The repeated cytogenetic analysis of subjects occupationally exposed to nanoparticles: a pilot study

Abstract

The application of nanomaterials has been rapidly increasing during recent years. Inhalation exposure to nanoparticles (NP) may result in negative toxic effects but there is a critical lack of human studies, especially those related to possible DNA alterations. We analyzed pre-shift and post-shift a group of nanocomposite researchers with a long-term working background (17.8 ± 10.0 years) and matched controls. The study group consisted of 73.2% males and 26.8% females. Aerosol exposure monitoring during a working shift (involving welding, smelting, machining) to assess the differences in exposure to particulate matter (PM) including nanosized fractions <25–100 nm, and their chemical analysis, was carried out. A micronucleus assay using Human Pan Centromeric probes, was applied to distinguish between the frequency of centromere positive (CEN+) and centromere negative (CEN−) micronuclei (MN) in the binucleated cells. This approach allowed recognition of the types of chromosomal damage: losses and breaks. The monitoring data revealed differences in the exposure to NP related to individual working processes, and in the chemical composition of nanofraction. The cytogenetic results of this pilot study demonstrated a lack of effect of long-term (years) exposure to NP (total frequency of MN, P = 0.743), although this exposure may be responsible for DNA damage pattern changes (12% increase of chromosomal breaks—clastogenic effect). Moreover, short-term (daily shift) exposure could be a reason for the increase of chromosomal breaks in a subgroup of researchers involved in welding and smelting processes (clastogenic effect, P = 0.037). The gender and/or gender ratio of the study participants was also an important factor for the interpretation of the results. As this type of human study is unique, further research is needed to understand the effects of long-term and short-term exposure to NP.

Introduction

A tremendous increase in the use of nanomaterials (NM) has been witnessed during the last decade in many areas of human life, including the chemical industry, cosmetics, biomedicine and food technology. Despite the many benefits of engineered NM, the huge diversity of nanoparticles (NP), their unique properties, almost ubiquitous presence together with ambient ultrafine particles, the size range of 1–100 nm and still not fully understood interactions with biological systems via a large number of consumer products or specific working processes, NM as well as NP, has raised the interest of toxicologists (1).

Current evidence obtained from exposure to NP in various biological systems, ranging from prokaryotes to higher eukaryotes, reveals that exposure to NP may result in numerous toxic effects. In general, in vivo and in vitro studies in model systems suggest that exposure to NP leads to: an inflammatory response, DNA damage, oxidative stress, lipid peroxidation, apoptosis, micronuclei (MN) formation, altered gene expression and methylation, genotoxicity, cytotoxicity and reproductive toxicity (2–6). Regardless of that, there is a critical lack of human biomonitoring studies focusing on the effects of NP exposure in humans, even though the size range of NP is connected with an increased risk of deposition in the alveolar space of the lungs (7). Several studies from the Czech Republic have concentrated on the biological impacts of occupational exposure to nano titanium dioxide and iron oxide and observed elevated oxidative damage to DNA, lipids and proteins in the exhaled breath condensate (EBC) (8–11), in addition to markers of inflammation (12). The studies by Liou et al. from Taiwan also found a significantly higher 8-isoprostane in EBC and 8-OHdG in the urine of workers with occupational exposure to metal oxide nanomaterials (13,14), in addition to global DNA methylation changes. A recent study from China that focused on nano titanium dioxide exposure also suggested cardiopulmonary effects (15); whereas a study from Sweden reported changes in inflammatory markers due to exposure to welding-fumes and derived NP aggregates (16).

Interestingly, cytogenetic methods that allow the detection of DNA alterations (chromosomal breaks and/or losses) have never been applied in human biomonitoring in populations exposed to NP. Investigation of total MN by cytokinesis-block micronucleus assay (CBMN assay) in binucleated cells (BNC), is a traditional method (17), successfully used for the evaluation of the effect of exposure to many chemicals (reviewed in Nersesyan et al. (18)). Despite this fact, no human studies focused on MN formation following the exposure to NP have been reported (19).

