Publications by authors named "Jana Tulinska"

31 Publications

Six-week inhalation of CdO nanoparticles in mice: The effects on immune response, oxidative stress, antioxidative defense, fibrotic response, and bones.

Food Chem Toxicol 2020 Feb 9;136:110954. Epub 2019 Nov 9.

Institute of Analytical Chemistry of Czech Academy of Sciences, Veveri 97, 60200, Brno, Czech Republic.

Due to the growing number of applications of cadmium oxide nanoparticles (CdO NPs), there is a concern about their potential deleterious effects. The objective of our study was to investigate the effect of CdO NPs on the immune response, renal and intestine oxidative stress, blood antioxidant defence, renal fibrotic response, bone density and mineral content. Six-week-old female ICR mice were exposed to CdO NPs for 6 weeks by inhalation (particle size: 9.82 nm, mass concentration: 31.7 μg CdO/m, total deposited dose: 0.195 μg CdO/g body weight). CdO NPs increased percentage of thymus CD3eCD8a cells and moderately enhanced splenocyte proliferation and production of cytokines and chemokines. CdO NPs elevated pro-fibrotic factors (TGF-β2, α-SMA and collagen I) in the kidney, and concentrations of AGEs in the intestine. The ratio of GSH and GSSG in blood was slightly reduced. Exposure to CdO NPs resulted in 10-fold higher Cd concentration in tibia bones. No differences were found in bone mass density, mineral content, bone area values, bone concentrations of Ca, P, Mg and Ca/P ratio. Our findings indicate stimulation of immune/inflammatory response, oxidative stress in the intestine, starting fibrotic response in kidneys and accumulation of CdO NPs in bones of mice.
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http://dx.doi.org/10.1016/j.fct.2019.110954DOI Listing
February 2020

Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

Arch Toxicol 2019 04 12;93(4):1095-1139. Epub 2019 Feb 12.

Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589, Berlin, Germany.

In 2012, a controversial study on the long-term toxicity of a Roundup herbicide and the glyphosate-tolerant genetically modified (GM) maize NK603 was published. The EC-funded G-TwYST research consortium tested the potential subchronic and chronic toxicity as well as the carcinogenicity of the glyphosate-resistant genetically modified maize NK603 by performing two 90-day feeding trials, one with GM maize inclusion rates of 11 and 33% and one with inclusion rates of up to 50%, as well as a 2-year feeding trial with inclusion rates of 11 and 33% in male and female Wistar Han RCC rats by taking into account OECD Guidelines for the testing of chemicals and EFSA recommendations on the safety testing of whole-food/feed in laboratory animals. In all three trials, the NK603 maize, untreated and treated once with Roundup during its cultivation, and the conventional counterpart were tested. Differences between each test group and the control group were evaluated. Equivalence was assessed by comparing the observed difference to differences between non-GM reference groups in previous studies. In case of significant differences, whether the effects were dose-related and/or accompanied by changes in related parameters including histopathological findings was evaluated. It is concluded that no adverse effects related to the feeding of the NK603 maize cultivated with or without Roundup for up to 2 years were observed. Based on the outcome of the subchronic and combined chronic toxicity/carcinogenicity studies, recommendations on the scientific justification and added value of long-term feeding trials in the GM plant risk assessment process are presented.
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http://dx.doi.org/10.1007/s00204-019-02400-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261740PMC
April 2019

Equivalence limit scaled differences for untargeted safety assessments: Comparative analyses to guard against unintended effects on the environment or human health of genetically modified maize.

Food Chem Toxicol 2019 Mar 5;125:540-548. Epub 2019 Feb 5.

Slovak Medical University (SZU), Faculty of Medicine, Limbová 12, 83303, Bratislava, Slovakia.

Safety assessments guard against unintended effects for human health and the environment. When new products are compared with accepted reference products by broad arrays of measurements, statistical analyses are usually summarised by significance tests or confidence intervals per endpoint. The traditional approach is to test for statistical significance of differences. However, absence or presence of significant differences is not a statement about safety. Equivalence limits are essential for safety assessment. We propose graphs to present the results of equivalence tests over the array of endpoints. It is argued that plots of the equivalence limit scaled difference (ELSD) are preferable over plots of the standardised effect size (SES) used previously for similar assessments. The ELSD method can be used either with externally specified equivalence limits or with equivalence limits estimated from (historical) data. The method is illustrated with two examples: first, environmental safety of MON810 Bt maize was assessed using field trial count data of arthropods; second, human safety of herbicide tolerant NK603 maize was assessed using haematological, biochemical and organ weight data from a 90-day rat feeding study. All assessed endpoints were classified in EFSA equivalence categories I or II, implying full equivalence or equivalence more likely than not.
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http://dx.doi.org/10.1016/j.fct.2019.02.007DOI Listing
March 2019

Toxicity evaluation of monodisperse PEGylated magnetic nanoparticles for nanomedicine.

Nanotoxicology 2019 05 1;13(4):510-526. Epub 2019 Feb 1.

a Institute of Macromolecular Chemistry, Czech Academy of Sciences , Prague , Czech Republic.

