Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM₁₀ (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination.

No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample.

Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m³), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice  Prague < Sofia.

Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.     

Oxidative and nitrosative stress markers in bus drivers

Exposure to ambient air pollution is associated with many diseases. Oxidative and nitrosative stress are believed to be two of the major sources of particulate matter (PM)-mediated adverse health effects. PM in ambient air arises from industry, local heating, and vehicle emissions and poses a serious problem mainly in large cities. In the present study we analyzed the level of oxidative and nitrosative stress among 50 bus drivers from Prague, Czech Republic, and 50 matching controls. We assessed simultaneously the levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP) and 8-oxodeoxyguanosine (8-oxodG) in urine and protein carbonyl groups and 3-nitrotyrosine (NT) in blood plasma. For the analysis of all four markers we used ELISA techniques. We observed significantly increased levels of oxidative and nitrosative stress markers in bus drivers. The median levels (min, max) of individual markers in bus drivers versus controls were as follows: 8-oxodG: 7.79 (2.64-12.34)nmol/mmol versus 6.12 (0.70-11.38)nmol/mmol creatinine (p<0.01); 15-F(2t)-IsoP: 0.81 (0.38-1.55)nmol/mmol versus 0.68 (0.39-1.79)nmol/mmol creatinine (p<0.01); carbonyl levels: 14.1 (11.8-19.0)nmol/ml versus 12.9 (9.8-16.6)nmol/ml plasma (p<0.001); NT: 694 (471-3228)nmol/l versus 537 (268-13833)nmol/l plasma (p<0.001). 15-F(2t)-IsoP levels correlated with vitamin E (R=0.23, p<0.05), vitamin C (R=-0.33, p<0.01) and cotinine (R=0.47, p<0.001) levels. Vitamin E levels also positively correlated with 8-oxodG (R=0.27, p=0.01) and protein carbonyl levels (R=0.32, p<0.001). Both oxidative and nitrosative stress markers positively correlated with PM2.5 and PM10 exposure. In conclusion, our study indicates that exposure to PM2.5 and PM10 results in increased oxidative and nitrosative stress.     

Targeting of gene expression by siRNA in CML primary cells

Development of array methods contributes to elucidation of many genes expressed during oncogenesis. Our array-based analyses of gene expression in patients with chronic myeloid leukemia (CML) revealed several genes (MMP8, MMP9, PCNA, JNK2, MAPK p38) with significant increased expression. We suppose that the genes may be implicated in the disease development and a siRNA-suppression can elucidate their functions in leukemogenesis. One of the crucial requirements for this purpose is a high efficiency of siRNA delivery into CML primary cells. Using fluorescein-labeled siRNAs we systematically tested a variety of physical and chemical non-vector based transfection methods in order to evaluate which of them gave the most suitable transfer. Chemically synthesized siRNAs against mentioned genes were transfected into the cells and level of knockdown was determined by real time RT-PCR. Chemical transfection reagents (Oligofectamine, Metafectene, siPORT Amine) commonly used to transfect siRNAs in CML cell lines showed very low siRNA delivery in CML primary cells—mRNA levels decreased at the most to 76%. Electroporation achieved better results (suppression to 63%) but it was associated with high degree of cell death (more than 60%). In the study we obtained the best transfection efficiency using nucleofector technology. Gene expressions ranged 22–37% that remained from original levels. According to our results, nucleofection appears to be the only suitable non-viral method for siRNA delivery into the hard-to-transfect CML primary cells.     

Tracing past human male movements in northern/eastern Africa and western Eurasia: new clues from Y-chromosomal haplogroups E-M78 and J-M12