To fill these gaps, we performed long-term monitoring (starting in 2015, planned to 2020) of the researchers, exposed during the nanocomposite producing processes. In 2016, we collected the biological material for the analysis of the frequency of MN from peripheral blood lymphocytes (PBL) with the aim to use an advanced version of this assay enriched with the centromere hybridisation method (20), to distinguish the clastogenic and aneugenic effects of exposure. We decided to use this approach as there are evidences suggesting a pivotal role of aneuploidy in the cytotoxicity and genotoxicity exerted by NPs in human primary lymphocytes and murine macrophages (21). The main goal of our study was to discover the potential negative effects of long-term (years) and short-term (daily) NP exposure on level and type of DNA damage, in a group of nanocomposite processing researchers. In our hypothesis, we built upon the recently published observations from a set of air pollution biomonitoring studies where we observed no effect of long-term increased air pollution exposure on the frequency of total MN together with substantial differences in DNA methylation pattern (22). We presumed versatility of these findings related to various environmental stressors, also including long-term NP exposure.

Materials and Methods

Long-term project

This study is part of a long-term monitoring project, repeatedly examining a group of researchers studying and processing nanocomposites with new characteristics, in parallel with their occupational environment. The main aim of the project was to investigate both the long-term (years), and short-term (pre-shift vs. post-shift) effects of exposure to NP. The study subjects were exposed in the workshops for 2.5 h. They spent the remainder of the total 8-h shift in their offices. During sampling, the following steps were carried out: (i) detailed monitoring of the occupational environment including PM in the range of <25 nm–10 µm, (ii) completion of a detailed questionnaire concerning health status and exposure information and (iii) whole venous blood collection (two samplings in 1 day, the first before the working shift and the second after the working shift in Workshop 1 or 2).

Study populations

A total of 41 participants [mean age ± standard deviation (SD): 40.2 ± 10.3 years; median (range): 39 (20–63) years] were involved in the collection of biological material performed in September 2016. To avoid a break in NP exposure during weekends that would confound analyses of short-term effects, the samples were obtained on Tuesdays and Wednesdays. The sample set included a group of 20 occupationally exposed nanocomposite processing researchers, working long-term [mean ± SD: 17.8 ± 10.0 years; median (range) 15 (5–40)] in nanocomposite research, sampled twice (pre-shift and post-shift, including processes such as welding mild steel S355J2 and smelting in Workshop 1; and machining including the milling and grinding of epoxide resin with nanoSiO₂, and geopolymer nanocomposites in Workshop 2) and 21 control volunteers living in the same location. The researchers did not use any respirators or other personal protective equipment. Both genders, with a prevalence of 15 men, were involved in each subgroup. More details on the comparison of the basic characteristics of the studied groups are presented in the results section.

Sixty-one blood samples were collected and transported from Liberec to Prague (a distance of approximately 110 km). All blood samples were thoroughly mixed with heparin and kept at 4°C–10°C until the whole venous blood cultures were established within 24 h. All participants signed an informed consent form and had the opportunity to withdraw from participation at any time during the study, according to the Helsinki II declaration. The ethical committee of the General University Hospital in Prague and First Medical Faculty, Charles University approved the study.

