Genotoxic effects of occupational exposure to glass fibres - A human biomonitoring study.

As part of a large human biomonitoring study, we conducted occupational monitoring in a glass fibre factory in Slovakia. Shopfloor workers (n = 80), with a matched group of administrators in the same factory (n = 36), were monitored for exposure to glass fibres and to polycyclic aromatic hydrocarbons (PAHs). The impact of occupational exposure on chromosomal aberrations, DNA damage and DNA repair, immunomodulatory markers, and the role of nutritional and lifestyle factors, as well as the effect of polymorphisms in metabolic and DNA repair genes on genetic stability, were investigated. The (enzyme-modified) comet assay was employed to measure DNA strand breaks (SBs) and apurinic sites, oxidised and alkylated bases. Antioxidant status was estimated by resistance to H2O2-induced DNA damage. Base excision repair capacity was measured with an in vitro assay (based on the comet assay). Exposure of workers to fibres was low, but still was associated with higher levels of SBs, and SBs plus oxidised bases, and higher sensitivity to H2O2. Multivariate analysis showed that exposure increased the risk of high levels of SBs by 20%. DNA damage was influenced by antioxidant enzymes catalase and glutathione S-transferase (measured in blood). DNA repair capacity was inversely correlated with DNA damage and positively with antioxidant status. An inverse correlation was found between DNA base oxidation and the percentage of eosinophils (involved in the inflammatory response) in peripheral blood of both exposed and reference groups. Genotypes of XRCC1 variants rs3213245 and rs25487 significantly decreased the risk of high levels of base oxidation, to 0.50 (p = 0.001) and 0.59 (p = 0.001), respectively. Increases in DNA damage owing to glass fibre exposure were significant but modest, and no increases were seen in chromosome aberrations or micronuclei. However, it is of concern that even low levels of exposure to these fibres can cause significant genetic damage.

Lack of mutagenicity of TiO2 nanoparticles in vitro despite cellular and nuclear uptake.

The potential genotoxicity of titanium dioxide (TiO2) nanoparticles (NPs) is a conflictive topic because both positive and negative findings have been reported. To add clarity, we have carried out a study with two cell lines (V79-4 and A549) to evaluate the effects of TiO2 NPs (NM-101), with a diameter ranging from 15 to 60 nm, at concentrations 1-75 µg/cm2. Using two different dispersion procedures, cell uptake was determined by Transmission Electron Microscopy (TEM). Mutagenicity was evaluated using the Hprt gene mutation test, while genotoxicity was determined with the comet assay, detecting both DNA breaks and oxidized DNA bases (with formamidopyrimidine glycosylase - Fpg). Cell internalization, as determined by TEM, shows TiO2 NM-101 in cytoplasmic vesicles, as well as close to and inside the nucleus. Such internalization did not depend on the state of agglomeration, nor the dispersion used. In spite of such internalization, no cytotoxicity was detected in V79-4 cells (relative growth activity and plating efficiency assays) or in A549 cells (AlamarBlue assay) after exposure lasting for 24 h. However, a significant decrease in the relative growth activity was detected at longer exposure times (48 and 72 h) and at the highest concentration 75 µg/cm2. When the modified enzyme-linked alkaline comet assay was performed on A549 cells, although no significant induction of DNA damage was detected, a positive concentration-effects relationship was observed (Spearman's correlation = 0.9, p 0.0001). Furthermore, no significant increase of DNA oxidized purine bases was observed. When the frequency of Hprt gene mutants was determined in V79-4 cells, no increase was observed in the exposed cells, relative to the unexposed cultures. Our general conclusion is that, under our experimental conditions, TiO2 NM-101 exposure does not exert mutagenic effects despite the evidence of NP uptake by V79-4 cells.

DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death.

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan-Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06-1.90) for overall mortality, and 1.94 (1.04-3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.

DNA Repair Gene Polymorphisms and Chromosomal Aberrations in Exposed Populations.

DNA damage and unrepaired or insufficiently repaired DNA double-strand breaks as well as telomere shortening contribute to the formation of structural chromosomal aberrations (CAs). Non-specific CAs have been used in the monitoring of individuals exposed to potential carcinogenic chemicals and radiation. The frequency of CAs in peripheral blood lymphocytes (PBLs) has been associated with cancer risk and the association has also been found in incident cancer patients. CAs include chromosome-type aberrations (CSAs) and chromatid-type aberrations (CTAs) and their sum CAtot. In the present study, we used data from our published genome-wide association studies (GWASs) and extracted the results for 153 DNA repair genes for 607 persons who had occupational exposure to diverse harmful substances/radiation and/or personal exposure to tobacco smoking. The analyses were conducted using linear and logistic regression models to study the association of DNA repair gene polymorphisms with CAs. Considering an arbitrary cutoff level of 5 × 10-3, 14 loci passed the threshold, and included 7 repair pathways for CTA, 4 for CSA, and 3 for CAtot; 10 SNPs were eQTLs influencing the expression of the target repair gene. For the base excision repair pathway, the implicated genes PARP1 and PARP2 encode poly(ADP-ribosyl) transferases with multiple regulatory functions. PARP1 and PARP2 have an important role in maintaining genome stability through diverse mechanisms. Other candidate genes with known roles for CSAs included GTF2H (general transcription factor IIH subunits 4 and 5), Fanconi anemia pathway genes, and PMS2, a mismatch repair gene. The present results suggest pathways with mechanistic rationale for the formation of CAs and emphasize the need to further develop techniques for measuring individual sensitivity to genotoxic exposure.

