Cytotoxic and genotoxic evaluation of different synthetic amorphous silica nanomaterials in the V79 cell line.

The nature of occupational risks and hazards in industries that produce or use synthetic amorphous silica (SAS) nanoparticles is still under discussion. Manufactured SAS occur in amorphous form and can be divided into two main types according to the production process, namely, pyrogenic silica (powder) and precipitated silica (powder, gel or colloid). The physical and chemical properties of SAS may vary in terms of particle size, surface area, agglomeration state or purity, and differences in their toxicity potential might therefore be expected. The aim of this study was to compare the cytotoxicity and genotoxicity of representative manufactured SAS samples in Chinese hamster lung fibroblasts (V79 cells). Five samples from industrial SAS producers were evaluated, that is, two pyrogenic SAS powders (with primary particle sizes of 20 nm and 25/70 nm), one precipitated SAS powder (20 nm) and two precipitated SAS colloids (15 and 40/80 nm). V79 cell cultures were treated with different concentrations of SAS pre-dispersed in bovine serum albumin -water medium. Pyr (pyrogenic) 20, Pre (precipitated) 20 and Col (colloid) 15 significantly decreased the cell viability after 24 h of exposure, whilst Pyr 25/70 and Col 40/80 had negligible effects. The cytotoxicity of Pyr 20, Pre 20 and Col 15 was revealed by the induction of apoptosis, and Pyr 20 and Col 15 also produced DNA damage. However, none of the SAS samples generated intracellular reactive oxidative species, micronuclei or genomic mutations in V79 cells after 24 h of exposure. Overall, the results of this study show that pyrogenic, precipitated and colloidal manufactured SAS of around 20 nm primary particle size can produce significant cytotoxic and genotoxic effects in V79 cells. In contrast, the coarser-grained pyrogenic and colloid SAS (approximately 50 nm) yielded negligible toxicity, despite having been manufactured by same processes as their finer-grained equivalents. To explain these differences, the influence of particle agglomeration and oxidative species formation is discussed.

Cytotoxicity and genotoxicity of panel of single- and multiwalled carbon nanotubes: in vitro effects on normal Syrian hamster embryo and immortalized v79 hamster lung cells.

Carbon nanotubes (CNTs) belong to a specific class of nanomaterials with unique properties. Because of their anticipated use in a wide range of industrial applications, their toxicity is of increasing concern. In order to determine whether specific physicochemical characteristics of CNTs are responsible for their toxicological effects, we investigated the cytotoxic and genotoxic effects of eight CNTs representative of each of the commonly encountered classes: single- SW-, double- DW-, and multiwalled (MW) CNTs, purified and raw. In addition, because most previous studies of CNT toxicity were conducted on immortalized cell lines, we decided to compare results obtained from V79 cells, an established cell line, with results from SHE (Syrian hamster embryo) cells, an easy-to-handle normal cell model. After 24 hours of treatment, MWCNTs were generally found to be more cytotoxic than SW- or DWCNTs. MWCNTs also provoked more genotoxic effects. No correlation could be found between CNT genotoxicity and metal impurities, length, surface area, or induction of cellular oxidative stress, but genotoxicity was seen to increase with CNT width. The toxicity observed for some CNTs leads us to suggest that they might also act by interfering with the cell cycle, but no significant differences were observed between normal and immortalized cells.

In vitro cytotoxicity and transforming potential of industrial carbon dust (fibers and particles) in syrian hamster embryo (SHE) cells.

Carbon fibers have many applications, mainly in high-tech industries such as the aviation industry. Eleven carbon samples (fibers and particles) coming from an aeronautic group were tested for their cytotoxicity and carcinogenic potential using in vitro short-term assays in Syrian hamster embryo cells. These samples were taken during each important step of the process, i.e. from the initial heating of polyacrylonitrile fibers to pure carbon fibers. They were compared to an asbestos fiber, an amorphous silica, and two commercial graphite powders. Their physical-chemical characteristics and their capacity to release reactive oxygen species (ROS) were determined. This study showed that none of the carbon samples was able to generate ROS as measured by Electron Paramagnetic Resonance analysis, and in our biological assays, they demonstrated no morphological transformation potential and low cytotoxicity compared to positive control (chrysotile asbestos).

N-methylation of anthracyclines modulates their cytotoxicity and pharmacokinetic in wild type and multidrug resistant cells.