Exposure monitoring

Full details on the methods of the general monitoring of the study group, including the results, were published previously (11). In brief, the offline Berner Low Pressure Impactor (BLPI) (HAUKE Gmbh., Austria) was used to sample aerosol particles onto 10 stages up to 13.6 µm including two nanoscales (25–56 and 56–100 nm) (23). These samples were consecutively analyzed by gravimetry, ion chromatography (24) and scanning electron microscope (SEM) (Tescan Indusem, Czech Republic) equipped by energy-dispersive X-ray spectroscopy (EDS) (XFlash detector 5010, Bruker, Germany) to analyze the elemental composition of size-resolved aerosol fractions. The relative ratios of individual elements obtained from SEM/EDS were recalculated to absolute concentrations using absolute concentrations of sulphates from ion chromatography (IC) and the relative content of S from EDS, using the assumption that all S is contained in the form of sulphates. Furthermore, additional online monitoring during the shift for individual processes and prior to it (background), included two standard aerosol spectrometers scanning mobility particle sizer (SMPS) (TSI SMPS 3936L, USA) and Aerodynamic Particle Sizer (APS) (TSI APS 3321, USA) which were used to obtain more details in the nanoscale range from 6 nm up. Additional data related to the airborne environmental contamination by particulate matter (PM) of aerodynamic diameter <2.5 µm (PM2.5) and <10 µm (PM10) in the city of Liberec (distance from the site of biological sample collection was ~3.0 km), during sample collection in September 2016, were obtained from the website of the Czech Hydrometeorological Institute (http://portal.chmi.cz/?l=en). Both sets of values, obtained from the radiometry beta ray absorption measuring method, were below the permissible annual limits of 25 and 40 µg/m³ for PM2.5 and PM10, respectively.

Micronucleus test

Whole venous blood samples collected into sodium-heparinised vacutainers (Greiner Bio-One GmbH, Austria) were used to set up cell cultures in duplicate. Each culture contained 0.5 ml of whole blood in a total of 7.5 ml cultivation medium [RPMI, containing l-glutamine and NaHCO₃ (Sigma, USA), 25% fetal bovine serum Superior (Biochrom AG, Germany) and 1% phytohaemagglutinin PHA-L (Biochrom AG, Germany)].The cultures were grown at 37°C as previously described (25). After 44 h of incubation, cytochalasin B (Sigma, USA) was added to a final concentration of 5 µg/ml (17). After 72 h, the cultures were harvested by centrifugation, treated with a hypotonic solution of KCl (0.075 M) and fixed repeatedly with methanol/acetic acid solution (3:1). Prior to performing the FISH analysis, nuclear division index (NDI) was calculated in 500 viable cells to determine the frequency of cells with 1, 2, 3 and 4 nuclei using the formula NDI = (M₁ + 2M₂ + 3M₃ + 4M₄)/N, where M₁–M₄ represents the number of cells with 1–4 nuclei and N is the total number of scored viable cells (26). Finally, slides with a density of 1500 BNC per square (22 × 22 mm) were prepared using the air-dry method and stored at room temperature until fluorescent staining of centromeres was performed.

The protocol to perform fluorescent in situ hybridisation (FISH) with fluorescein-5-isothiocyanate (FITC) labelled Human Pan Centromeric probes has been adapted from the manufacturer’s protocol provided by Cambio (Cambridge, UK). In brief: slides were dehydrated through a series of alcohol washes (70%, 90%, 100%) for 2 min each and dried at room temperature. Denaturation was carried out in 70% formamide in 2×SSC for 2 min at 72°C; slides were immersed in ice-cold 70% ethanol and again dehydrated through a series of alcohol washes and dried at room temperature. Probes for hybridisation were denatured for 10 min at 85°C and immediately chilled on ice. Ten microlitres of ready to use probe per 22 × 22 mm area was applied and left to hybridise for approximately 16 h at 37°C in a humidified chamber. The slides were washed twice the following day at 37°C in 50% formamide and 2×SSC for 5 min each, and twice in 2×SSC, again for 5 min each. The counterstaining, following the washes, was with DAPI (4, 6-diamidino-2-phenylindole) (Sigma, USA) mixed with Vectashield mounting medium (Vector Laboratories, USA) at a final concentration of 0.24 µg/ml. The slides were stored in the dark at 4°C–10°C until microscopic analysis.