Innovative nanotechnology aims to develop particles that are small, monodisperse, smart, and do not cause unintentional side effects. Uniform magnetic FeO nanoparticles (12 nm in size) were prepared by thermal decomposition of iron(III) oleate. To make them colloidally stable and dispersible in water and cell culture medium, they were modified with phosphonic acid- (PA) and hydroxamic acid (HA)-terminated poly(ethylene glycol) yielding PA-PEG@FeO and HA-PEG@FeO nanoparticles; conventional γ-FeO particles were prepared as a control. Advanced techniques were used to evaluate the properties and safety of the particles. Completeness of the nanoparticle coating was tested by real-time polymerase chain reaction. Interaction of the particles with primary human peripheral blood cells, cellular uptake, cytotoxicity, and immunotoxicity were also investigated. Amount of internalized iron in peripheral blood mononuclear cells was 72, 38, and 25 pg Fe/cell for HA-PEG@FeO, γ-FeO, and PA-PEG@FeO, respectively. Nanoparticles were localized within the cytoplasm and in the extracellular space. No cytotoxic effect of both PEGylated nanoparticles was observed (0.12-75 μg/cm) after 24 and 72-h incubation. Moreover, no suppressive effect was found on the proliferative activity of T-lymphocytes and T-dependent B-cell response, phagocytic activity of monocytes and granulocytes, and respiratory burst of phagocytes. Similarly, no cytotoxic effect of γ-FeO particles was observed. However, they suppressed the proliferative activity of T-lymphocytes (75 μg/cm, 72 h) and also decreased the phagocytic activity of monocytes (15 μg/cm, 24 h; 3-75 μg/cm, 72 h). We thus show that newly developed particles have great potential especially in cancer diagnostics and therapy.
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http://dx.doi.org/10.1080/17435390.2018.1555624DOI Listing
May 2019

Consumption of a dark roast coffee blend reduces DNA damage in humans: results from a 4-week randomised controlled study.

Eur J Nutr 2019 Dec 17;58(8):3199-3206. Epub 2018 Nov 17.

Norgenotech AS, Skreia, Norway.

Purpose: To determine the DNA protective effects of a standard coffee beverage in comparison to water consumption.

Methods: The single-blind, randomised controlled study with parallel design included healthy women (n = 50) and men (n = 50) recruited from the general Central European population. The subjects were randomised in a coffee and a control group, with stratification for sex and body mass index. The study comprised two periods of 4 weeks: a preconditioning period, with daily consumption of at least 500 ml water but no coffee, nor tea, nor any other caffeine-containing product. During the subsequent intervention period the coffee group consumed 500 ml of freshly brewed dark roast coffee blend per day, the control group consumed water instead. On the last day of each period, blood was drawn and analysed by comet assay (single-cell gel electrophoresis) to assess the level of DNA damage (strand breakage).

Results: At the end of the intervention period the mean level of DNA strand breaks in the coffee group has decreased in comparison to the control group [difference in means 0.23% TI (tail intensity), p = 0.028]. The mean change from baseline (delta value) was - 23% in the coffee group (p = 0.0012). Effects of coffee intake were similar for men and women. During intervention, neither group showed any significant change in body weight or calorie intake.

Conclusions: Our results indicate that regular consumption of a dark roast coffee blend has a beneficial protective effect on human DNA integrity in both, men and women.
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http://dx.doi.org/10.1007/s00394-018-1863-2DOI Listing
December 2019

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

Arch Toxicol 2018 07 31;92(7):2385-2399. Epub 2018 May 31.

Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany.

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.
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http://dx.doi.org/10.1007/s00204-018-2230-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015625PMC
July 2018

Immunotoxicity, genotoxicity and epigenetic toxicity of nanomaterials: New strategies for toxicity testing?

Food Chem Toxicol 2017 Nov 26;109(Pt 1):797-811. Epub 2017 Aug 26.

Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia. Electronic address:

The unique properties of nanomaterials (NMs) are beneficial in numerous industrial and medical applications. However, they could also induce unintended effects. Thus, a proper strategy for toxicity testing is essential in human hazard and risk assessment. Toxicity can be tested in vivo and in vitro; in compliance with the 3Rs, alternative strategies for in vitro testing should be further developed for NMs. Robust, standardized methods are of great importance in nanotoxicology, with comprehensive material characterization and uptake as an integral part of the testing strategy. Oxidative stress has been shown to be an underlying mechanism of possible toxicity of NMs, causing both immunotoxicity and genotoxicity. For testing NMs in vitro, a battery of tests should be performed on cells of human origin, either cell lines or primary cells, in conditions as close as possible to an in vivo situation. Novel toxicity pathways, particularly epigenetic modification, should be assessed along with conventional toxicity testing methods. However, to initiate epigenetic toxicity screens for NM exposure, there is a need to better understand their adverse effects on the epigenome, to identify robust and reproducible causal links between exposure, epigenetic changes and adverse phenotypic endpoints, and to develop improved assays to monitor epigenetic toxicity.
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http://dx.doi.org/10.1016/j.fct.2017.08.030DOI Listing
November 2017

The Cell Surface Markers Expression in Postmenopausal Women and Relation to Obesity and Bone Status.

Int J Environ Res Public Health 2017 07 11;14(7). Epub 2017 Jul 11.

