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Abstract: Objectives: Maternal smoking has a negative effect on all stages of pregnancy. Tobacco smoke-related defects are well established at the clinical level; however, less is known about molecular mechanisms underlying these pathologic conditions. We thus performed a comprehensive analysis of transcriptome alterations induced by smoking in maternal and fetal cells. Study design: Samples of peripheral blood (PB), placenta (PL), and cord blood (UCB) were obtained from pregnant smokers (n = 20) and gravidas without significant exposure to tobacco smoke (n = 52). Gene expression profiles were assayed by Illumina Expression Beadchip v3 for analysis of 24,526 transcripts. The quantile method was used for normalization. Differentially expressed genes were analyzed in the Limma package and the P-values were corrected for multiple testing. Unsupervised hierarchical clustering was performed using average linkage and Euclidean distance. The enrichment of deregulated genes in biological processes was analyzed in DAVID database. Results: Comparative analyses defined significant deregulation of 193 genes in PB, 329 genes in PL, and 49 genes in UCB of smokers. The deregulated genes were mainly related to xenobiotic metabolism, oxidative stress, inflammation, immunity, hematopoiesis, and vascularization. Notably, functional annotation of the affected genes identified several deregulated pathways associated with autoimmune diseases in the newborns of smokers. Conclusions: The study demonstrated maternal smoking causes significant changes in transcriptome of placental and fetal cells that deregulate numerous biological processes important for growth and development of the fetus. An activation of fetal CYP genes showed a limited ability of the placenta to modulate toxic effects of maternal tobacco use. [Copyright &y& Elsevier]

Abstract: Smoking in pregnancy increases a woman''s risk of preterm delivery resulting in serious neonatal health problems and chronic lifelong disabilities for the children (e.g., mental retardation, learning problems). To study the effects of tobacco smoke on the placental transcriptome, we performed gene expression profiling on placentas from women exposed to tobacco smoke in pregnancy (N = 12) and from those without significant exposure (N = 64). Gene expression profiles were determined by Illumina HumanRef-8 v2 Expression BeadChips with 18,216 gene probes. Microarray data were normalized by quantile method and filtered for a detection P-value <0.01. Differential gene expression was determined by moderated t-statistic. A linear model was fitted for each gene given a series of arrays using lmFit function. Multiple testing correction was performed using the Benjamini and Hochberg method. Abundant levels of transcripts were found for genes encoding placental hormones (CSH1, CSHL1), pregnancy-specific proteins (PSG3, PSG4, PAPPA), and hemoglobins (HBB, HBG, HBA). Comparative analysis of smokers vs nonsmokers revealed the differential expression of 241 genes (P < 0.05). In smoker cohort, we detected high up-regulation of xenobiotic genes (CYP1A1, CYP1B1, CYB5A, COX412), collagen genes (e.g., COL6A3, COL1A1, COL1A2), coagulation genes (F5, F13A1) as well as thrombosis-related genes (CD36, ADAMTS9, GAS6). In smokers, we identified deregulated genes that show tissue non-specific induction and may be considered as general biomarkers of tobacco smoke exposure. Further, we also found genes specifically deregulated in the exposed placentas. Functional annotation analysis suggested processes and pathways affected by tobacco smoke exposure that may represent molecular mechanisms of smoke-induced placental abnormalities. [Copyright &y& Elsevier]