The analysis of 1000 BNC per blood sample (a total of 2000 BNC per study subject in case both pre-shift and post-shift samples are considered) was performed using a fluorescence microscope (Axioskop; Zeiss, Germany) equipped with filters for DAPI (blue) and FITC (green) and an immersion oil objective lens for a final magnification of ×1000. All observations [BNC with MN: either centromere positive (CEN+) or centromere negative (CEN−)] were recorded and collected by ISIS software, version 5.0 (MetaSystems, Germany). See Figure 1A–I for an overview of representative images (scored and non-scored) from microscopic analysis. The results of total MN were expressed as frequency MN/1000 BNC and as a percentage of aberrant cells (%AB.C.), i.e. the % of BNC with one or more MN.

Statistical analysis

Basic descriptive statistics [mean, SD, median and range (minimum and maximum)], were calculated using Microsoft Excel 2013. An advanced statistical analysis was performed using the IBM SPSS Software (Chicago, USA) version 22.0. The Kolmogorov–Smirnov test was used to test normality of the data distribution. For the independent groups (exposed vs. controls), t-test (normally distributed variables) or the nonparametric Mann–Whitney U test (non-normally distributed variables) were used for the comparison of the studied parameters (total MN/1000 BNC, %AB.C., CEN+ MN/1000 BNC and CEN− MN/1000 BNC). For the paired values (pre-shift vs. post-shift), paired sample t-test or the Wilcoxon signed-rank test, depending on the data distribution, were used for the same studied parameters. We further applied bivariate and multivariate logistic regression analysis, to assess the impact of the studied parameters (age, gender, body mass index [BMI] and exposure) on the frequency of the total and individual types of MN. Logistic regression was performed separately: (i) for controls and pre-shift data with the aim to evaluate the effect of long-term exposure and (ii) for controls and post-shift data with the aim to evaluate the effect of short-term exposure. For logistic regression, all dependent variables were transformed into a two-level scale using medians. These results were expressed as odds ratios with a 95% confidence interval and significance level (P value).

Results

The entire study was performed within a group of researchers, with long-term working backgrounds in nanocomposite research, and matched controls from the same location. Both the short-term and long-term effects of exposure to the DNA damage level were analyzed by an improved micronucleus assay, combined with Pan-centromeric FISH technique (examples of representative images from microscopic analysis are in Figure 1A–I).

In Table 1, the general characteristics of the study subjects including subgroups are presented [number of study subjects (N), mean, SD, median, minimum and maximum). The subgroups are stratified by age, gender, BMI and lifestyle characteristics (smoking and occasional alcohol consumption). The data are based on information obtained from questionnaires during a sampling period in September 2016. There was no significant difference between the exposed and control groups for any of the selected characteristics. There was also no difference between the exposed subgroups (exposed in Workshop 1 vs. exposed in Workshop 2) for age and gender (P = 0.270 and P = 0.436, respectively). A younger/older ratio (6:5 and 5:4 for the exposed group in Workshop 1 and Workshop 2, respectively; data not shown) is comparable. Small, non-significant differences for BMI, which tended to be higher among the exposed, were found. The ratios of underweight: normal weight: overweight: obese participants were 1:5:8:6 and 0:11:8:2 in the exposed and control groups, respectively. Owing to the small number of underweight, obese and smoking participants (weak smokers, smoking 2.5–10 packs/year), these factors were not included separately in the analyses. The same was true for occasional alcohol consumption (non-periodical weak consumers drinking on special occasions only) which was found in the vast majority of participants of the study in both groups.

Additional time exposure characteristics obtained from questionnaires (years of exposure, common exposure time/day) as well as exposure time/monitoring day, are presented in Table 2. The exposed group was split into two subgroups according to the workplace. The subgroups did not significantly differ by the time of exposure as well as by age (Table 1). The subjects worked separately in two workshops substantially differing by working processes and exposure characteristics. Specifically, in Workshop 1, welding and smelting, and in Workshop 2, machining including milling and grinding were performed. The data generally show a very long exposure history of the whole exposed group (5–40 years) that involved on average 101.4 min exposure during the common working shift. There was about 50% higher exposure (up to 156.3 min on average) in comparison with a standard shift which was connected with the monitoring day. Some differences although not significant, particularly in the length of standard daily exposure, were found in the exposure history data of researchers from Workshops 1 and 2.