Department of Clinical and Experimental Pharmacology, Faculty of Medicine, Slovak Medical University, 83303 Bratislava, Slovakia.

The age-related changes and hormonal deprivation in postmenopausal women are associated with the immune response alteration. The excessive fat accumulation, local and systemic inflammation may lead to dysregulation in immune function and relevant health problems, including obesity and osteoporosis. We analyzed the expression of cell surface markers in the venous blood specimens, stained with fluorophores-conjugated monoclonal antibodies and analysed by multicolour flow cytometry. The significant changes of cytotoxic, naive, and memory T-lymphocytes, plasmacytoid dendritic cells (DCs) were in postmenopausal women versus fertile women. Body mass index (BMI) affected markedly the cell surface expression of CD265/RANK. Osteoporosis is linked to reduced percentage of plasmacytoid DCs, and elevated natural Treg cells ( < 0.05). The confounding factors such as women age, BMI, bone mineral density (BMD), waist size and tissue fat affect the expression of RANK on myeloid DCs and CD40L on T-lymphocytes that might be the immunophenotypic modulators after menopause.
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http://dx.doi.org/10.3390/ijerph14070751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551189PMC
July 2017

The effect of core and lanthanide ion dopants in sodium fluoride-based nanocrystals on phagocytic activity of human blood leukocytes.

J Nanopart Res 2017 13;19(2):68. Epub 2017 Feb 13.

Medical Faculty, Department of Immunology and Immunotoxicology, Slovak Medical University, Limbova 12, 833 03 Bratislava, Slovakia.

Sodium fluoride-based β-NaLnF4 nanoparticles (NPs) doped with lanthanide ions are promising materials for application as luminescent markers in bio-imaging. In this work, the effect of NPs doped with yttrium (Y), gadolinium (Gd), europium (Eu), thulium (Tm), ytterbium (Yb) and terbium (Tb) ions on phagocytic activity of monocytes and granulocytes and the respiratory burst was examined. The surface functionalization of <10-nm NPs was performed according to our variation of patent pending ligand exchange method that resulted in meso-2,3-dimercaptosuccinic acid (DMSA) molecules on their surface. Y-core-based NCs were doped with Eu ions, which enabled them to be excited with UV light wavelengths. Cultures of human peripheral blood ( = 8) were in vitro treated with five different concentrations of eight NPs for 24 h. In summary, neither type of nanoparticles is found toxic with respect to conducted test; however, some cause toxic effects (they have statistically significant deviations compared to reference) in some selected doses tested. Both core types of NPs (Y-core and Gd-core) impaired the phagocytic activity of monocytes the strongest, having minimal or none whatsoever influence on granulocytes and respiratory burst of phagocytic cells. The lowest toxicity was observed in Gd-core, Yb, Tm dopants and near-infrared nanoparticles. Clear dose-dependent effect of NPs on phagocytic activity of leukocytes and respiratory burst of cells was observed for limited number of samples.
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http://dx.doi.org/10.1007/s11051-017-3779-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5306425PMC
February 2017

One-year oral toxicity study on a genetically modified maize MON810 variety in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

Arch Toxicol 2016 Oct 20;90(10):2531-62. Epub 2016 Jul 20.

Institute of Veterinary Biochemistry, Freie Universität Berlin, Oertzenweg 19b, 14163, Berlin, Germany.

The GRACE (GMO Risk Assessment and Communication of Evidence; www.grace-fp7.eu ) project was funded by the European Commission within the 7th Framework Programme. A key objective of GRACE was to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of a 1-year feeding trial with a GM maize MON810 variety, its near-isogenic non-GM comparator and an additional conventional maize variety are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 452. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after a chronic exposure.
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http://dx.doi.org/10.1007/s00204-016-1798-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043003PMC
October 2016

Automotive airborne brake wear debris nanoparticles and cytokinesis-block micronucleus assay in peripheral blood lymphocytes: A pilot study.

Environ Res 2016 07 29;148:443-449. Epub 2016 Apr 29.

Health Effects Laboratory, Department of Environmental Chemistry, NILU-Norwegian Institute for Air Research, Instituttveien 18, 2007 Kjeller, Norway.

Motor vehicle exhaust and non-exhaust processes play a significant role in environmental pollution, as they are a source of the finest particulate matter. Emissions from non-exhaust processes include wear-products of brakes, tires, automotive hardware, road surface, and traffic signs, but still are paid little attention to. Automotive friction composites for brake pads are composite materials which may consist of potentially hazardous materials and there is a lack of information regarding the potential influence of the brake wear debris (BWD) on the environment, especially on human health. Thus, we focused our study on the genotoxicity of the airborne fraction of BWD using a brake pad model representing an average low-metallic formulation available in the EU market. BWD was generated in the laboratory by a full-scale brake dynamometer and characterized by Raman microspectroscopy, scanning electron microscopy, and transmission electron microscopy showing that it contains nano-sized crystalline metal-based particles. Genotoxicity tested in human lymphocytes in different testing conditions showed an increase in frequencies of micronucleated binucleated cells (MNBNCs) exposed for 48h to BWD nanoparticles (NPs) (with 10% of foetal calf serum in culture medium) compared with lymphocytes exposed to medium alone, statistically significant only at the concentration 3µg/cm(2) (p=0.032).
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http://dx.doi.org/10.1016/j.envres.2016.04.022DOI Listing
July 2016

Functionalized porous silica&maghemite core-shell nanoparticles for applications in medicine: design, synthesis, and immunotoxicity.

