In-vitro evaluation of copper/copper oxide nanoparticles cytotoxicity and genotoxicity in normal and cancer lung cell lines

BackgroundNanotoxicology is a major field of study that reveals hazard effects of nanomaterials on the living cells.MethodsIn the present study, Copper/Copper oxide nanoparticles (Cu/CuO NPs) were prepared by the chemical reduction method and characterized by different techniques such as: X-Ray Diffraction, Transmission and Scanning Electron Microscopy. Evaluation of the toxicity of Cu/CuO NPs was performed on 2 types of cells: human lung normal cell lines (WI-38) and human lung carcinoma cell (A549). To assess the toxicity of the prepared Cu/CuOs NPs, the two cell types were exposed to Cu/CuO NPs for 72 h. The half-maximal inhibitory concentration IC₅₀ of Cu/CuO NPs for both cell types was separately determined and used to examine the cell genotoxicity concurrently with the determination of some oxidative stress parameters: nitric oxide, glutathione reduced, hydrogen peroxide, malondialdehyde and superoxide dismutase.ResultsCu/CuO NPs suppressed proliferation and viability of normal and carcinoma lung cells. Treatment of both cell types with their IC₅₀’s of Cu/CuO NPs resulted in DNA damage besides the generation of reactive oxygen species and consequently the generation of a state of oxidative stress.ConclusionOverall, it can be concluded that the IC₅₀'s of the prepared Cu/CuO NPs were cytotoxic and genotoxic to both normal and cancerous lung cells.     

Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes

In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7alpha-position for Delta(8)-THC and the 8beta-position for Delta(9)-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7alpha-(Delta(8)-THC), 8beta-(Delta(9)-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Delta(8)-THC (7.60 nmol/min/nmol CYP), Delta(9)-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7alpha-(5.34 nmol/min/nmol CYP) and 7beta-hydroxylation (1.39 nmol/min/nmol CYP) of Delta(8)-THC, the 8beta-hydroxylation (6.10 nmol/min/nmol CYP) and 9alpha,10alpha-epoxidation (1.71 nmol/min/nmol CYP) of Delta(9)-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7alpha- and 7beta-hydroxylation of Delta(8)-THC, and the 9alpha,10alpha-epoxidation of Delta(9)-THC.     

Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells

We introduce a method to follow DNA repair that is suitable for both clinical and laboratory samples. An episomal construct with a unique 8-oxoguanine (8-oxoG) base at a defined position was prepared in vitro using single-stranded phage harboring a 678-bp tract from exons 5 to 9 of the human P53 gene. Mixing curve experiments showed that the real-time PCR method has a linear response to damage, suggesting that it is useful for DNA repair studies. The episomal construct with a unique 8-oxoG base was introduced into AD293 cells or human peripheral blood mononuclear cells, and plasmids were recovered as a function of time. The quantitative real-time PCR assay demonstrated that repair of the 8-oxoG was 80% complete in less than 48 h in AD293 cells. Transfection of small interfering RNAs down-regulated OGG1 expression in AD293 cells and reduced the repair of 8-oxoG to 30%. Transfection of the episome into unstimulated white blood cells showed that 8-oxoG repair had a half-life of 2 to 5 h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type.     

Sensitivity to microcystins: a comparative study in human cell lines with and without multidrug resistance phenotype

Multidrug resistance (MDR) is an obstacle in cancer treatment. An understanding of how tumoral cells react to oxidants can help us elucidate the cellular mechanism involved in resistance. Microcystins are cyanobacteria hepatotoxins known to generate oxidative stress. The aim of this study was to compare the sensitivity to microcystins of human tumoral cell lines with (Lucena) and without (K562) MDR phenotype. Endpoints analyzed were effective microcystins concentration to 50% of exposed cells (EC50), antioxidant enzyme activity, lipid peroxidation, DNA damage, reactive oxygen species (ROS) concentration, and tubulin content. Lucena were more resistant and showed lower DNA damage than K562 cells (P<0.05). Although microcystins did not alter catalase activity, a higher mean value was observed in Lucena than in K562 cells. Lucena cells also showed lower ROS concentration and higher tubulin content. The higher metabolism associated with the MDR phenotype should increase ROS concentration and make for an improved antioxidant defense against the toxic effects of microcystins.     