Detailed online and offline monitoring during the shift for individual working processes, as well as monitoring of the background levels of exposure with an emphasis on nanofractions, were completed in this study. The proportions of the three PM fractions (ranging from nano to 10 µm: (i) <25–100 nm, (ii) 100 nm–1 µm and (iii) 1–10 µm) measured by online monitoring (SMPA and APS) during the shift, related to the individual working processes including background values, are presented in Table 3. The presented data show the highest proportion (94.64%) of nanofraction (PM < 25–100 nm) related to the smelting process carried out in Workshop 1. The highest proportion of 100 nm–1 µm fraction was connected with the welding process performed in the same workshop. In contrast, data for Workshop 2 (milling and grinding processes) with a mildly increased 100 nm–1 µm fraction, seemed to be similar to a 15 h night background proportion of PM fractions. The results of the analysis of SEM/EDS relative elemental composition (%) and absolute elemental mass concentration (µg/m³) of the two nanosized fractions (25–56 and 56–100 nm) related to individual working processes obtained by BLPI, are summarised in Table 4 [relative elemental composition (%) and absolute mass concentration (µg/m³), respectively]. The absolute mass concentration data are the most important for understanding of the real exposure of the study participants. Generally, individual processes differed in the chemical composition of the nanosized fraction with a dominant content of Fe common to all processes. Other common elements included Si, S and Cl. The production of Mn and Na was specific for welding and smelting; Al for smelting and machining; and K was detected in the smelting process only. The presented data clearly show that the machining processes were associated with the production of a substantially different pattern of chemical compounds in comparison with the welding and smelting processes [Table 4, relative elemental composition (%)]. For welding, a substantially higher absolute elemental mass concentration of the nanosized fraction was observed when compared with smelting and machining [Table 4, absolute mass concentration (µg/m³)], despite the fact that the proportion of nanofraction among PM fractions was the lowest for this process (Table 3). Specifically, the process of welding produced a 9.55× and 107× higher total level of nanosized fractions (25–56 + 56–100 nm) in comparison with smelting and machining, respectively [Table 4, absolute mass concentration (µg/m³)].

Cytotoxicity (NDI) and genotoxicity data (the total MN frequencies per 1000 BNC and %AB.C.) in the studied group, stratified by the general and exposure characteristics of the study subjects, are shown in Table 5. All the cytotoxicity data presented by NDI show homogenous viability of cells in individual groups and subgroups without significant differences (mean ± SD: 2.02 ± 0.08; median (range): 2.02 (1.8 – 2.16) for the pooled data). The genotoxicity results presented in Table 5 [general characteristics (group/subgroups^X)] represent pooled data from the exposed pre-shift and control subjects. These data were analyzed with the aim to enlarge the dataset given the fact that no significant differences were observed between the exposed and control subjects for general characteristics (Table 1) as well as the total MN/1000 BNC [Table 5, exposure characteristics (group/subgroups)]. The pooled data showed no differences in the DNA damage levels in the subgroups stratified by age and BMI, whereas the impact of gender was significant. Data related to the effects of long-term and short-term exposure to NP for all participants of the study, are summarised in Table 5 [exposure characteristics (group/subgroups)]. These data show similar results for the controls and long-term exposed researchers, analyzed pre-shift for both total MN/1000 BNC and %AB.C. (P = 0.753 and P = 0.948, respectively), and show a trend of an increase of the DNA damage levels after short-term exposure for a comparison of the pre-shift and post-shift data (P = 0.087 and P = 0.192, for total MN/1000 BNC and %AB.C., respectively). Interestingly, short-term exposure to products of machining of surfaces (including milling and grinding) in workroom 2 analyzed separately for males, showed significant differences for total MN/1000 BNC (P = 0.042; data not shown). Generally, the comparison of pre-shift and post-shift data on an individual basis revealed a trend of increased or the same frequency of total MN per 1000 BNC in 85% of cases (data not shown).