Croat Med J 2016 Apr;57(2):165-78

Daniel Horak, Department of Polymer Particles, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Sq. 2 , 162 06 Prague 6, Czech Republic,

Aim: To determine cytotoxicity and effect of silica-coated magnetic nanoparticles (MNPs) on immune response, in particular lymphocyte proliferative activity, phagocytic activity, and leukocyte respiratory burst and in vitro production of interleukin-6 (IL-6) and 8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF).

Methods: Maghemite was prepared by coprecipitation of iron salts with ammonia, oxidation with NaOCl and modified by tetramethyl orthosilicate and aminosilanes. Particles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). Cytotoxicity and lymphocyte proliferative activity were assessed using [3H]-thymidine incorporation into DNA of proliferating human peripheral blood cells. Phagocytic activity and leukocyte respiratory burst were measured by flow cytometry; cytokine levels in cell supernatants were determined by ELISA.

Results: γ-Fe2O3&SiO2-NH2 MNPs were 13 nm in size. According to TEM, they were localized in the cell cytoplasm and extracellular space. Neither cytotoxic effect nor significant differences in T-lymphocyte and T-dependent B-cell proliferative response were found at particle concentrations 0.12-75 μg/cm2 after 24, 48, and 72 h incubation. Significantly increased production of IL-6 and 8, and GM-CSF cytokines was observed in the cells treated with 3, 15, and 75 µg of particles/cm2 for 48 h and stimulated with pokeweed mitogen (PHA). No significant changes in TNF-α and IFN-γ production were observed. MNPs did not affect phagocytic activity of monocytes and granulocytes when added to cells for 24 and 48 h. Phagocytic respiratory burst was significantly enhanced in the cultures exposed to 75 µg MNPs/cm2 for 48 h.

Conclusions: The cytotoxicity and in vitro immunotoxicity were found to be minimal in the newly developed porous core-shell γ-Fe2O3&SiO2-NH2 magnetic nanoparticles.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856187PMC
http://dx.doi.org/10.3325/cmj.2016.57.165DOI Listing
April 2016

Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

Arch Toxicol 2014 Dec 2;88(12):2289-314. Epub 2014 Oct 2.

Slovak Medical University, Limbová 12, 83303, Bratislava, Slovakia.

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu ) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event.
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http://dx.doi.org/10.1007/s00204-014-1374-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247492PMC
December 2014

Hydrophobic sodium fluoride-based nanocrystals doped with lanthanide ions: assessment of in vitro toxicity to human blood lymphocytes and phagocytes.

J Appl Toxicol 2014 Nov 1;34(11):1220-5. Epub 2014 Sep 1.

Institute of Physics, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370, Wroclaw, Poland.

In vitro immunotoxicity of hydrophobic sodium fluoride-based nanocrystals (NCs) doped with lanthanide ions was examined in this study. Although there is already a significant amount of optical and structural data on NaYF4 NCs, data on safety assessment are missing. Therefore, peripheral whole blood from human volunteers was used to evaluate the effect of 25 and 30 nm hydrophobic NaYF4 NCs dissolved in cyclohexane (CH) on lymphocytes, and of 10 nm NaYF4 NCs on phagocytes. In the concentration range 0.12-75 µg cm(-2) (0.17-106 µg ml(-1) ), both 25 and 30nm NaYF4 NCs did not induce cytotoxicity when measured as incorporation of [(3) H]-thymidine into DNA. Assessment of lymphocyte function showed significant suppression of the proliferative activity of T-lymphocytes and T-dependent B-cell response in peripheral blood cultures (n = 7) stimulated in vitro with mitogens phytohemagglutinin (PHA) and pokeweed (PWM) (PHA > PWM). No clear dose-response effect was observed. Phagocytic activity and respiratory burst of leukocytes (n = 5-8) were generally less affected. A dose-dependent suppression of phagocytic activity of granulocytes in cultures treated with 25 nm NCs was observed (vs. medium control). A decrease in phagocytic activity of monocytes was found in cells exposed to higher doses of 10 and 30 nm NCs. The respiratory burst of phagocytes was significantly decreased by exposure to the middle dose of 30 nm NCs only. In conclusion, our results demonstrate immunotoxic effects of hydrophobic NaYF4 NCs doped with lanthanide ions to lymphocytes and to lesser extent to phagocytes. Further research needs to be done, particularly faze transfer of hydrophobic NCs to hydrophilic ones, to eliminate the solvent effect.
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http://dx.doi.org/10.1002/jat.3050DOI Listing
November 2014

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles.

Nanotoxicology 2015 May 14;9 Suppl 1:44-56. Epub 2013 Nov 14.

Health Effects Laboratory, Department of Environmental Chemistry, NILU, Norwegian Institute for Air Research , Kjeller , Norway .

Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.
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http://dx.doi.org/10.3109/17435390.2013.847505DOI Listing
May 2015

Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model.

Nanotoxicology 2015 May;9 Suppl 1:33-43

Department of Immunology and Immunotoxicology and Department of Experimental and Applied Genetics, Slovak Medical University , Bratislava , Slovakia .

A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 μg/cm(2) exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n = 9-13). PLGA-PEO NPs were not toxic up to dose 3 μg/cm(2); dose of 75 μg/cm(2) displays significant decrease in [(3)H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM > CON A, and no changes in PHA cultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) > monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised DNA bases in PLGA-PEO-treated cells. To conclude on immuno- and genotoxicity of PLGA-PEO NPs, more experiments with various particle size, charge and composition need to be done.
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http://dx.doi.org/10.3109/17435390.2013.816798DOI Listing
May 2015

Health effects of selected nanoparticles in vivo: liver function and hepatotoxicity following intravenous injection of titanium dioxide and Na-oleate-coated iron oxide nanoparticles in rodents.

Nanotoxicology 2015 May;9 Suppl 1:95-105

Slovak Medical University , Bratislava , Slovakia .

The study determined the effect of intravenous administration of acutely toxic or sub-lethal doses of Na-oleate-coated Fe3O4 (OC-Fe3O4) nanoparticles (NPs) on liver structure and function in Wistar rats, compared to titanium dioxide (TiO2) NPs and saline-injected controls. The acute study, using a modified OECD 425 progressive dosing procedure, found LD50 values of 59.22 and 36.42 mg/kg for TiO2 and OC-Fe3O4 NPs, respectively. In the sub-lethal study, rats were either injected with saline (negative controls), a sub-lethal reference (0.592 mg/kgTiO2 NPs, equal to 1% of LD50 on a body weight basis) or OC-Fe3O4 NPs in doses equivalent to 0.1, 1 or 10% of the LD50, respectively (corresponding to 0.0364, 0.364 and 3.64 mg Fe3O4/kg body weight). Animals were sampled 24 h, 1, 2 and 4 weeks post-injection for adverse effects. Mitochondrial respiration was significantly increased 2 weeks after injection of 10% OC-Fe3O4 NPs compared to controls, but the effect was transient. Cholesterol and triacylglycerol concentrations in the liver tissue did not increase in any treatment. There were some disturbances to antioxidant enzymes after OC-Fe3O4 NPs treatment in the livers of animals 1 week post-exposure; with the most sensitive changes occurring in glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities. Lipidosis and mild necrosis with changes in sinusoid space were also observed in histological sections of the liver. Overall, these data suggest that the liver likely retains functional integrity with acute and sub-lethal doses of OC-Fe3O4 NPs, albeit with some stimulation of redox defences and evidence of some tissue injury.
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http://dx.doi.org/10.3109/17435390.2013.815285DOI Listing
May 2015

Comprehensive assessment of nephrotoxicity of intravenously administered sodium-oleate-coated ultra-small superparamagnetic iron oxide (USPIO) and titanium dioxide (TiO2) nanoparticles in rats.

Nanotoxicology 2014 Mar 21;8(2):142-57. Epub 2013 Jan 21.

Institute of Molecular Biomedicine, Medical Faculty, Comenius University , Bratislava , Slovakia.

As a main excretory organ, kidney is predisposed to direct/indirect injury. We addressed the potential nephrotoxic effects following expositions of healthy rats to nanoparticle (NP) loads relevant to humans in a situation of 100% bioavailability. Up to 4 weeks after administration, a single iv bolus of oleate-coated ultra-small superparamagnetic iron oxide NPs (in dose of 0.1%, 1.0% and 10.0% of LD50) or TiO2 NPs (1.0% of LD50) did not elicit decline in renal function, damage to proximal tubules, alterations in: renal histology or expression of pro-inflammatory/pro-fibrotic genes, markers of systemic or local renal micro-inflammation or oxidative damage. Antioxidant enzyme activities in renal cortex, mildly elevated at 24 h, completely restored at later time points. Data obtained by multifaceted approach enable the prediction of human nephrotoxicity during preclinical studies, and may serve as comparison for alternative testing strategies using in vitro and in silico methods essential for the NP-nephrotoxicity risk assessment.
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http://dx.doi.org/10.3109/17435390.2012.763147DOI Listing
March 2014

Association between the human immune response and body mass index.

Hum Immunol 2012 May 6;73(5):480-5. Epub 2012 Mar 6.

Department of Immunology and Immunotoxicology, Faculty of Medicine, Slovak Medical University in Bratislava, Limbova 12, 833 03 Bratislava, Slovak Republic.

The aim of this study was to determine the strength of the association between the human immune response and body mass index (BMI) and whether differences exist in the effects of obesity on selected immune parameters between men and women. Two hundred ninety participants were divided into groups according to sex and BMI. Parameters CD3, CD4, CD8, CD16+56, CD19, HLADR, CD11b, CD11c, and CD54 were quantified. Leukocyte and differential counts were performed. We observed elevation with regard to the normal weight group in the parameters of white blood cells, neutrophils, monocytes, CD3, CD4, CD19, and CD11b for the whole study group. A decrease was observed in the expression of CD16+56. The effect of BMI on the immune system was much more apparent in women. BMI was correlated with the majority of the measured parameters, reflecting a strong association between BMI and the human immune system.
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http://dx.doi.org/10.1016/j.humimm.2012.02.023DOI Listing
May 2012

Changes in immunologic parameters of humoral immunity and adipocytokines in obese persons are gender dependent.