Anthracyclines are the most commonly used classes of anticancer agents in chemotherapy. Development of resistance to these molecules is one of the major reasons for treatment failure. The overexpression of the membrane transporter P-glycoprotein (P-gp) is among the principal mechanisms involved in this phenomenon. This pump, which is responsible for the multidrug resistance (MDR) phenotype, decreases the toxicity of a wide range of unrelated anticancer drugs by increasing their cellular efflux. Structure-activity relationship experiments have shown that the positively charged amino group of the anthracyclines could be responsible for their transport by P-gp. Here, we used three new anthracyclines that shared the same chromophore but differed by the degree of N-methylation of their sugar moiety. Oxaunomycin (OXN) possessed a non-methylated amino group, while LB-1 was monomethylated and beta-clamycin T (BCT) was dimethylated. In sensitive cells (FLC), reduced cytotoxicity was related to the level of N-methylation; whereas in resistant cells (DOX-RFLC(1) and DOX-RFLC(2)) overexpressing different levels of P-gp, increased N-methylation enhanced anthracycline cytotoxicity. Decreased resistance in DOX-RFLCs was associated with an increased drug accumulation due to a reduced cellular efflux. As expected, the MDR modulator verapamil decreased resistance to these anthracyclines by increasing the cellular accumulation. These results suggest that N-methylation of anthracyclines circumvents resistance by diminishing drug transport by P-gp in MDR-positive cells. These observations could be the consequence of the steric hindrance created by the methyl group(s) which may impair the interaction between the positively charged amino group and the active site of P-gp.

Prevention of pathologies associated with oxidative stress and dietary intake deficiencies: folate deficiency and requirements.

Folate (folic acid, folacin) is an essential vitamin that is found in nature. Folates contain the core chemical structure of pteroylglutamic acid, but vary in their state of reduction, the single carbon moiety they bear and/or the length of the glutamate chain. At least 50% of whole body folate is stored in the liver. The influence of intracellular folate concentration depends largely on dietary intake. The supply of folate depends primarily on the quantity and bioavailability of ingested folate and the rate of loss by urinary and fecal routes and through catabolism.

Iron: deficiencies and requirements.

A report from the World Health Organization estimates that 46% of the world's 5- to 14-year-old children are anemic. In addition, 48% of the world's pregnant women are anemic. A majority of these cases of anemia are due to iron deficiency. Our aim here is to review the latest data on iron regulatory mechanisms, iron sources and requirements. Human and animal studies have shown that amino acids and peptides influence iron absorption from the intestinal lumen. Inter-organ transport and uptake of nonheme iron is largely performed by the complex transferring-transferring receptor system. Moreover, the discovery of cytoplasmic iron regulatory proteins (IRPs) has provided a molecular framework from which we understand the coordination of cellular iron homeostasis in mammals. IRPs and the iron responsive elements (IREs) to which they bind allow mammals to make use of the essential properties of iron while reducing its potentially toxic effect. Physiologic iron requirements are three times higher in pregnancy than they are in menstruating women (approximately 1200 mg must be acquired from the body's iron store or from the diet by the end of pregnancy). The administration of iron supplements weekly instead of daily in humans has been proposed and is being actively investigated as a viable means of controlling iron deficiency in populations, including pregnant women.

Pharmacologic or genetic manipulation of glutathione S-transferase P1-1 (GSTpi) influences cell proliferation pathways.

Glutathione S-transferase P1-1 (GSTpi) is an abundant and ubiquitously expressed protein in normal and malignant mammalian tissues and possesses catalytic and ligand binding properties. Our present data suggest that the protein contributes to the regulation of cell proliferation. Mouse embryo fibroblasts (MEFs) isolated from mice with a GSTP1-1 [glutathione S-transferase P1-1 (isozyme in nonhepatic tissue)] null genotype (GSTpi(-/-)) doubled their population in 26.2 h versus 33.6 h for the wild type (GSTpi(+/+)). Retroviral transfection of GSTP1-1 into GSTpi(-/-) MEF cells slowed the doubling time to 30.4 h. Both early passage and immortalized MEF cells from GSTpi(-/-) animals expressed significantly elevated activity of extracellular signal-regulated kinases ERK1/ERK2, kinases linked to cell proliferation pathways. In vivo, GSTpi(-/-) mice had higher basal levels of circulating white blood cells compared with GSTpi(+/+). Administration of a peptidomimetic inhibitor of GSTP1-1, TLK199, (gamma-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester), stimulated both lymphocyte production and bone marrow progenitor (colony-forming unit-granulocyte macrophage) proliferation, but only in GSTpi(+/+) and not in GSTpi(-/-) animals. Selection of a resistant clone of an HL60 tumor cell line through chronic exposure to TLK199 resulted in cells with elevated activities of c-Jun NH2 terminal kinase (JNK1) and ERK1/ERK2, and allowed the cells to proliferate under stress conditions that induced high levels of apoptosis in the wild type cells. The in vitro and in vivo data are consistent with the principle that GSTP1-1 influences cell proliferation.

Oxidative stress induced in pathologies: the role of antioxidants.