Little is known about the impact of genetic variation on the genetic damage induced by urban air pollution or environmental tobacco smoke (ETS) in exposed populations. The levels of bulky DNA adducts (32P-postlabelling, nuclease P1 enrichment) and chromosomal aberrations were measured in lymphocytes of 194 non-smoking students living in the city of Athens, and the rural region of Halkida, Greece. In these individuals personal exposure to PAH was also measured. Furthermore, genetic polymorphisms were examined in cytochromes P450 1A1, 1B1, in the GSTM1, GSTP1 and GSTT1 as well as in microsomal epoxy hydrolase (EPHX) genes. Subjects with the CYP1&ast;2A mutant genotype also suffering significant ETS exposure tended to exhibit higher adduct levels and % aberrant cells. In contrast, CYP1B1 polymorphisms seemed to have an impact on the DNA adduct levels only among individual with negligible ETS exposure. Subjects carrying both the CYP1&ast;2A mutant genotype and the GSTM1 null genotype tended to have higher DNA adduct levels. A similar effect was also observed with the combined CYP1A1&ast;2A/GSTP1 (Ile/Val) and the CYP1A1&ast;2A/mEH “slow” polymorphisms. In both cases, the effect was more pronounced among subjects with higher levels of ETS exposure. Stepwise restriction of the observations to subjects characterised by (a) GSTP1 mutant, (b) GSTM1 null, (c) mEH “slow” (His139His) genotypes and (d) ETS exposure resulted in a significant trend of increasing DNA adduct levels only among individuals with at least one CYP1A1&ast;2A mutated allele, illustrating the importance and complexity of gene-exposure and gene–gene interactions in determining the level of genotoxic damage on an individual levels. [Copyright &y& Elsevier]

DNA damage can impair normal cellular functions and result in various pathophysiological processes including cardiovascular diseases and cancer. We compared the genotoxic potential of diverse DNA damaging agents, and focused on their effects on the DNA damage response (DDR) and cell fate in human lung cells BEAS-2B. Polycyclic aromatic hydrocarbons [PAHs; benzo[ a ]pyrene (B[ a ]P), 1-nitropyrene (1-NP)] induced DNA strand breaks and oxidative damage to DNA; anticancer drugs doxorubicin (DOX) and 5-bromo-2′-deoxyuridine (BrdU) were less effective. DOX triggered the most robust p53 signaling indicating activation of DDR, followed by cell cycle arrest in the G2/M phase, induction of apoptosis and senescence, possibly due to the severe and irreparable DNA lesions. BrdU not only activated p53, but also increased the percentage of G1-phased cells and caused a massive accumulation of senescent cells. In contrast, regardless the activation of p53, both PAHs did not substantially affect the cell cycle distribution or senescence. Finally, a small fraction of cells accumulated only in the G2/M phase and exhibited increased cell death after the prolonged incubation with B[ a ]P. Overall, we characterized differential responses to diverse DNA damaging agents resulting in specific cell fate and highlighted the key role of DNA lesion type and the p53 signaling persistence. • PAHs induced considerably higher level of DNA damage than DOX and BrdU. • DOX treatment was more toxic and led to G2 arrest, senescence and apoptosis. • BrdU triggered senescence possibly due to an alternative mechanism. • Despite slight increase of apoptosis, PAHs displayed better survival of cells. [ABSTRACT FROM AUTHOR]

Abstract TiO 2 along with nano-TiO 2 are commonly found in consumer products. In vivo studies have observed an accumulation of nano-TiO 2 in macrophages. However, characteristics of nano-TiO 2 determining toxicity remain unclear. In our study, the cytotoxic effects of 14 diverse nano-TiO 2 on THP-1 macrophage-like cells were measured by 3 cytotoxicity assays (MTS, WST-1 and LDH). Total averaged cytotoxicity was calculated using principal component analysis. Characteristics of all 14 nano-TiO 2 included hydrodynamic diameter, zeta potential, shape, polydispersity index (PDI) and concentration; moreover, crystal form, specific surface area and crystallite size were measured for 10 nano-TiO 2. The variables affecting cytotoxicity were chosen using LASSO (least absolute shrinkage and selection operator). Except for concentration, PDI in media measured within 1 h after preparation of the nanomaterial dispersion was selected as a variable affecting cytotoxicity: stable dispersion resulted in higher cytotoxic effects. Crystallite size has been shown to have nonlinear effects (particles of sizes between 20 and 60 nm were cytotoxic while smaller and larger ones were not) and thus it has been excluded from LASSO. The shape (particles/fibre) and crystal form did not affect the cytotoxicity. PDI and the nonlinear effect of size could be an explanation for the inconsistencies of the cytotoxicity of nano-TiO 2 in various studies. Graphical abstract Unlabelled Image Highlights • Physical factors affecting cytotoxicity of nano-TiO 2 in THP-1 cells were studied • Concentration and polydispersity index measured within 1 h affected cytotoxicity • Particles of 20–60 nm were cytotoxic while smaller and larger ones were not • No significant difference between anatase/rutile crystallinity was found • Overall cytotoxic effects of nano-TiO 2 in THP-1 macrophage-like cells were low [ABSTRACT FROM AUTHOR]