Hum Immunol 2012 May 17;73(5):486-92. Epub 2012 Feb 17.

Department of Immunology and Immunotoxicology, Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia.

The aim of this study was to investigate several immunologic parameters using of immunonephelometry and adipocytokines by the enzyme immunoassay and their changes in different states of obesity. Obesity is considered to involve a state of chronic low-grade inflammation, with links between adipose cells and the immune system. We found significantly higher complement C3 levels in all obese subjects. Levels of the complement C4 were significantly higher in obese women, but not in men, when compared with the corresponding group of normal weight subjects. The increase in C-reactive protein concentrations was significant in both obese and morbidly obese women, but only in morbidly obese men. No significant differences in tumor necrosis factor-α, interleukin-6, interleukin-10, and soluble intercellular adhesion molecule-1 were found. sE-selectin levels were higher in both overweight and obese women but only in morbidly obese men. We found decreased adiponectin concentrations in obese and morbidly obese women. Concentrations of leptin were significantly higher only in obese men (p < 0.05), whereas in women the increase in leptin levels was significant in overweight, obese, and morbidly obese subjects. In conclusion, our results demonstrate elevated levels of C3, C-reactive protein, sE-selectin, and leptin in obese women and men. In obese women, we also observed increased concentrations of C4 and decreased levels of adiponectin.
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http://dx.doi.org/10.1016/j.humimm.2012.02.006DOI Listing
May 2012

Occupational exposure to mineral fibres. Biomarkers of oxidative damage and antioxidant defence and associations with DNA damage and repair.

Mutagenesis 2008 Jul 14;23(4):249-60. Epub 2008 Feb 14.

Research Base of Slovak Medical University, Limbová 12, 833 03 Bratislava, Slovakia.

In order to study the effect of mineral wool exposure on oxidative DNA damage and lipid peroxidation, an epidemiological study was conducted in a mineral wool factory in Slovakia. Altogether 141 subjects were investigated (21-58 years old), 43 controls (20 men and 23 women: 27 non-smokers, 16 smokers) and 98 exposed (75 men and 23 women: 61 non-smokers, 37 smokers). We found higher malondialdehyde (MDA) levels in the group of all exposed workers (P = 0.025) and in exposed non-smokers (P = 0.003) and a significantly suppressed activity of ceruloplasmin oxidase (P = 0.02, P < 0.02, respectively) and catalase (CAT) (P = 0.04, P = 0.01, respectively) in these groups. The activity of glutathione S-transferase (GST) was affected by exposure to mineral wool; levels were significantly lower in all exposed subjects (P = 0.04), in the exposed non-smokers (P = 0.03) and in exposed men (P < 0.01). Concentrations of vitamin C in plasma and the ferric-reducing activity of plasma (FRAP) were not affected by the mineral wool exposure. There was a significant negative correlation between the activity of glutathione peroxidase (GPX) and MDA in the whole group (P < 0.01) and in the exposed group and between CAT activity and MDA in all subjects (P < 0.01). GST activity correlated inversely with oxidized pyrimidines in lymphocyte DNA, in almost all subgroups. We found significant negative correlations between DNA repair and GPX in all subjects (P = 0.03) as well as in control men (P < 0.03) and between DNA repair and CAT in all control subjects (P < 0.02) and in control men (P < 0.01). Interestingly, we found a positive correlation between DNA repair and MDA in all subjects (P < 0.01) and in all exposed subjects (P < 0.03). The presented results indicate that mineral wool exposure induces an increase in oxidative damage to biomolecules especially in the group of male non-smokers. However, optimal levels of antioxidants could have a protective effect. Biomarkers such as MDA, antioxidant enzymes and antioxidant vitamins measured in blood may be useful biomarkers of oxidative stress and antioxidant protection. We do not recommend FRAP as a marker of antioxidant status as interference from other constituents can provide false or confusing results. Our study supports the idea that there might also be other mechanisms by which antioxidant enzymes (especially GST) protect cells against oxidative DNA damage.
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http://dx.doi.org/10.1093/mutage/gen004DOI Listing
July 2008

Cell and antibody mediated immunity induced by vaccination with novel Candida dubliniensis mannan immunogenic conjugate.

Int Immunopharmacol 2007 Oct 15;7(10):1325-33. Epub 2007 Jun 15.

Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia.