Exposure to oxidant molecules issued from the environment (pollution, radiation), nutrition, or pathologies can generate reactive oxygen species (ROS for example, H2O2, O2-, OH). These free radicals can alter DNA, proteins and/or membrane phospholipids. Depletion of intracellular antioxidants in acute oxidative stress or in various diseases increases intracellular ROS accumulation. This in turn is responsible for several chronic pathologies including cancer, neurodegenerative or cardiovascular pathologies. Thus, to prevent against cellular damages associated with oxidative stress it is important to balance the ratio of antioxidants to oxidants by supplementation or by cell induction of antioxidants.

Comparison of annamycin to adriamycin in cardiac and MDR tumor cell systems.

Based on the response of a wide variety of tumors to the anthracycline, Adriamycin, numerous studies have been initiated to find an even more effective analog. In this pursuit two of the obstacles that have been necessary to overcome are a unique dose dependent Adriamycin-induced cardiotoxicity reported in patients treated with this chemotherapeutic agent as well as p-gp-mediated multi drug resistance (MDR) which has been found in tumor cells exposed to Adriamycin in vitro and in vivo as well as in human tumor samples. Using an in vitro cardiac cell system and MDR+ and MDR- Friend leukemia cell lines we find that a relatively new anthracycline, Annamycin, has reduced cardiotoxic activity but is more effective in inhibiting the growth of MDR+ cells than Adriamycin. The reduced cardiotoxicity of Annamycin is approximately 10 fold lower than Adriamycin whereas the increased efficacy against the MDR+ Friend leukemia tumor cell line is about 2 fold. The observation that Adriamycin preferentially accumulates in cardiac-muscle (CM) but not in cardiac non-muscle (NM) cells while Annamycin accumulates equally in both, may explain in part the reduced cardiotoxicity of Annamycin. Moreover, the cytosolic accumulation of Annamycin vs the nuclear localization of Adriamycin suggests a different target site for each drug.

Apoptosis inhibition and ornithine decarboxylase superinduction as early epigenetic events in morphological transformation of Syrian hamster embryo cells exposed to 2-methoxyacetaldehyde, a metabolite of 2-methoxyethanol.

We have conducted a study to determine the carcinogenic potential of ethylene glycol monomethyl ether (EGME), a member of the glycol ether family, as compared to its reactive metabolite 2-methoxy-acetaldehyde (MALD). Since disruption of equilibrium between cell proliferation and cell death is thought to play a key role in multistage carcinogenesis, we investigated, in Syrian hamster embryo (SHE) cells exposed to various doses of EGME and MALD, impairment in apoptosis rate and in ornithine decarboxylase (ODC) metabolism. The activity of this rate-limiting enzyme of polyamine biosynthesis is closely related to cell proliferation and cell transformation. At the end-point, comparative action of the two products on SHE cell morphological transformation frequency was evaluated. One-stage exposure of SHE cells to 2 mM EGME and 200 microM MALD for 5 h did not change basal apoptotic level, whereas 0.16 microM phorbol ester (TPA) decreased it. Using two-stage exposure protocol (1 h xenobiotic followed by 5 h TPA), MALD strongly inhibited apoptosis more than did TPA alone; the parent compound EGME did not have any effect on TPA inhibiting action. Western blotting analysis showed that sequential treatment (MALD/TPA) increased Bcl-2 oncoprotein expression, whereas Bcl-XL and Bax proteins were not changed. The same staged exposure of SHE cells to MALD/TPA strongly induced ODC activity, and the rate was higher than that obtained with TPA alone: this was accompanied by an increase of ODC protein level. This ODC superinduction was not observed with EGME/TPA treatment. In long-term SHE-cell morphological transformation assay, staged exposure to MALD (800 microM or 1 mM for 24 h) followed by TPA applications increased the number of transformed colonies at the seventh day. Such early cooperative events as apoptosis inhibition and ODC superinduction, followed by the increase of SHE-cell transformation frequency, are highly indicative of a carcinogenic potential for the metabolite, MALD.

Impact of dietary supplement of Crassostrea gigas extract (JCOE) on glutathione levels and glutathione S-transferase activity in rat tissues.

Male Sprague Dawley rats received various amounts of extract of Crassostrea gigas by gavage every day for 2 weeks or one month. At these times, groups of animals were sacrificed and samples of major organs analyzed for levels of glutathione (GSH) and glutathione S-transferase (GST) activities. Following the two week protocol, GSH levels were significantly increased in the mucosa of the large intestine; at one month the small intestine and spleen were elevated. GST activity increased in liver under both schedules and at one month, activity was also elevated in kidney and small intestine. Since the Crassostrea gigas extract contains high levels of a variety of important amino acids, it is concluded that biologically available peptides are taken up in target organs and stimulate GSH metabolism. Enhanced levels of GSH and associated enzymes may contribute to a more effective detoxification phenotype, thus providing enhanced chemoprotective capacity.