Abstract: Methylated chrysenes (MeChry) are important cigarette smoke constituents and 5-MeChry has been listed as possibly carcinogenic to humans. Although a major attention has been in past paid especially to mutagenic, tumor-initiating effects of MeChry, little is known about toxic effects of MeChry related to tumor promotion. As the position of methyl group has been repeatedly observed to determine genotoxic effects of MeChry, we examined both genotoxic and nongenotoxic effects of MeChry, using rat liver cell lines as experimental models. All six MeChry were relatively efficient aryl hydrocarbon receptor (AhR) agonists, with 3- and 6-MeChry being the most potent inducers of the AhR-mediated reporter gene activity. All six compounds disrupted contact inhibition in rat liver epithelial WB-F344 cells, a process previously reported to be AhR-dependent, suggesting that MeChry may interfere with cell cycle control in an AhR-dependent manner. In contrast, only 5- and 6-MeChry were found to acutely inhibit gap junctional intercellular communication (GJIC), another parameter correlating with tumor promoting effects of xenobiotics. Both 5- and 6-MeChry were efficient inducers of mRNA expression of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons, including cytochromes P450 1A1/1B1 and aldo-keto reductase 1C9. However, only 5-MeChry, and not 6-MeChry, induced significant formation of DNA adducts in rat liver epithelial cells, which corresponded with its ability to induce high accumulation of cells in S-phase. On the other hand, 5-MeChry induced neither apoptosis related to DNA damage nor phosphorylation of p53 tumor suppressor. Taken together, our results suggest that methyl group position may affect both genotoxic and nongenotoxic effects of MeChry, such as formation of DNA adducts and inhibition of GJIC. All MeChry showed a potency to disrupt cell proliferation control, while 5-MeChry was a single compound inducing DNA damage, disruption of cell cycle control and inhibition of GJIC in rat liver cells. [Copyright &y& Elsevier]

Abstract: Polycyclic aromatic hydrocarbons (PAHs) with molecular weight 278 are a group of PAHs that are mostly not covered by the current monitoring programs, despite their relative abundance in environmental samples and possible carcinogenicity. Although benzo[g]chrysene (BgChry) and dibenz[a,h]anthracene (DBahA) have been for a long time studied as genotoxic, tumour-initiating compounds, little is known about the potential tumour-promoting effects of this group of PAHs. In the present study, we investigated their impact on activation of the aryl hydrocarbon receptor (AhR), induction of enzymes involved in metabolic activation of PAHs, disruption of cell cycle control in confluent cell population and inhibition of gap junctional intercellular communication (GJIC), using the rat liver epithelial cell line WB-F344 as a model of liver progenitor cells. We found that BgChry was the weakest inducer of the AhR-mediated activity, while relative potencies of benzo[b]chrysene (BbChry) and benzo[c]chrysene (BcChry) were comparable to the previously reported values for dibenzanthracenes. All compounds increased expression of cytochromes P450 1A1 and 1B1, and aldo-keto reductase 1C9. BgChry was found to induce high amounts of DNA adducts, which corresponded with induction of p53 phosphorylation at Ser15, apoptosis and accumulation of cells in S-phase of cell cycle, leading to a decrease in cell numbers. All other compounds were found to stimulate cell proliferation in contact-inhibited WB-F344 cells in a dose-dependent manner. We found that only BgChry, and to a lesser extent also BcChry, inhibited GJIC at high concentrations. Taken together, dibenzanthracenes and benzochrysenes, with exception of BgChry, seem to act primarily through deregulation of cell proliferation in liver epithelial cells, which is related to their relatively high AhR-mediated activity. The disruption of cell cycle control might contribute to their carcinogenic effects, as well as to carcinogenicity of complex environmental mixtures containing high levels of PAHs with molecular weight 278. [Copyright &y& Elsevier]