The Dose- and Time-Dependent Cytotoxic Effect of Graphene Nanoplatelets: In Vitro and In Vivo Study.

Graphene-based nanomaterials received attention from scientists due to their unique properties: they are highly conductive, mechanically resistant and elastic. These materials can be used in different sectors of society from electronic energy storage in industry to biomedical applications. This study evaluates the influence of graphene nanoplatelets in vitro and in vivo. The toxicological influence of graphene nanoplatelets (GPs) was analyzed by cytotoxic methods, the change of cell proliferation was assessed in real-time, and the effect of GPs on a living organism was evaluated in an animal model using histopathological examination. We analyzed two types of GP administration: intratracheal and peroral. We found dose- and time-dependent cytotoxic effects of GPs in vitro; the concentration above 50 µg/mL increased the cytotoxicity significantly. The real-time analysis confirmed these data; the cells exposed to a high concentration of GPs for a longer time period resulted in a decrease in cell index which indicated lower cell viability. Histopathological examination revealed thickened alveolar septa and accumulation of GPs in the endocardium after intratracheal exposure. Peroral administration did not reveal any morphological changes. This study showed the dose- and time-dependent cytotoxic potential of graphene nanoplatelets in in vitro and in vivo models.

Chromosomal aberrations in environmentally exposed population in relation to metabolic and DNA repair genes polymorphisms.

The capital city of Prague is one of the most polluted localities of the Czech Republic. Therefore, the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed onto respirable air particles (<2.5mum) on chromosomal aberrations was studied in a group of policemen (males, aged 22-50 years) working in the downtown area of Prague and spending daily >8h outdoors (N=53). Age- and sex-matched healthy volunteers spending >90% daily time indoors were chosen as controls (N=52). Ambient air particles (PM10, PM2.5) and c-PAHs were monitored using versatile air pollution sampler (VAPS), and personal exposure was evaluated using personal samplers during working shift. Chromosomal aberrations were analyzed by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH). Urinary cotinine plasma levels of vitamins A, E and C, folate, total cholesterol, HDL, LDL cholesterols and triglycerides were also analyzed as possible effect modifiers. Genotypes CYP1A1*2A, CYP1A1*2C, GSTM1, GSTP1, GSTT1, EPHX1, NAT2, hOGG1, XRCC1, XPD, p53 BstI, p53 MspI, MTHFR677, and MS2656 were determined by PCR-based RFLP assays. The following levels of air pollution were recorded during the study period (mean from HiVol sampling): PM10 62.6microg/m(3), c-PAHs 24.7ng/m(3), B[a]P 3.50ng/m(3). The conventional cytogenetic analysis did not reveal any differences between the group of policemen exposed to the ambient air pollution and the control group. The cytogenetic analysis by FISH analysis used the whole chromosome painting probes for chromosomes #1 and #4 (Cambio, UK). It detected a significant increase in all studied endpoints in the policemen compared to controls (% AB.C.=0.33+/-0.25 versus 0.24+/-0.18, p<0.05, F(G)/100=1.72+/-1.57 versus 1.25+/-1.11, p<0.05, AB/1000 (aberrations/1000 cells)=5.58+/-4.62 versus 3.90+/-3.06, p<0.05). CYP1A1*2C (Ile/Ile), XPD 23 (Lys/Lys), and XPD 6 (CC) genotypes were associated with an increase of aberrant cells by conventional method. Factors associated with an increased level of translocations by FISH included age, smoking, B[a]P-like DNA adducts (corresponding to the exposure of c-PAHs), folate, polymorphisms of CYP1A1*2C, GSTP1, EPHX1, p53 MspI and MTHFR. Ambient air exposure to c-PAHs significantly increased FISH cytogenetic parameters in nonsmoking policemen. We may conclude that FISH indicates that the city policemen in Prague represent a group of increased genotoxic risk. This is the first study that has reported a relationship between DNA adducts (biomarker of exposure) and chromosomal aberrations by FISH (biomarker of effect).

Human biomonitoring system in the Czech Republic.

The human biomonitoring (HBM) is an integral part of Environmental Health Monitoring System in the Czech Republic since 1994. Selected biomarkers of the internal dose (heavy metals, PCBs) and cytogenetic analysis of peripheral lymphocytes as a biomarker of the exposure/effect to/of environmental genotoxic factors are systematically followed up in the blood and urine of adults (blood donors), in children aged 8 to 10 years, and in the breast milk of nursing mothers. Selected outputs documented the declining trend of blood lead levels, with the recent reference value of 80 mg/l for men, and the rising trend of blood selenium levels in adults, but not in children. PCBs and chlorinated pesticides in human milk show a long-term downward trend, but still higher than in neighbouring countries. The frequency of aberrant cells revealed a downward trend, but the increase obtained in the last monitored period needs to be explained. Further HBM activities are required to demonstrate the corresponding trends and to reduce human exposure and health risks.

