NRU) assay with CHO (Chinese Hamster Ovary) cells; and (3) genotoxicity using the micronucleus assay with CHO cells and short-term exposures (3-h ± S9). The Ames assay (TA100 + S9) and the NRU assay were also applied to the gas/vapour phase of the MSS that passed through the Cambridge pad. On a per-milligram-nicotine basis, the preferred way of comparing toxicities of different types of tobacco products, the MSS from cigarillos was not less toxic, and in some cases more toxic (TPM fraction TA98 + S9, NRU), than the MSS from cigarettes. Thus, our findings support our prior work on smoke mutagenicity that showed MSS from cigarillos was not less toxic than MSS from cigarettes.