Toxicity evaluation of monodisperse PEGylated magnetic nanoparticles for nanomedicine.

Innovative nanotechnology aims to develop particles that are small, monodisperse, smart, and do not cause unintentional side effects. Uniform magnetic Fe3O4 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@Fe3O4 and HA-PEG@Fe3O4 nanoparticles; conventional γ-Fe2O3 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@Fe3O4, γ-Fe2O3, and PA-PEG@Fe3O4, 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/cm2) 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-Fe2O3 particles was observed. However, they suppressed the proliferative activity of T-lymphocytes (75 µg/cm2, 72 h) and also decreased the phagocytic activity of monocytes (15 µg/cm2, 24 h; 3-75 µg/cm2, 72 h). We thus show that newly developed particles have great potential especially in cancer diagnostics and therapy.

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).

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.

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

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.

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

Sodium fluoride-based ß-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 (n = 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers.

Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m³) and high (above 50 mg/m³)) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R=-0.38, p=0.001); SSBs were also significantly higher in men (p=0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34±1.00 SSB/109 Da), followed by high exposure group (0.72±0.81 SSB/109 Da) and controls (0.65±0.82 SSB/109 Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p<0.001) and positively with SSBs (p<0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p<0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

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

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.

The effect of ceramic fibers on the immune system.

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.

Markers of individual susceptibility and DNA repair rate in workers exposed to xenobiotics in a tire plant.

Workers employed in tire plants are exposed to a variety of xenobiotics, such as 1,3-butadiene (BD), soots containing polycyclic aromatic hydrocarbons, and other organic chemicals (e.g., styrene). In the present study, we investigated markers of genotoxicity [chromosomal aberrations (CAs) and single-strand breaks (SSBs)] in a cohort of 110 tire plant workers engaged in jobs with different levels of xenobiotic exposure in relation to various polymorphisms in genes coding for biotransformation enzymes (CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, and GSTT1) and in genes involved in DNA repair (XPD exon 23, XPG exon 15, XPC exon 15, XRCC1 exon 10, and XRCC3 exon 7). In addition, the expression of CYP2E1, a gene playing a key role in BD metabolism, was determined by real-time PCR in peripheral blood lymphocytes, and the capacity of lymphocytes to repair gamma-ray-induced SSBs and to convert 8-oxoguanine in HeLa cell DNA into SSBs was assessed using in vitro assays. No positive associations were detected between the CA frequency or SSB induction and levels of workplace exposure; however, a nonsignificant twofold higher irradiation-specific DNA repair rate was found among highly exposed workers. In evaluations conducted with the markers of individual susceptibility, workers with low-EPHX1-activity genotypes exhibited a significantly higher CA frequency as compared to those with medium and high-EPHX1-activity genotypes (P = 0.050). CA frequencies were significantly lower in individuals homozygous for the XPD exon 23 variant allele in comparison to those with the wild-type CC genotype (P = 0.003). Interestingly, CAs were higher in individuals with higher CYP2E1 expression levels, but the association was nonsignificant (P = 0.097). The results from this study suggest the importance of evaluating markers of individual susceptibility, since they may modulate genotoxic effects induced by occupational exposure to xenobiotics.

Immunomodulatory effects of mineral fibres in occupationally exposed workers.

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.

Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers.

The effect of occupational exposure to styrene on frequencies of chromosomal aberrations and binucleated cells with micronuclei and on single-strand break levels in peripheral blood lymphocytes was studied in 86 reinforced plastic workers and 42 control individuals (including 16 maintenance workers with intermittent, low-dose exposure). In these individuals, the irradiation-specific DNA repair rates and the repair rates of 8-oxoguanines were investigated. We assessed the exposure by measuring the concentrations of styrene in air and in blood and of mandelic acid, phenylglyoxylic acid, 4-vinyl phenol conjugates and regioisomeric phenyl hydroxyethyl mercapturic acids in urine. All these parameters correlated with one another. No clear relationship was found between the styrene exposure and the frequencies of chromosomal aberrations. Binucleated cells with micronuclei were moderately related to the parameters of styrene exposure. We found a negative correlation between all exposure parameters and single-strand breaks. The positive correlation between exposure parameters and DNA repair rates suggests that particular DNA repair pathways may be induced by styrene exposure.

Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates, chromosomal aberrations and single-strand breaks in DNA.

We analysed the associations between genetic polymorphisms in genes coding for DNA repair enzymes XPD (exon 23 A --> C, K751Q), XPG (exon 15 G --> C, D1104H), XPC (exon 15 A --> C, K939Q), XRCC1 (exon 10 G --> A, R399Q) and XRCC3 (exon 7 C --> T, T241 M) and the levels of chromosomal aberrations (CAs) and single-strand breaks (SSBs) in peripheral lymphocytes in a central European population. We also measured the irradiation-specific DNA repair rates and the repair rates of 8-oxoguanines in these individuals. An elevated frequency of CAs was observed in individuals with the XPD exon 23 A allele (AA and AC) genotypes (F = 3.6, P = 0.028, ANOVA). In multifactorial analysis of variance, the XPD exon 23 polymorphism appeared as a major factor influencing CAs (F = 4.2, P = 0.017). SSBs in DNA, on the other hand, were modulated by XPD (F = 4.3, P = 0.023), XPG (F = 4.3, P = 0.024) and XRCC1 genotypes (F = 3.0, P = 0.064). Irradiation-specific DNA repair rates (reflecting mainly base excision repair activity) were affected by XRCC1 (F = 5.9, P = 0.010) and XPC polymorphisms (F = 4.2, P = 0.046, MANOVA). Our results from this study suggest that markers of genotoxicity are associated with polymorphisms in genes encoding DNA repair enzymes.

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

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.

New aspects in the biomonitoring of occupational exposure to styrene.

OBJECTIVE: Based on our previous studies we analysed DNA and haemoglobin adducts, single-strand breaks in DNA, chromosomal aberrations and HPRT mutant frequencies in styrene-exposed workers, in relation to employment time, with respect to markers of individual susceptibility. In our study the risk assessment of genotoxic styrene in occupationally exposed humans is reviewed in the light of adaptation and/or population selection. Various styrene-induced DNA adducts (representing important integral measures of DNA damage) are discussed as potential powerful biomarkers of styrene exposure, with respect to DNA repair. METHODS: The complexity of followed markers required multiple methodological approaches, including modified (32)P-post-labelling assay, PCR-based methods for genotyping, cytogenetic analysis, T-cell cloning assay, comet assay and tests for DNA repair capacity, and statistical tests. RESULTS: Haemoglobin and DNA adducts, single-strand breaks in DNA, chromosomal aberrations and mutant frequencies at the HPRT gene were assessed with respect to the duration of exposure. No apparent accumulation of these biomarkers was found to be dependent on the years of employment, suggesting that adaptive processes play a role in chronically exposed workers. Statistically significant differences between the exposed and control individuals were found for the deduced epoxide hydrolase activity ( P=0.041) and the frequency of heterozygous Ala/Val genotype in GSTP1, exon 5 ( P=0.025). Significantly increased DNA repair capacity was found in styrene-exposed laminators compared with control individuals. CONCLUSIONS: In the light of present knowledge well-designed population studies on styrene genotoxicity are needed. Such studies should cover important areas of individual susceptibility (metabolising and repair enzymes, distributions of genetic polymorphisms) and possible role of adaptation, as well as the crucial role of DNA repair. Additionally, genotoxic effects of in-vivo formed 3,4-styrene oxide should be also addressed.