We further analyzed the frequency of CEN+ and CEN− MN per 1000 BNC (Table 6). Highly significant differences in the presence of chromosomal losses, for the comparison between genders (5.03 ± 2.64 and 11.00 ± 5.45, for males and females respectively, P = 0.001) are shown in Table 6 [general characteristics (group/subgroups^X)] (pooled data include 25% females from the exposed and 29% females from the control group). Interestingly, a lower BMI seems to be associated with a higher frequency of CEN+ MN (P = 0.090); this result was significant in males (P = 0.041, data not shown). A short-term increase of CEN+ MN seems to be associated with an individual working process [Table 6, exposure characteristics (group/subgroups)]. Post-shift significant increase of chromosomal breaks in a group of participants in Workshop 1 when compared with the controls was found (P = 0.037). The increase of chromosomal breaks was also observed for males when analyzed separately (P = 0.025 and P = 0.037; for the males exposed pre-shift and post-shift in Workshop 1 compared with the controls, respectively; data not shown). In opposite, a non-significant trend to a post-shift increase of aneuploidy was found in Workshop 2 in comparison with Workshop 1 (P = 0.073) in the study group that includes both genders. However, this trend was not observed in males only (P = 0.673, data not shown). Generally, long-term exposure during NP producing processes was associated with an increase of chromosomal breaks as indicated from the CEN+/CEN− ratio for the pre-shift exposed and controls (52/48% and 64/36%, respectively). However, this observation is strongly dependent on the particular working process. A role of gender was also found: a 12%, 15% and 6% increase of breaks was seen for the whole group, males and females, respectively (data for individual genders not shown).

Tables 7 and 8 summarise the results from bivariate and multivariate logistic regression of the studied parameters and their impact on the frequency of total, CEN+ and CEN− MN per 1000 BNC. The data presented in Table 7 concern the impact of age, gender, BMI and long-term exposure, by including the parameters for the controls and pre-shift samples. In Table 8, the short-term effects of exposure are presented. Data from the controls and post-shift samples were used here in the analysis. The results confirmed the previous observations. Gender was the most significant factor affecting the frequency of total and CEN+ MN per 1000 BNC. In addition, the effects of long-term and short-term exposure confirmed the trends detected by Mann–Whitney Sum U test in Table 5. There is no effect of long-term exposure on the total MN frequency (Table 7) consistent with the hypothesis mentioned in the Introduction. A trend of increased frequency of the total MN/1000 BNC, as well as chromosomal breaks represented by CEN− MN/1000 BNC after short-term exposure, was also confirmed (Table 8).

Discussion

We analyzed the impact of exposure characteristics (long-term, short-term) on the frequency and type of MN in a group of nanocomposite processing researchers. We further studied the effects of general characteristics (age, gender, BMI) which are controlled in many studies as possible confounders. The analyzed samples obtained from PBL included 20 long-term exposed participants (exposure term: 5–40 years) examined twice during 1 day (pre-shift and post-shift, i.e. post exposure in the workshops lasting about 2.5 h) and 21 controls matching all the general characteristics. A total of 73.2% males and 26.8% females participated in this study. A comparison of pre-shift and control data was used to estimate the effect of chronic exposure, whereas the comparison of pre-shift and post-shift or control data was intended to estimate the effect of acute exposure. Such cytogenetic analysis of a human population exposed to NP, monitoring of both chromosomal breaks and losses and involving detailed monitoring of PM exposure including nanofraction and their elemental composition, has never been published.

Our data from exposure monitoring, comparing three working processes (welding, smelting and machining) revealed substantial differences in representation of individual PM fractions (<25–100 nm, 100 nm–1 µm, 1–10 µm) as well as in quantity and chemical composition of the nanosized fraction. The welding process was associated with higher risk of exposure to both nanosized fractions (25–56, 56–100 nm) with a majority of Fe. In addition, each process was characterised by a unique exposure pattern represented by a specific chemical composition (e.g. K was specific for smelting only, Al for smelting and machining, Na for smelting and welding).