The hCOMET project: International database comparison of results with the comet assay in human biomonitoring. Baseline frequency of DNA damage and effect of main confounders.

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.

DNA repair gene polymorphisms and chromosomal aberrations in healthy, nonsmoking population.

Nonspecific structural chromosomal aberrations (CAs) can be found at around 1% of circulating lymphocytes from healthy individuals but the frequency may be higher after exposure to carcinogenic chemicals or radiation. The frequency of CAs has been measured in occupational monitoring and an increased frequency of CAs has also been associated with cancer risk. Alterations in DNA damage repair and telomere maintenance are thought to contribute to the formation of CAs, which include chromosome type of aberrations and chromatid type of aberrations. In the present study, we used the result of our published genome-wide association studies to extract data on 153 DNA repair genes from 866 nonsmoking persons who had no known occupational exposure to genotoxic substances. Considering an arbitrary cut-off level of P< 5 × 10-3, single nucleotide polymorphisms (SNPs) tagging 22 DNA repair genes were significantly associated with CAs and they remained significant at P < 0.05 when adjustment for multiple comparisons was done by the Binomial Sequential Goodness of Fit test. Nucleotide excision repair pathway genes showed most associations with 6 genes. Among the associated genes were several in which mutations manifest CA phenotype, including Fanconi anemia, WRN, BLM and genes that are important in maintaining genome stability, as well as PARP2 and mismatch repair genes. RPA2 and RPA3 may participate in telomere maintenance through the synthesis of the C strand of telomeres. Errors in NHEJ1 function may lead to translocations. The present results show associations with some genes with known CA phenotype and suggest other pathways with mechanistic rationale for the formation of CAs in healthy nonsmoking population.

Impact of genetic polymorphisms in kinetochore and spindle assembly genes on chromosomal aberration frequency in healthy humans.

Genomic instability is a characteristic of a majority of human malignancies. Chromosomal instability is a common form of genomic instability that can be caused by defects in mitotic checkpoint genes. Chromosomal aberrations in peripheral blood are also indicative of genotoxic exposure and potential cancer risk. We evaluated associations between inherited genetic variants in 33 mitotic checkpoint genes and the frequency of chromosomal aberrations (CAs) in the presence and absence of environmental genotoxic exposure. Associations with both chromosome and chromatid type of aberrations were evaluated in two cohorts of healthy individuals, namely an exposed and a reference group consisting of 607 and 866 individuals, respectively. Binary logistic and linear regression analyses were performed for the association studies. Bonferroni-corrected significant p-value was 5 × 10-4 for 99 tests based on the number of analyzed genes and phenotypes. In the reference group the most prominent associations were found with variants in CCNB1, a master regulator of mitosis, and in genes involved in kinetochore function, including CENPH and TEX14, whereas in the exposed group the main association was found with variants in TTK, also an important gene in kinetochore function. How the identified variants may affect the fidelity of mitotic checkpoint remains to be investigated, however, the present study suggests that genetic variation may partly explain interindividual variation in the formation of CAs.

Effects of Titanium Dioxide Nanoparticles on the Hprt Gene Mutations in V79 Hamster Cells.

The genotoxicity of anatase/rutile TiO2 nanoparticles (TiO2 NPs, NM105 at 3, 15 and 75 µg/cm2) was assessed with the mammalian in-vitro Hypoxanthine guanine phosphoribosyl transferase (Hprt) gene mutation test in Chinese hamster lung (V79) fibroblasts after 24 h exposure. Two dispersion procedures giving different size distribution and dispersion stability were used to investigate whether the effects of TiO2 NPs depend on the state of agglomeration. TiO2 NPs were fully characterised in the previous European FP7 projects NanoTEST and NanoREG2. Uptake of TiO2 NPs was measured by transmission electron microscopy (TEM). TiO2 NPs were found in cytoplasmic vesicles, as well as close to the nucleus. The internalisation of TiO2 NPs did not depend on the state of agglomeration and dispersion used. The cytotoxicity of TiO2 NPs was measured by determining both the relative growth activity (RGA) and the plating efficiency (PE). There were no substantial effects of exposure time (24, 48 and 72 h), although a tendency to lower RGA at longer exposure was observed. No significant difference in PE values and no increases in the Hprt gene mutant frequency were found in exposed relative to unexposed cultures in spite of evidence of uptake of NPs by cells.