The antioxidant effects of Crassostrea gigas extract (JCOE) in human volunteers.

Since several in vitro and animal studies of an extract from Crassostrea gigas (JCOE) have demonstrated its antioxidant properties and other interesting effects, a preliminary human trial was carried out. Seven healthy male volunteers aged 23-37 received orally 3 x 2 capsules of JCOE per day for 8 days. On days 0, 1, 4, 8 and 15 (7 days after completion of the schedule) blood samples were drawn and the antioxidant potential of serum was tested. A statistically significant increase in the buffering effects of serum against hemoglobin (Hb) and lactate dehydrogenase (LDH) release from red blood cells treated with the free radical generator azobis amidino propane (AAPH) was found following JCOE treatment. At 8 days, the oxidative effects were reduced by > 90% of the pretreatment values. In these same individuals, serum levels of reduced glutathione were increased by an average of 1.5-fold over the time course of treatment. It is concluded that in normal human volunteers, JCOE capsules provide an orally available formula for enhancing the antioxidant capacity of blood serum. While the extract is known to contain some direct acting antioxidant components, at least a portion of the protective effect is facilitated by enhancement of GSH biosynthesis.

Two-stage exposure of Syrian-hamster-embryo cells to environmental carcinogens: superinduction of ornithine decarboxylase correlates with increase of morphological-transformation frequency.

As part of environmental toxicology, it is important to assess both the carcinogenic potential of xenobiotics and their mode of action on target cells. Since dysregulation of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, is considered as an early and essential component in the process of multistage carcinogenesis, we have studied the mode of ODC induction in Syrian-hamster-embryo(SHE) cells stage-exposed to carcinogens and to non-carcinogens. One-stage (5 hr) treatment of SHE cells with 50 microM clofibrate (CLF), a non-genotoxic carcinogen, or with 0.4 microM benzo(a)pyrene (BaP), a genotoxic carcinogen, slightly decreased basal ODC activity. Using the 2-stage exposure, 1 hr to carcinogen, then replacement by TPA for 5 hr, the ODC activity was higher than that obtained with TPA alone. This ODC superinduction was not observed when SHE cells were similarly pre-treated with non-carcinogenic compounds. Several environmental chemicals, pesticides, solvents, oxidizers and drugs were investigated with this SHE cell model. With one-stage exposure, some xenobiotics decreased basal ODC activity, while for others ODC changes were not noticeable. With 2-stage exposure (chemical followed by TPA), all carcinogens amplified the TPA-inducing effect, resulting in ODC superinduction. Comparative studies of the action of carcinogens and of non-carcinogens, using 2-stage exposure protocols, clearly show a close relationship between ODC induction rate and morphological transformation frequency.

Dysregulation of ornithine decarboxylase activity, apoptosis and Bcl-2 oncoprotein in Syrian hamster embryo cells stage-exposed to di(2-ethylhexyl)phthalate and tetradecanoylphorbol acetate.

Perturbations of cell proliferation and death are considered as essential events in the process of carcinogenesis. Thus, two parameters, ornithine decarboxylase (ODC), an enzyme closely related to cell proliferation and transformation, and apoptotic phenomenon are profoundly modified. Using Syrian hamster embryo (SHE) cells, we have examined in the framework of two-stage carcinogenesis (initiation-promotion) the effects of a non-genotoxic [diethylhexylphthalate (DEHP)] or genotoxic [benzo[a]pyrene (BaP)] carcinogen or a non-carcinogenic compound [phthalic anhydride (AP)] on these parameters. Immunoreactive Bcl-2 and Bcl-xL proteins were also investigated following two-stage exposures. Whereas exposures to BaP, DEHP or AP alone did not provoke any modification of ODC activity, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly increased it. Using two-stage exposure protocol (xenobiotics first, then replacement by TPA-promoter), the ODC activity was higher than that obtained with TPA alone. This superinduction of ODC activity was observed only with the carcinogenic compounds DEHP and BaP. Following the same exposure protocol, spontaneous cellular apoptosis was decreased. Furthermore, Bcl-2 oncoprotein was also upregulated approximately 8- and 11-fold for BaP and DEHP respectively; meanwhile Bcl-xL protein rate did not change. The non-carcinogenic compound AP slightly inhibited spontaneous SHE cell death without ODC superinduction. Exogenous polyamines, putrescine, spermidine and spermine diluted in the medium did not inhibit spontaneous apoptosis. Although inhibition of apoptosis was not specific of carcinogenic compound, both superinduction of ODC activity and inhibition of apoptosis via Bcl-2 upregulation, may cooperate during early stages of the carcinogenic process.