Biomarkers of exposure and effect—interpretation in human risk assessment

The effect of exposure to carcinogenic polycyclic aromatic hydrocarbons adsorbed onto respirable air particles (PM2.5, diameter < 2.5 μm) on DNA adducts and chromosomal aberrations was repeatedly studied in Prague, Czech Republic, in groups of policemen working in the downtown area and in bus drivers. Personal exposure was evaluated using personal samplers during working shifts. DNA adducts were analyzed in lymphocytes by the 32P-postlabeling assay and chromosomal aberrations were analyzed by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH). The impact of environmental pollution on DNA adducts and chromosomal aberrations was studied in a total of 950 subjects. Our results suggest that the environmental exposure of nonsmokers to concentrations higher than 1 ng benzo[a]pyrene/m3 represents a risk of DNA damage, as indicated by an increase in DNA adducts and the genomic frequency of translocations determined by FISH

Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 &lt; NM103 and NM302 &lt; NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties

Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (-S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (-S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced

Western blotting analyses of the levels of XPE, XPC and XPA proteins.

<p>Protein expression was measured after the 24 h treatment of HEL12469 cells with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) in the absence (–S9) and presence (+S9) of the microsomal S9 fraction. A representative result of two independent experiments is shown. Amido Black-stained proteins were used as a loading control.</p

PAH content in extractable organic matter (EOM) from individual samplings.

*<p>Significant differences (p<0.001) in PAH concentrations between EOMs from Prague-summer vs. Ostrava-summer and Prague-winter vs. Ostrava-winter.</p

Bulky DNA adduct levels/10⁸ nucleotides detected in DNA extracted from HEL12469 cells.

<p>The cells were treated for 24 h with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) in the absence (–S9) and presence (+S9) of the microsomal S9 fraction. P–W = Prague-winter, O–W = Ostrava-winter, P–S = Prague-summer, O–S = Ostrava-summer, N.D. – not detectable.</p

Final concentration of benzo[a]pyrene (B[a]P) in culture media after treatment of the cells with various concentrations of EOM.

<p>Final concentration of benzo[a]pyrene (B[a]P) in culture media after treatment of the cells with various concentrations of EOM.</p