Although we also analyzed the impact of age, gender and BMI as possible confounders affecting the frequency of MN, smoking, was excluded from our analyses due to only a small number of smokers in the study. Heavy smokers, a group associated with a risk of increased genotoxic damage (27), were not present in our study. The sampling season was also repeatedly reported to affect MN frequency (25,28), but our samples were collected within the same season (September 2016). We also analyzed the effect of age on the frequency of the total as well as CEN+ and CEN− MN, as this association was reported (29); however, we did not observe any significant results. This can be explained beside a relatively small sample set (N = 41), by the presence of both genders with a prevalence of younger females (age below median). Moreover, gender was the strongest confounder in our study. A significantly higher frequency of the total MN (P < 0.01), caused by a dramatic increase of CEN+ MN which represented aneuploidy (P < 0.001), was observed in pooled female data when compared with males. These results are in agreement with the previously published data which also indicated increased MN frequency associated with the presence of the inactive chromosome X in females’ lymphocytes (29–31). In our study we observed a tendency of increased aneuploidy with a lower BMI. We found such a trend in our previous report (20). Although the impact of BMI was evident in our study group, more data is needed to confirm the general validity of this observation (32).

The strength of our study is the fact that apart from the total MN which is predominantly analyzed in most biomonitoring studies, two basic classes of MN based on the presence of centromere (CEN+ and CEN−) were recorded. Although it is also possible that a MN contains both one or more fragments, and a whole chromosome, this event is relatively rare in non-cancerous cells (31). Several studies have indicated the proportion of CEN+ MN to be in the range from 30% to 80% (31,33,34). Our data agree with these reports, along with the impact of gender, as discussed earlier. The proportion of CEN+/CEN− MN obtained for males was 52/48%, respectively. This observation was in support of our previous study (20) where we found corresponding ratios of 50/50% and 54/46% in samples collected in spring and fall, respectively. In contrast, the data for females were strongly shifted to chromosomal losses of up to 68/32% ratio for CEN+ and CEN− MN, respectively. Even though our study groups were matched by gender, this effect must be taken into consideration in inter-study comparisons.

With regard to the CEN+/CEN− ratio, we found its substantial shift towards the increase of chromosomal breaks related to long-term (years) exposure to NP (from 64/36% for controls to 52/48% for exposed, analyzed pre-shift). However, the particular working process characterised by ‘exposure pattern’ (composed by different ratios of individual fractions and chemical compounds) could be the reason for subgroup differences. Especially, a group of researchers in Workshop 1 compared with the controls showed a significant increase of chromosomal breaks after short-term exposure. The processes in Workshop 1 were characterised by exposure to high concentrations of Fe. Overall, these results suggested differences in DNA damage profiles (chromosomal breaks and losses) associated with both the particle size and the chemical composition of PM.

In addition to the separate analysis of CEN+ and CEN− MN/1000 BNC, we also studied the levels of total MN/1000 BNC including %AB.C. Our results showed no effect of long-term professional NP exposure. This is supported by frequencies of both parameters (total MN/1000 BNC a %AB.C.), which demonstrate a high similarity between exposed pre-shift samples and the controls (P = 0.753 and P = 0.948). The short-term exposure, also reflecting exposure of the previous day(s), as the sampling was performed on Tuesdays and Wednesdays, was evaluated in two ways: (i) the comparison of pre-shift and post-shift data of exposed subgroups and (ii) the comparison of post-shift data of exposed groups with the controls. Both approaches demonstrated a trend towards increased levels of total DNA damage analyzed by the MN assay. Moreover, the effects of short-term exposure in this study group were also manifested by significant elevations of lipid peroxidation and inflammatory markers, as well as by a significant decrease of fractional exhaled nitric oxide in EBC (11,35).