Sequence-specific DNA damage induced by carcinogenic danthron and anthraquinone in the presence of Cu(II), cytochrome P450 reductase and NADPH

The mechanism of metal-mediated DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone was investigated by the DNA sequencing technique using ³²P-labeled human DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Danthron caused DNA damage particularly at guanines in the 5′-GG-3′, 5-GGGG-3′, 5′-GGGGG-3′ sequences (damaged bases are underlined) in the presence of Cu(II), cytochrome P450 reductase and the NADPH-generating system. The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H₂O₂ and Cu(I). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine increased with increasing concentration of danthron. On the other hand, carcinogenic anthraquinone induced less oxidative DNA damage than danthron. Electron spin resonance study showed that the semiquinone radical could beproduced by P450 reductase plus NADPH-mediated reduction of danthron, while little signal was observed with anthraquinone. These results suggest that danthron is much more likely to be reduced by P450 reductase and generate reactive oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated metabolites with similar reactivity to danthron may participate in DNA damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone seems to be relevant for the expression of their carcinogenicity.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

Increased cytotoxicity of food-borne mycotoxins toward human cell lines in vitro via enhanced cytochrome p450 expression using the MTT bioassay

Eight food-borne mycotoxins epidemiologically implicated in human disease were tested for their cytotoxic effects on human cells previously immortalised and transfected to introduce human cytochrome p450(CYP 450) genes. Such cells retain many characteristics of normal cell growth and differentiation while simultaneously having the potential of either increasing or decreasing the metabolic activity (cytotoxicity) of the challenging mycotoxins. The MTT assay provided an indication of cytotoxicity. Of the nine CYP 450s introduced CYP1A2 was most effective,rendering the cells 540 times more sensitive than the control cells to aflatoxin B₁, 28 times more sensitive to aflatoxin G₁ and 8-fold more sensitive to ochratoxin A. CYP3A4 resulted in the cells being 211 times more toxic to aflatoxin B₁ and 8-fold more toxic to aflatoxin G₁ while CYP 2A6, CYP 3A5 and CYP 2E1 also produced observable effects. No increase in metabolic activity was found using cyclopiazonic acid, deoxynivalenol,fumonisin B₁, patulin or T-2 toxin. CD5Os were calculated for the mycotoxins against the non-CYP-introduced control cells. There was almost a five order of magnitude difference between the most toxic,T-2 toxin (CD50 0.0057 g/ml) and the least toxic, fumonisin ₁(CD50 476.2 g/ml). In vitro biological assays thus provide an excellent system for quantifying the often low CD50s expressed bymycotoxins in foods.This revised version was published online in October 2005 with corrections to the Cover Date.     

Disruption of EARLY LESION LEAF 1, encoding a cytochrome P450 monooxygenase,induces ROS accumulation and cell death in rice

Lesion-mimic mutants (LMMs) provide a valuable tool to reveal the molecular mechanisms determining programmed cell death (PCD) in plants. Despite intensive research, the mechanisms behind PCD and the formation of lesions in various LMMs still remain to be elucidated. Here, we identified a rice (Oryza sativa) LMM, early lesion leaf 1 (ell1), cloned the causal gene by map-based cloning, and verified this by complementation. ELL1 encodes a cytochrome P450 monooxygenase, and the ELL1 protein was located in the endoplasmic reticulum. The ell1 mutant exhibited decreased chlorophyll contents, serious chloroplast degradation, upregulated expression of chloroplast degradation-related genes, and attenuated photosynthetic protein activity, indicating that ELL1 is involved in chloroplast development. RNA sequencing analysis showed that genes related to oxygen binding were differentially expressed in ell1 and wild-type plants; histochemistry and paraffin sectioning results indicated that hydrogen peroxide (H₂O₂) and callose accumulated in the ell1 leaves, and the cell structure around the lesions was severely damaged, which indicated that reactive oxygen species (ROS) accumulated and cell death occurred in the mutant. TUNEL staining and comet experiments revealed that severe DNA degradation and abnormal PCD occurred in the ell1 mutants, which implied that excessive ROS accumulation may induce DNA damage and ROS-mediated cell death in the mutant. Additionally, lesion initiation in the ell1 mutant was light dependent and temperature sensitive. Our findings revealed that ELL1 affects chloroplast development or function, and that loss of ELL1 function induces ROS accumulation and lesion formation in rice.     

The effect of hyperthermia on cell viability,oxidative damage,and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

The purpose of the study was to investigate the effects of mild hyperthermia on cell viability, release of lactate dehydrogenase (LDH), superoxide dismutase (SOD) activity, malondialdehyde (MDA) formation, total antioxidant capacity (T-AOC), and the relative mRNA levels of heat shock protein (HSP60, 70, and 90) in hepatic cells of grass carp (Ctenopharyngodon idellus) before and after temperature stress. Cultured cells were exposed to thermal stress (32 °C) for 0.5, 1, 2, 4, and 8 h. The results showed that hyperthermia stress significantly reduced cell viability (P<0.01) and increased LDH release at 0.5 and 1 h (P<0.05). Additionally, hyperthermia stress led to oxidative stress as evidenced by significantly decreased T-AOC after treating cells for 0.5 and 8 h (P<0.05). SOD activity also significantly decreased after 1 h of stress (P<0.05), but MDA formation increased after 8 h of stress (P<0.05). This may be partly responsible for the lower cell viability and higher LDH release we observed. The differences between SOD activity, MDA formation, and T-AOC between the 2 h treatment group and the control were smaller than that of other groups. This indicated that cellular antioxidant enzyme systems play an important role in the defense against oxidative stress. Further tests showed that the expression of HSP60 at 1, 2, and 4 h (P<0.05), HSP70 at 0.5 and 1 h (P<0.01), and HSP90 at all time points after stress were higher (P<0.01) than pre-stress levels. This suggested that HSPs possess the ability to modulate cellular anti-stress responses and play key roles in protecting organisms from heat stress. In conclusion, hyperthermia inhibits cell proliferation, induces cell oxidative stress, and enhances HSP expression in hepatic cells of grass carp.     

