In this review we discuss the effects of exposure to complex PAH mixtures in vitro and in vivo on mechanisms related to carcinogenesis. Of particular concern regarding exposure to complex PAH mixtures is how interactions between different constituents can affect the carcinogenic response and how these might be included in risk assessment. Overall the findings suggest that the responses resulting from exposure to complex PAH mixtures is varied and complicated. More- and less-than additive effects on bioactivation and DNA damage formation have been observed depending on the various mixtures studied, and equally dependent on the different test systems that are used. Furthermore, the findings show that the commonly used biological end-point of DNA damage formation is insufficient for studying mixture effects. At present the assessment of the risk of exposure to complex PAH mixtures involves comparison to individual compounds using either a surrogate or a component-based potency approach. We discuss how future risk assessment strategies for complex PAH mixtures should be based around whole mixture assessment in order to account for interaction effects. Inherent to this is the need to incorporate different experimental approaches using robust and sensitive biological endpoints. Furthermore, the emphasis on future research should be placed on studying real life mixtures that better represent the complex PAH mixtures that humans are exposed to.

The widespread production and use of nanoparticles calls for faster and more reliable methods to assess their safety. The main aim of this study was to investigate the genotoxicity of three reference TiO2 nanomaterials (NM) within the frame of the FP7-NANoREG project, with a particular focus on testing the applicability of mini-gel comet assay and micronucleus (MN) scoring by flow cytometry. BEAS-2B cells cultured under serum-free conditions were exposed to NM100 (anatase, 50–150nm), NM101 (anatase, 5–8nm) and NM103 (rutile, 20–28nm) for 3, 24 or 48h mainly at concentrations 1–30 μg/ml. In the mini-gel comet assay (eight gels per slide), we included analysis of (i) DNA strand breaks, (ii) oxidised bases (Fpg-sensitive sites) and (iii) light-induced DNA damage due to photocatalytic activity. Furthermore, MN assays were used and we compared the results of more high-throughput MN scoring with flow cytometry to that of cytokinesis-block MN cytome assay scored manually using a microscope. Various methods were used to assess cytotoxic effects and the results showed in general no or low effects at the doses tested. A weak genotoxic effect of the tested TiO2 materials was observed with an induction of oxidised bases for all three materials of which NM100 was the most potent. When the comet slides were briefly exposed to lab light, a clear induction of DNA strand breaks was observed for the anatase materials, but not for the rutile. This highlights the risk of false positives when testing photocatalytically active materials if light is not properly avoided. A slight increase in MN formation for NM103 was observed in the different MN assays at the lower doses tested (1 and 5 μg/ml). We conclude that mini-gel comet assay and MN scoring using flow cytometry successfully can be used to efficiently study cytotoxic and genotoxic properties of nanoparticles.