Antigen-specific humoral response, as well as the induction of cellular immunity generated by Candida dubliniensis mannan-human serum albumin (HSA) conjugate, a novel proposed immunogenic structure for subcellular vaccine, were evaluated in rabbits. Mannan-HSA conjugate-induced specific IgG and IgA increased significantly after boosters (IgG: P<0.001 and IgA: P<0.01). Mannan-HSA conjugate up-regulation of cell-surface expression of B-lymphocyte and granulocyte activation antigens CD25 and CD11b indicated the effective activation. Immunogenic effect of conjugate on T lymphocytes was demonstrated via inductive increase of CD4+ T lymphocyte subset and CD4+/CD8+ ratio and via induction of T(H)1 cytokines. Immunogenic effectiveness of mannan-HSA conjugate at a dose of 0.25 mg of mannan antigenic moiety overcame that of the mannan alone and of yeast whole cells, thus promising further application in Candida vaccine development.
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http://dx.doi.org/10.1016/j.intimp.2007.05.014DOI Listing
October 2007

An in vitro study of the toxic effects of Stachybotrys chartarum metabolites on lung cells.

Altern Lab Anim 2007 Mar;35(1):47-52

Research Base of Slovak Medical University, Bratislava, Slovakia.

During a study of indoor fungal colonisation, several isolates of Stachybotrys chartarum were recovered, and the effects of metabolites from four isolates on lung epithelial Type II cells and alveolar macrophages were studied in vitro. All the isolates showed toxic effects on both cell types, and they differed only in the extent of the changes induced. In Type II cells, the number of alkaline phosphatase positive cells was reduced, the pattern of Maclura pomifera agglutinin (MPA) binding was changed, and acid phosphatase activity in alveolar macrophages was diminished. In both cell types, the production of monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor-alpha (TNF-alpha) was changed, and parameters related to antioxidant status (superoxide dismutase, glutathione peroxidase, glutathione) were decreased.
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http://dx.doi.org/10.1177/026119290703500115DOI Listing
March 2007

Genetic predisposition and health effect of occupational exposure to asbestos.

Neuro Endocrinol Lett 2006 Dec;27 Suppl 2:100-3

Research Base of Slovak Medical University, Bratislava, Slovakia.

Objectives: As asbestos presents a direct genetic hazard for humans, a small-scale molecular epidemiological study was conducted to monitor 61 subjects long-term exposed to asbestos in comparison with 49 town controls and 21 control subjects from administration of the same factory.

Results: Asbestos exposed workers had significantly higher numbers of chromosomal aberrations compared with both control groups (P=0.003). Clinical examination showed that 44.3% of exposed workers developed symptoms of asbestosis. We were interested in the relationship between the risk of asbestos-coupled diseases and individual variability in biotransformation enzymes, especially in glutathione S-transferases and microsomal epoxide hydrolase. GSTP1*105Val allele appeared less in the group of workers with asbestosis compared to those without asbestosis (18.5% vs 34.7%, P=0.044), and in subjects with developed asbestosis coupled with bronchitis compared to those without bronchitis (0% vs 25%, P=0.048). Similarly, the genotype corresponding to low activity of microsomal epoxide hydrolase was significantly decreased in workers with fibrotic plaques compared to those without plaques (26.7% vs 56.3%, P=0.045).

Conclusions: Our results suggest that GSTP1*105Val allele and low EPHX1 activity genotype may be protective for people occupationally exposed to asbestos. However, more extensive studies are needed to confirm our results.
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December 2006

The effect of ceramic fibers on the immune system.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2005 Dec;149(2):397-9

Laboratory of Immunotoxicology, Department of Immunology and Immunotoxicology, Research Base of Slovak Medical University - Institute of Preventive and Clinical Medicine, Limbova 14, Bratislava, Slovakia.

Male Sprague-Dawley rats were treated by intratracheal instillation with 1 mg/animal of refractory ceramic fibers. Intratracheal exposure to ceramic fibers led to significant changes of immune response. Results of proliferative activity of spleen lymphocytes showed significantly decreased proliferative activity of T-cells in response to mitogens phytohemagglutinin and concanavalin A in animals given ceramic fibers in comparison with control rats. Similarly, T-dependent B-cell response to pokeweed mitogen was significantly suppressed. Spontaneous proliferative activity of lymphocytes in non-stimulated spleen cell cultures did not differ in exposed and control rats. No significant changes were found among groups in percentage of phagocytic blood polymorphonuclear leukocytes and percentage of cells with respiratory burst.
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December 2005

Immunological monitoring in workers occupationally exposed to asbestos.

Toxicology 2005 Jan;206(2):299-308

Research Base of the Slovak Medical University, Institute of Preventive and Clinical Medicine, Department of Clinical Immunology, Limbova 12, 833 01 Bratislava, Slovak Republic.

Occupational exposure to asbestos is strongly associated with pulmonary diseases, cancer and immunotoxic effects. Both systemic and local immunity may play an important role in the pathogenesis of these events. Immune cells appear to be influenced by asbestos exposure, either through direct effects or as a result of the host's protective response to exposure. In this study several immune system parameters were assessed in workers (n = 61) with at least 5 years' exposure to asbestos at an industrial plant. Workers exposed to asbestos fibres had significantly increased levels of immunoglobulin E and concentrations of interleukin-6 and -8 in comparison with two sets of controls (in-plant and town control groups). The levels of soluble adhesion molecule ICAM-1 were higher in the exposed group compared to the town control group. Significantly increased levels of IgA were found in asbestos-exposed group in comparison to the town control. Evaluation of the expression of adhesion molecules on lymphocytes, monocytes and granulocytes by flow cytometry showed significant increases in the class of selectins CD62L on monocytes and granulocytes. Moreover, significantly increased expression of markers CD69 and CD66b on eosinophils was found among workers exposed to asbestos. In conclusion, exposure to asbestos fibres was found to have several effects on immune system. Alterations of these immune parameters may indicate hypersensitivity (increased levels of IgE, increased expression of activation markers CD66b and CD69 on eosinophils) and an elevated inflammatory status (increased levels of interleukins--IL-6, IL-8) in exposed workers.
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http://dx.doi.org/10.1016/j.tox.2004.09.004DOI Listing
January 2005

Immunomodulatory effects of mineral fibres in occupationally exposed workers.