Distinct pathways associated with chromosomal aberration frequency in a cohort exposed to genotoxic compounds compared to general population.

Non-specific structural chromosomal aberrations (CAs) observed in peripheral blood lymphocytes of healthy individuals can be either chromosome-type aberrations (CSAs) or chromatid-type aberrations (CTAs) depending on the stage of cell division they are induced in and mechanism of formation. It is important to study the genetic basis of chromosomal instability as it is a marker of genotoxic exposure and a predictor of cancer risk. For that purpose, we conducted two genome-wide association studies (GWASs) on healthy individuals in the presence and absence of apparent genotoxic exposure from the Czech Republic and Slovakia. The pre-GWAS cytogenetic analysis reported the frequencies of CSA, CTA and total CA (CAtot). We performed both linear and binary logistic regression analysis with an arbitrary cut-off point of 2% for CAtot and 1% for CSA and CTA. Using the statistical threshold of 1.0 × 10-5, we identified five loci with in silico predicted functionality in the reference group and four loci in the exposed group, with no overlap between the associated regions. A meta-analysis on the two GWASs identified further four loci with moderate associations in each of the studies. From the reference group mainly loci within genes related to DNA damage response/repair were identified. Other loci identified from both the reference and exposed groups were found to be involved in the segregation of chromosomes and chromatin modification. Some of the discovered regions in each group were implicated in tumourigenesis and autism.

Titanium dioxide nanoparticles tested for genotoxicity with the comet and micronucleus assays in vitro, ex vivo and in vivo.

The genotoxicity of TiO2 nanoparticles (NPs) was assessed with the cytokinesis-block micronucleus (CBMN) assay in TK6 lymphoblastoid cells, lymphocytes from human volunteers, and bone marrow erythrocytes from rats exposed in vivo; and with the comet assay (detecting both strand breaks and oxidised purines) in human and rat peripheral blood mononuclear cells (PBMCs). NPs were dispersed using three different methods giving different size distribution and stability. On average, TiO2 NPs caused no increase in micronuclei in TK6 cells, rat bone marrow erythrocytes or human lymphocytes (though lymphocytes from 3 out of 13 human subjects showed significant increases). PBMCs from rats treated in vivo with a single dose of NPs dispersed by a method with low agglomeration showed an increase in strand breaks after 1 day. TiO2 NPs dispersed in a stable, non-agglomerated state induced DNA strand breaks at 75 µg/cm2 after 4 h exposure of human PBMCs and at 15 µg/cm2 and 75 µg/cm2 after 24 h exposure, but no increase in DNA oxidation was seen. Overall, NPs in an agglomerated state did not cause DNA damage. However, at the individual level, significant increases in strand breaks were seen in PBMCs from most of the volunteers. Cells from one volunteer showed positive effects in all conditions and both tests, while cells from another volunteer appeared to be completely resitant to TiO2 NPs. The implication is that some individuals may be more sensitive than others to effects of this nanomaterial. Differences seen in results obtained with the micronucleus and the comet assay may be due to the mechanisms underlying the genotoxic effects of TiO2 NPs and the different endpoints represented by the two assays.

Genetic variation associated with chromosomal aberration frequency: A genome-wide association study.

Chromosomal aberrations (CAs) in human peripheral blood lymphocytes (PBL) measured with the conventional cytogenetic assay have been used for human biomonitoring of genotoxic exposure for decades. CA frequency in peripheral blood is a marker of cancer susceptibility. Previous studies have shown associations between genetic variants in metabolic pathway, DNA repair and major mitotic checkpoint genes and CAs. We conducted a genome-wide association study on 576 individuals from the Czech Republic and Slovakia followed by a replication in two different sample sets of 482 (replication 1) and 1288 (replication 2) samples. To have a broad look at the genetic susceptibility associated with CA frequency, the sample sets composed of individuals either differentially exposed to smoking, occupational/environmental hazards, or they were untreated cancer patients. Phenotypes were divided into chromosome- and chromatid-type aberrations (CSAs and CTAs, respectively) and total chromosomal aberrations (CAtot). The arbitrary cutoff point between individuals with high and low CA frequency was 2% for CAtot and 1% for CSA and CTA. The data were analyzed using age, sex, occupation/cancer and smoking history as covariates. Altogether 11 loci reached the P-value of 10-5 in the GWAS. Replication 1 supported the association of rs1383997 (8q13.3) and rs2824215 (21q21.1) in CAtot and rs983889 (5p15.1) in CTA analysis. These loci were found to be associated with genes involved in mitosis, response to environmental and chemical factors and genes involved in syndromes linked to chromosomal abnormalities. Identification of new genetic variants for the frequency of CAs offers prediction tools for cancer risk in future. Environ. Mol. Mutagen. 60:17-28, 2019. © 2018 Wiley Periodicals, Inc.