Chromosomal aberration frequencies determined by conventional methods: Parallel increases over time in the region of a petrochemical industry and throughout the Czech Republic.

The rationale for cytogenetic monitoring to determine if safe maximum allowable concentrations (MAC) of genotoxic chemicals are being maintained in a workplace is that exposure levels that do not increase chromosomal aberration frequencies are without harmful effects. Such monitoring, widely used in occupational health programs in the Czech Republic (CR), includes workers exposed to 1,3-butadiene (BD) or other chemicals. Studies of BD exposed workers in the years 1992, 1993, 1994, 1998, and 2004 compared mean frequencies of cells carrying chromosomal aberrations (frequency of aberrant cells=%AB.C.) in exposed workers with those in non-exposed matched controls in the same plant or in other individuals living in the region of the same petrochemical industry. Workers potentially exposed to acrylonitrile at this site were also evaluated in 2000, along with another unexposed matched control group. The %AB.C. values of exposed workers and their controls were also compared with reference values determined for normal individuals (ages 20-59 years) throughout the CR. Substantial discrepancies were noted between subjects in the region of the petrochemical industry (exposed workers and controls) for the years 2000 and 2004 and the reference CR-wide normal values that had been determined during an earlier time period. The matched non-exposed controls at the petrochemical industry site showed a mean %AB.C. value of 1.56+/-1.23% (N=25) in 1998; this rose to a mean of 2.65+/-2.29% (N=33) in 2000. In 2004, values for non-exposed matched controls at the industry site were 2.64+/-1.75% for males (N=25) and 2.38+/-1.74% (N=26) for females. However, the earlier determined CR-wide %AB.C. mean reference values for normal individuals were 1.77+/-1.16% (N=1305) for the interval 1977-1988 and 1.45+/-1.17% (N=2140) for the interval 1991-1999. As both reference values are substantially lower than those determined in 2000 and 2004 for the non-exposed matched controls at the petrochemical industry site, an analysis of the CR-wide mean normal individual reference values for this same 2000-2004 period was conducted. Unexpectedly, it was found that this reference value too had risen to 1.95+/-1.36% (N=1045) and was comparable to the concurrent matched control values at the petrochemical industry site where the monitoring studies were conducted. This substantial increase in %AB.C. values in 2000 and 2004, therefore, has occurred throughout the CR and is probably unrelated to chemicals uniquely present at the petrochemical industry site.

Possible genetic damage in the Czech nuclear power plant workers.

The aim of our study was to identify occupational risk of irradiation exposure in the Czech nuclear power plant workers. We analyzed levels of chromosomal aberrations, a well-known biomarker of early biological effects and a predictor of cancer risk. We applied the conventional method of cytogenetic analysis and fluorescence in situ hybridization (FISH, whole chromosome painting for chromosomes 1 and 4, combined with a pancentromeric probe) to three groups: 123 subjects in the Temelin nuclear power plant (2 years in use), 114 subjects in the Dukovany nuclear power plant (20 years in use), and 53 matched controls from Ceske Budejovice. Nuclear power plant workers were divided into two groups: subjects with admittance into the monitored zone, and others. Following factors were also analyzed: GSTM1, GSTT1, GSTP1, XPD, XRCC1, hOGG1, p53, MTHFR, and MS gene polymorphisms, levels of vitamins A, C, E, and folate in plasma, and level of cotinine in urine. Long-term exposure to ionizing radiation in the monitored zone was 0.47+/-1.50 mSv (miliSievert) in the Temelin nuclear power plant and 5.74+/-9.57 mSv in the Dukovany nuclear power plant. Using the conventional cytogenetic analysis, we observed 1.90+/-0.95 and 1.82+/-1.19% AB.C. (percent of aberrant cells) in the Temelin nuclear power plant, and 2.39+/-1.01 and 2.33+/-1.04% AB.C. in the Dukovany nuclear power plant, for monitored zone workers and others, respectively. In the control group, we found 2.25+/-0.82% AB.C. Genomic frequency of translocations F(G)/100 measured by FISH was 1.89+/-1.40 and 2.01+/-1.68 in the Temelin nuclear power plant, and 2.48+/-1.93 and 2.14+/-1.62 in the Dukovany nuclear power plant for monitored zone workers and others, respectively. In the control group, F(G)/100 was 1.83+/-1.19. Following factors were identified as potential confounders by the conventional cytogenetic analysis: XPD-6, by the FISH: age, GSTP1 and p53Bst genotypes, long-term use of medication, alcohol consumption, and smoking. No association between the dose of irradiation and the level of chromosomal aberrations in any nuclear power plant was detected either by the conventional cytogenetic analysis or by FISH.