Mutat Res 2004 Sep;553(1-2):111-24

Research Base of the Slovak Medical University, Institute of Preventive and Clinical Medicine, Limbova 12, 833 03 Bratislava, Slovak Republic.

In the context of a large-scale molecular epidemiology study, the possible immunomodulatory effects of mineral fibres, in workers occupationally exposed to asbestos, rockwool and glass fibres, were examined. In each plant, 61, 98 and 80 exposed workers and 21, 43 or 36 control clerical subjects, respectively, were recruited. In the case of the asbestos-exposed subjects, an additional town-control group of 49 people was included. Evidence of pulmonary fibrosis was found in 42% of the asbestos-exposed workers, while evidence of pleural fibrosis was found in 24%. The asbestos-exposed cohort had significantly decreased forced vital capacity of lungs as well as forced expiratory volume per first second. Our findings indicate that exposure to all three types of fibres examined modulates to different degrees the immune response. Suppression of T-cell immunity and to a lesser extent, B-cell immunity was found in the case of workers from a former asbestos cement plant, while stimulation of T-cell response was observed in rockwool workers, and stimulation of T- and B-cell response was seen in glass fibre workers. Depression of the percentage of lymphocyte subpopulation of CD 16+56 (natural killer cells) in peripheral blood was found in glass fibre workers. Statistical analysis showed increased levels of proinflammatory cytokines (IL-6 asbestos; IL-8 all three fibres), expression of adhesion molecule L-selectin on granulocytes and monocytes (asbestos), levels of soluble adhesion molecules (SAMs) in sera (ICAM-1 all three fibres; E-selectin glass fibres), increased levels of immunoglobulin E (asbestos and rockwool) and elevated expression of activation markers on eosinophils (CD66b asbestos, glass fibres; CD69 asbestos). Significant correlations were observed between lymphocyte proliferation and markers of DNA damage and repair. Increased levels of proinflammatory cytokines, SAMs, immunoglobulin E and elevated expression of activation markers on eosinophils was found in people with symptoms of hypersensitivity and an elevated inflammatory status.
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http://dx.doi.org/10.1016/j.mrfmmm.2004.06.030DOI Listing
September 2004

Immunotoxic and cancerostatic effects of ethyl-4-isothiocyanatobutanoate in female Lewis rats with implanted fibrosarcoma.

Int Immunopharmacol 2002 Nov;2(12):1681-91

Department of Biochemistry and Microbiology, Slovak University of Technology, Bratislava, Slovakia.

Isothiocyanates (ITCs) have been isolated from plants. Naturally occurring and synthetic ITCs are known as effective chemopreventive agents. Ethyl 4-isothiocyanatobutanoate (E-41B) is a derivative of gamma-aminobutyric acid. Immunotoxic and canocerostatic effects of E-41B in female inbred Lewis rats implanted with experimental fibrosarcoma BP6-TU2 was evaluated in this study. On day 5 after subcutaneous application of tumor cells, animals started to be treated intraperitoneally three times a week with two different doses of E-41B: 28 and 35 mg/kg/day during 28 days. High dose of E-41B was close to maximum tolerated dose (MTD). Control groups of rats with or without tumors injected intraperitoneally only saline or 70% dimethylsulphoxide were added. Administrating of E-41B resulted in suppression of thymus, popliteal lymph node, spleen weight and spleen cellularity. Hematologic evaluation displayed decreased erythrocyte (ERY) count and level of hemoglobin (HB) in rats treated withE-41B. Immune assays--the phagocytic activity of polymorphonuclear leukocytes (PMN) and monocytes, primary antibody response and in vitro proliferative activity of spleen lymphocytes (LY) to mitogens were not significantly affected by E-41B treatment E-41B moderately decreased tumor weights, but this decrease was not statistically significant in comparison with DMSO-exposed rats with tumors. The fibrosarcoma implantation itself increased significantly spleen weight and changed hematological parameters (decreased HB, increased mean cell volume of ERY, increased leukocyte count, increased % PMN, decreased % LY, decreased % EO). Moreover, moderate decreased percentage of CD161+ positive cells (NK cells) were found in peripheral blood. Immune assays showed decline in proliferation of lymphocytes and phagocytic activity of leukocytes. Our findings indicate that administration of E-41B displayed hematoxic effect in rats implanted with fibrosarcoma. Immunotoxic effect was shown as decreased lymphoid organ weight and spleen cytotoxicity although function of immune cells was not impaired.
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http://dx.doi.org/10.1016/s1567-5769(02)00164-9DOI Listing
November 2002