p66(Shc) gene has a pro-apoptotic role in human cell lines and it is activated by a p53-independent pathway

p66(Shc) protein has been proposed to be an indispensable factor for p53-dependent, mitochondria-mediated apoptosis in mice. Here, we show that p66(Shc) plays a pro-apoptotic role also in cell lines of human origin such as SaOs-2 and HeLa, where p53 is either absent or inactivated, thus, suggesting that p66(Shc) pro-apoptotic role is independent from the presence of a functional form of p53. The active form of p66(Shc) is phosphorylated in Serine 36. We confirm the importance of Serine 36 phosphorylation for p66(Shc) pro-apoptotic role, and our results suggest that the kinase involved in this process is activated independently from p53.     

DNA damage and cell necrosis induced by naphthalene due to the modulation of biotransformation enzymes in an estuarine crab Scylla serrata

The sublethal effect of naphthalene (2.5, 5, and 10 mg L(-1)) was studied in an estuarine crab Scylla serrata with reference to macromolecular changes. Biotransformation enzymes such as cytochrome P450, cytochrome b(5), NADPH cytochrome P450 reductase, aryl hydrocarbon hydroxylase, glutathione-S-transferase, and UDP-glucuronyl transferase were elevated in the hepatopancreas of naphthalene-exposed crabs in comparison with control. Remarkable amount of DNA damage and cell necrosis was observed in hepatopancreas, hemolymph, and ovary of the crabs exposed to naphthalene, when compared with control. For all the parameters studied, a concentration-dependent gradient of the changes was observed. The expression of DNA damage and cell necrosis suggests an increased production of oxidants during naphthalene metabolism.     

The radiosensitizing effect of fluorocyclopentenyl-cytosine (RX-3117) in ovarian and lung cancer cell lines

ABSTRACT

RX-3117 (fluorocyclopentenyl-cytosine) is a novel cytidine analog currently being evaluated in a Phase Ib clinical trial in cancer patients with solid tumors. The radiosensitizing effect of RX-3117 was studied in A2780 ovarian cancer cells and non-small cell lung cancer cell lines and related to cell survival and the effect on cell cycle and cell cycle proteins. RX-3117 has a schedule-dependent radiosensitizing effect, but only at pre-incubation (dose modifying factors: 1.4–1.8), observed at pulse and fractionated irradiation. Radiosensitizion was also seen in a three-dimensional spheroid model. At the low radiosensitizing concentration, RX-3117 in combination with radiation led to an accumulation of cells in S-phase, which was accompanied with an increase of cell cycle proteins such as p-Chk2 and p-cdc25C. In addition, RX-3117 caused DNA damage and increased apoptosis. In conclusion, our in vitro experiments showed a radiosensitizing effect of RX-3117.     

Selection, establishment and characterization of cell lines derived from a chemically-induced rat mammary heterogeneous tumor, by flow cytometry, transmission electron microscopy, and immunohistochemistry

Summary In order to isolate, characterize, and establish culture cell lines with different diagnostic and prognostic significance, derived from multiclonal neoplasms, a ductal infiltrating mammary tumor was induced in rats by 7,12-dimethylbenz[a]anthracene. Clones with different DNA/protein content, being the DI of 1.16, 1.30, and 1.60, respectively, were observed in the primary tumor. Biparametric flow cytometry suggested that the clone at 1.30 is made up of two subpopulations with different protein and slightly different DNA contents. The culture, after a few passages, exhibited the presence of aneuploid cells and the absence of diploid components, demonstrating that only tumor cells survived. The limiting dilution method gave rise to four lines with DI of 1.16, 1.25, 1.30, and 1.50; a mean chromosome number of 45, 46, 47, and 88, respectively; and different morphological and ultrastructural features. These characteristics were stable during the experimental procedure, that is, for about 20 passages. Conversely, the detection of cytoskeletal proteins indicated that the tumor epithelial cells underwent early dedifferentiation into sarcoma-like cells showing markers of stromal cell type and thus exhibiting phenotypic instability in vitro, a feature reported in many advanced human breast cancers in vivo. In conclusion, this cellular model represents the in vivo situation and appears suitable for in vitro studies of tumor cell characteristics and might be used to predict clinical behavior.     