The polycyclic aromatic hydrocarbons (PAHs) dibenzo[a,l]pyrene (DBP) and benzo[a]pyrene (BP) are widespread environmental contaminants and potent carcinogens. The fjord-region DBP is considerably more carcinogenic than the bay-region BP. This fact can be ascribed to differences in DNA binding efficiency of their ultimate carcinogenic diol epoxide (DE) intermediates, differences in structural features of the DNA adducts, and differences in DNA adduct recognition and the subsequent lesion removal by nucleotide excision repair (NER). We have compared the formation and removal of adducts as a function of time formed by the carcinogenic metabolites (-)-anti-DBPDE and (+)-anti-BPDE in A549 human epithelial lung carcinoma cells. Cells were exposed to 0.1 or 1.0 microM (-)-anti-DBPDE and (+)-anti-BPDE, respectively. Adducts were measured at various post-treatment times (up to 6 h) by enzymatic DNA hydrolysis and a HPLC procedure that allows monitoring of all cis- and trans-nucleoside adducts of dA and dG. Treatment with 0.1 microM (-)-anti-DBPDE resulted in an initial increase of adducts to a maximal level of 144 pmol adducts/mg of DNA after 1 h of incubation. This was followed by an apparent, although not statistically significant, slow removal of adducts. After 6 h of incubation, at least 80% seems to remain. In cells treated with 1.0 microM (+)-anti-BPDE, the maximal level of 140 pmol adducts/mg of DNA was reached within 20 min of exposure. The formation was followed by an initial rapid decline in the adduct level (1.54 pmol adducts/mg of DNA/min) and a later statistically significant slower rate (0.14 pmol adducts/mg of DNA/min) of adduct removal. After 1 h of incubation, about 45% of the adducts are removed followed by 75% at 6 h. The biphasic pattern of BPDE removal has been observed previously in mammalian cells and, at least in part, may reflect the action of transcription-coupled repair (TCR) and the subsequent global genomic repair (GGR). Comparing the rate of removal of adducts derived from BPDE with those of DBPDE, the latter are obviously more refractory to the NER-coupled repair than the former. Furthermore, the apparent resistance of adducts from DBPDE to be eliminated may reflect the ability of such adducts to escape recognition and/or the subsequent removal by the NER machinery. Further analysis of DNA adduct distribution as a function of incubation time reveals that the dA/dG adduct ratio for BPDE was independent of time (4% dA, 96% dG), whereas the corresponding ratio for DBPDE was significantly increased from 2.9 (74% dA, 26% dG) at 20 min to 4.0 (80% dA, 20% dG) after 6 h of incubation. The results presented here on DNA adduct removal in mammalian cells are in part consistent with recent results on NER-coupled activity on bay- and fjord-region DE-modified oligonucleotides in vitro and further substantiate the hypothesis that the high carcinogenicity of the nonplanar PAHs arise from the ability of the preferentially formed dA adducts to escape recognition by surveillance systems and the subsequent NER...

Mammalian V79 cells stably expressing human glutathione transferase (GST) A1-1, M1-1, and P1-1 (the allelic variant with Val105 and Ala114) have been constructed and characterized. The cells have been used to study the capacity of individual GST isoenzymes in conjunction with GSH to detoxify diol epoxides from dibenzo[a,l]pyrene (DBPDE), the most carcinogenic polycyclic aromatic hydrocarbon (PAH) identified so far, and diol epoxides from benzo[a]pyrene (BPDE). The relationship between GSH-conjugation and DNA adduct-formation has been investigated as well as factors governing the accessibility of lipophilic diol epoxide substrates for the soluble GSTs in the cells. Relative to control cells, those expressing GSTA1-1 showed the highest rate (about 50-fold increase) to perform GSH-conjugation of (-)-anti-DBPDE (R-absolute configuration at the benzylic oxirane carbon in the fjord-region) followed by GSTM1-1 (25-fold increase) and GSTP1-1 (10-fold increase). GSTA1-1 was found to be strongly inhibited when expressed in cells (10% of fully functional protein). Taking this factor into account, the rates of conjugation found in the cells fairly well reflected the order of catalytic efficiencies (k(cat)/K(m)) obtained with the pure enzymes. Increased GSH conjugation of (-)-anti-DBPDE was associated with a reduction in DNA adduct formation. GSTA1-1 inhibited the formation of adducts more than 6-fold and GSTM1-1 and GSTP1-1 about 2-fold. With (+)-anti-BPDE, GSTP1-1-expressing cells demonstrated a substantially higher rate of GSH-conjugate formation than cells with GSTA1-1 and GSTM1-1 cells (33- and 10-fold increase, respectively). Relative to control cells, GSTM1-1 was found to inhibit DNA adduct formation of (+)-anti-BPDE most effectively followed by GSTP1-1 and GSTA1-1 (12-, 4-, and 3-fold, respectively). Values of k(cat)/K(m) and estimated oil/water partition coefficients of DBPDE and BPDE were used to calculate the concentration of free diol epoxides in solution and expected rates of GSH conjugate formation in cells, and these theoretical results were compared with the observed ones. With the highly reactive (+)-anti-BPDE, 1-2% of the expected activity was observed, whereas the corresponding values for the less reactive (-)-anti-DBPDE were up to 13%. The most obvious explanations for the low observed rate with (+)-anti-BPDE are rapid and competing reactions such as hydrolysis and/or more unspecific chemical and physical reactions with cellular constituents (proteins, lipids, nucleic acids, etc.). In addition, the difference between the theoretical and observed rates may also reflect participation of factors such as macromolecular crowding and reduced rates of diffusion, factors expected to further restrict the accessibility of GST and the diol epoxides in the intact cell.