In Vitro Approaches for Assessing the Genotoxicity of Nanomaterials.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Genetic variation in the major mitotic checkpoint genes associated with chromosomal aberrations in healthy humans.

Non-specific chromosomal aberrations (CAs) are microscopically detected in about 1% of lymphocytes drawn from healthy persons. Causes of CAs in general population are not known but they may be related to risk of cancer. In view of the importance of the mitotic checkpoint machinery on maintaining chromosomal integrity we selected 9 variants in main checkpoint related genes (BUB1B, BUB3, MAD2L1, CENPF, ESPL1/separase, NEK2, PTTG1/securin, ZWILCH and ZWINT) for a genotyping study on samples from healthy individuals (N = 330 to 729) whose lymphocytes had an increased number of CAs compared to persons with a low number of CAs. Genetic variation in individual genes played a minor importance, consistent with the high conservation and selection pressure of the checkpoint system. However, gene pairs were significantly associated with CAs: PTTG1-ZWILCH and PTTG1-ZWINT. MAD2L1 and PTTG1 were the most common partners in any of the two-way interactions. The results suggest that interactions at the level of cohesin (PTTG1) and kinetochore function (ZWINT, ZWILCH and MAD2L1) contribute to the frequency of CAs, suggesting that gene variants at different checkpoint functions appeared to be required for the formation of CAs.

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

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).

Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts.

Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12µg/cm(2) At these range we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure.

Interactions of DNA repair gene variants modulate chromosomal aberrations in healthy subjects.

Human cancers are often associated with numerical and structural chromosomal instability. Structural chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBL) arise as consequences of direct DNA damage or due to replication on a damaged DNA template. In both cases, DNA repair is critical and inter-individual differences in its capacity are probably due to corresponding genetic variations. We investigated functional variants in DNA repair genes (base and nucleotide excision repair, double-strand break repair) in relation to CAs, chromatid-type aberrations (CTAs) and chromosome-type aberrations (CSAs) in healthy individuals. Chromosomal damage was determined by conventional cytogenetic analysis. The genotyping was performed by both restriction fragment length polymorphism and TaqMan allelic discrimination assays. Multivariate logistic regression was applied for testing individual factors on CAs, CTAs and CSAs. Pair-wise genotype interactions of 11 genes were constructed for all possible pairs of single-nucleotide polymorphisms. Analysed individually, we observed significantly lower CTA frequencies in association with XPD Lys751Gln homozygous variant genotype [odds ratio (OR) 0.64, 95% confidence interval (CI) 0.48-0.85, P = 0.004; n = 1777]. A significant association of heterozygous variant genotype in RAD54L with increased CSA frequency (OR 1.96, 95% CI 1.01-4.02, P = 0.03) was determined in 282 subjects with available genotype. By addressing gene-gene interactions, we discovered 14 interactions significantly modulating CAs, 9 CTAs and 12 CSAs frequencies. Highly significant interactions included always pairs from two different pathways. Although individual variants in genes encoding DNA repair proteins modulate CAs only modestly, several gene-gene interactions in DNA repair genes evinced either enhanced or decreased CA frequencies suggesting that CAs accumulation requires complex interplay between different DNA repair pathways.

Metabolic gene variants associated with chromosomal aberrations in healthy humans.

Nonspecific chromosomal aberrations (CAs) are found in about 1% of lymphocytes drawn from healthy individuals. They include chromosome-type aberrations (CSAs), which are increased in exposure to ionizing radiation, and chromatid-type aberrations (CTAs) which in experimental systems are formed by DNA binding carcinogens and mutagens. The frequency of CAs is associated with the risk of cancer, but the causes of CAs in general population are unknown. Here, we want to test whether variants in metabolic genes associate with CAs in healthy volunteers. Cases were considered those whose total CA (CAtot) frequency was >2% and for CSA and CTA the limit was >1%. Controls had lower frequencies of CAs. Functional polymorphisms in seven genes were selected for analysis: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), each coding for phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. The number of volunteers genotyped for each gene varied from 550 to 1,500. Only EPHX1 was individually associated with CAtot; high activity genotypes decreased CAtot. A total of six significant (P < 0.01) pair-wise interactions were observed, most including a GST variant as one of the pair. In all genotype combinations with significant odds ratios for CAs a GST variant was involved. The present data provide evidence that variants in genes coding for metabolic enzymes, which individually have small effects, interact and are associated with CA frequencies in peripheral lymphocytes of healthy volunteers.

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.