Zinc toxicity in various lung cell lines is mediated by glutathione and GSSG reductase activity

In a previous work, it was shown that in cells after a decrease of cellular glutathione content, toxic zinc effects, such as protein synthesis inhibition or GSSG (glutathione, oxidized form) increases, were enhanced. In this study, zinc toxicity was determined by detection of methionine incorporation as a parameter of protein synthesis and GSSG increase in various lung cell lines (A549, L2, 11Lu, 16Lu), dependent on enhanced GSSG reductase activities and changed glutathione contents. After pretreatment of cells with dl-buthionine-[R,S]-sulfoximine (BSO) for 72 h, cellular glutathione contents were decreased to 15–40% and GSSG reductase activity was increased to 120–135% in a concentration-dependent manner. In BSO pretreated cells, the IC₅₀ values of zinc for methionine incorporation inhibition were unchanged as compared to cells not pretreated. The GSSG increase in BSO pretreated cells by zinc was enhanced in L2, 11Lu, and 16Lu cells, whereas in A549 cells, the GSSG increase by zinc was enhanced only after pretreatment with the highest BSO concentration. Inhibition of GSSG reductase in alveolar epithelial cells was observed at lower zinc concentrations than needed for methionine incorporation inhibition, whereas in fibroblastlike cells, inhibition of GSSG reductase occurred at markedly higher zinc concentrations as compared to methionine incorporation inhibition. These results demonstrate that GSSG reductase is an important factor in cellular zinc susceptibility. We conclude that reduction of GSSG is reduced in zinc-exposed cells. Therefore, protection of GSH oxidation by various antioxidants as well as enhancement of GSH content are expected to be mechanisms of diminishing toxic cellular effects after exposure to zinc.     

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO‐S, CHO‐K1 8 mM glutamine, and CHO‐K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO‐S and CHO‐K1, with the diversity increasing and new variants appearing, while CHO‐K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.     

Inhibition of thioredoxin reductase induces apoptosis in neuronal cell lines: role of glutathione and the MKK4/JNK pathway

The Thioredoxin (Trx)/Thioredoxin reductase (TrxR)-system has emerged as a crucial component of many cellular functions particularly antioxidant defence. We investigated the effect of the selective TrxR inhibitor 1-chloro-2,4-dinitrobenzene (CDNB) on survival and redox status in neuronal cell lines. CDNB was found to cause apoptosis without depletion of glutathione or loss of mitochondrial complex I-activity. Cells treated with CDNB displayed an early increase of reactive oxygen species and rapid activation of stress inducible protein kinases c-Jun N-terminal kinase (JNK) and mitogen activated protein kinase kinase 4 (MKK4). Thus TrxR inhibition by CDNB results in generation of reactive oxygen species and subsequent activation of stress-inducible kinases without impairment of the cellular antioxidant status or mitochondrial function. Inhibition of the specific kinases involved in cell death triggered by Trx/TrxR dysfunction could represent a novel and selective therapeutic approach in neurodegenerative disorders.     

Peroxisome proliferator-activated receptor gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2, and ciglitazone, induce growth inhibition and cell cycle arrest in hepatic oval cells

There is growing evidence to show that hepatic oval cells contribute to liver regeneration, dysplastic nodule formation, and hepato-carcinogenesis. Peroxisome proliferator-activated receptors (PPARs) and their ligands play an important role in cell growth, inflammatory responses, and liver pathogenesis including fibrosis and cancer. However, little is known about the role of PPARgamma/its ligands in the growth and differentiation of hepatic oval cells. In this study, we found that OC15-5, a rat hepatic oval cell line, expressed PPARgamma at mRNA and protein levels, and a natural ligand for PPARgamma, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), and a synthetic ligand, ciglitazone, inhibited growth of OC15-5 cells by arresting at G1-S in a dose-dependent manner. Apoptosis was also induced in OC15-5 cells by 15d-PGJ2 treatment. In OC15-5 cells treated with 15d-PGJ2, the expression of CDK inhibitor, p27(Kip1), was up-regulated, while that of p21(WAF1/Cip1), p18(INK4C) CDK2, CDK4, and cyclin E was unchanged. In addition, delayed up-regulation of AFP expression was observed in OC15-5 cells after 15d-PGJ2 or ciglitazone treatment. This is the first report to show that the PPARgamma ligand was involved in the growth, cell cycle, and differentiation of hepatic oval cells, raising the possibility that the PPARgamma ligands may regulate liver regeneration and hepato-carcinogenesis.