A comparison of our data with similar studies is difficult, as no relevant work concerning the same cytogenetic endpoints in relation to human NP exposure and monitoring has been published. Therefore, we focused on the studies analyzing various cytogenetic markers in welders where the exposure to PM, also in nanosize range, occurs (16). A study published in 1983 analyzed the effect of long-term exposure (mean 19 years) and similarly to our results the authors reported no significant results (36). A Mexican study which focused on the genetic damage in exfoliated oral mucosa cells, also did not observe differences between the cases and controls (37). A comparison of chromosomal alterations induction, including MN, between the nasal and buccal cells of welders, showed a higher sensitivity of the epithelial cells from the respiratory system (38). Finally, Iarmarcovai et al., (39) performed an additional identification of centromeres and analyzed genetics polymorphisms in DNA repair and detoxification genes. These authors reported significantly higher levels of chromosome/genome damage in welders, suggesting that the combined analysis of genetic polymorphisms and centromeres in MN may improve the sensitivity of the micronucleus assay in detecting genotoxic effects (39).

As mentioned in the Introduction, one of our hypotheses was based on recently published observations from a set of air pollution biomonitoring studies, suggesting epigenetic mechanisms which change the regulation function of genes and reduce the negative consequences of environmental stressors on DNA (22). The results of this small pilot study show no effect of long-term NP exposure on the total frequency of MN, but investigation of epigenetic changes remains the challenge for future research. However, besides the same total level of DNA damage after long-term exposure, we also observed a shift of the CEN+/CEN− ratio towards the increased frequency of breaks. We can speculate that epigenetic mechanisms leading to changes of functional activity of specific genes may contribute to this result. The short-term exposure during working shifts was about 50% higher than the standard daily exposure of the long-term exposed subjects. This suggests that even though there were no genotoxic effects of a long-term NP exposure, a short-term substantial increase in NP levels was an event causing a mild increase of DNA damage.

Although our results suggest trends in response to NP exposure, some challenges for future research in this field of toxicology studies, as well as some gaps in our study, should be mentioned. (i) It is important to accumulate more data from epidemiological biomonitoring studies concentrating on the effects of NP exposure in human populations (40), specifically focusing on separate analyses of both genders. (ii) Detailed monitoring of various occupation environments should be applied with the aim to select professions with an increased risk of exposure to NP (41). (iii) Personal monitoring of nanofraction exposure should be implemented to these types of studies. (iv) The data from published studies should be pooled with the aim to obtain a larger set of results for statistical analysis. (v) An advanced MN-FISH method (staining of centromeres, telomeres or individual chromosomes) should be applied more frequently so that it becomes a more routine application. (vi) More biomarkers and methods should be implemented into this research (e.g. comet assay, genetics polymorphisms or epigenetic markers). (vii) The effect of NP exposure in groups of subjects without previous exposure should also be analyzed.

Conclusions

The aim of this study was to fill the gap in human biomonitoring studies (19) and focus on the effects of exposure to NP on DNA damage in occupationally exposed subjects. The advanced version of a micronucleus assay, that allows the detection of both clastogenic and aneugenic effects separately, was used. No effect of long-term NP exposure on the total frequencies of MN was observed in this pilot study. We further observed a shift in the types of aberrations towards the increased levels of chromosomal breaks in the exposed group, suggesting a clastogenic effect NP exposure. The data on short-term exposure (pre-shift vs. post-shift) also showed a greater dynamic effect of acute rather than chronic exposure. Our data further suggest that particular working processes, characterised by certain nanosize fraction content and elemental composition, may cause differences in the type of DNA damage. Finally, the gender and/or gender ratio of the study participants are also important factors for the final interpretation of the results. In summary, publication of more data from human biomonitoring studies, with the aim to improve the understanding of the mechanisms and consequences of NP exposure, should be given high priority in future research.

Funding

This work was supported by the project of Grant Agency of the Czech Republic (18-02079S).

Acknowledgements

The authors thank the volunteers for their kind participation in this study. We would also like to thank Ms. Frances Zatrepalkova for language editing.

Conflict of interest statement: None declared.

References