Detailed population data were obtained on the distribution of novel biallelic markers that finely dissect the human Y-chromosome haplogroup E-M78. Among 6,501 Y chromosomes sampled in 81 human populations worldwide, we found 517 E-M78 chromosomes and assigned them to 10 subhaplogroups. Eleven microsatellite loci were used to further evaluate subhaplogroup internal diversification. The geographic and quantitative analyses of haplogroup and microsatellite diversity is strongly suggestive of a northeastern African origin of E-M78, with a corridor for bidirectional migrations between northeastern and eastern Africa (at least 2 episodes between 23.9-17.3 ky and 18.0-5.9 ky ago), trans-Mediterranean migrations directly from northern Africa to Europe (mainly in the last 13.0 ky), and flow from northeastern Africa to western Asia between 20.0 and 6.8 ky ago. A single clade within E-M78 (E-V13) highlights a range expansion in the Bronze Age of southeastern Europe, which is also detected by haplogroup J-M12. Phylogeography pattern of molecular radiation and coalescence estimates for both haplogroups are similar and reveal that the genetic landscape of this region is, to a large extent, the consequence of a recent population growth in situ rather than the result of a mere flow of western Asian migrants in the early Neolithic. Our results not only provide a refinement of previous evolutionary hypotheses but also well-defined time frames for past human movements both in northern/eastern Africa and western Eurasia.     

The crystal structure of the phosphatidylinositol 4‐kinase IIα

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4‐kinase type IIα (PI4K IIα), in complex with ATP solved by X‐ray crystallography at 2.8 Å resolution. The structure revealed a non‐typical kinase fold that could be divided into N‐ and C‐lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C‐lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.     

Seasonal variability of oxidative stress markers in city bus drivers. Part II. Oxidative damage to lipids and proteins

The aim of the present study was to investigate the seasonal variability of markers of oxidative damage to lipids (15-F2t-isoprostane, 15-F2t-IsoP) and proteins (protein carbonyl levels) in 50 bus drivers and 50 controls from Prague, Czech Republic, and to identify factors affecting oxidative stress markers. The samples were collected in three seasons with different levels of air pollution. The exposure to environmental pollutants (carcinogenic polycyclic aromatic hydrocarbons, c-PAHs, particulate matter, PM2.5 and PM10, and volatile organic compounds, VOC) was monitored by personal and/or stationary monitors. For the analysis of both markers, ELISA techniques were used. The median levels of individual markers in bus drivers versus controls were as follows: 15-F2t-IsoP (nmol/mmol creatinine): winter 2005, 0.81 versus 0.68 (p<0.01); summer 2006, 0.62 versus 0.60 (p=0.90); winter 2006, 0.76 versus 0.51 (p<0.001); carbonyl levels (nmol/ml plasma): winter 2005, 14.1 versus 12.9 (p=0.001); summer 2006, 17.5 versus 16.6 (p=0.26); winter 2006, 13.5 versus 11.7 (p<0.001). Multivariate logistic regression identified PM levels measured by stationary monitors over a period 25-27 days before urine collection as a factor positively associated with lipid peroxidation, while protein oxidation levels correlated negatively with both c-PAHs and PM levels. In conclusion, markers of oxidative damage to lipids and proteins were increased in bus drivers in winter seasons, but not in summer. Lipid peroxidation was positively correlated with c-PAHs and PM exposure; protein oxidation correlated negatively and was highest in summer suggesting another factor(s) affecting protein carbonyl levels.     

Community assembly in Nothobranchius annual fishes: Nested patterns,environmental niche and biogeographic history

The assembly of local communities from regional species pools is shaped by historical aspects of distribution, environmental conditions, and biotic interactions. We studied local community assembly patterns in African annual killifishes of the genus Nothobranchius (Cyprinodontiformes), investigating data from 168 communities across the entire range of regionally co‐existing species. Nothobranchius are small fishes associated with annually desiccating pools. We detected a nested pattern of local communities in one region (Southern Mozambique, with Nothobranchius furzeri as the core and dominant species), but no nestedness was found in the second region (Central Mozambique, with Nothobranchius orthonotus being the dominant species). A checkerboard pattern of local Nothobranchius community assembly was demonstrated in both regions. Multivariate environmental niche modeling revealed moderate differences in environmental niche occupancy between three monophyletic clades that largely co‐occurred geographically and greater differences between strictly allopatric species within the clades. Most variation among species was observed along an altitudinal gradient; N. furzeri and Nothobranchius kadleci were absent from coastal plains, Nothobranchius pienaari, Nothobranchius rachovii, and Nothobranchius krysanovi were associated with lower altitude and N. orthonotus was intermediate and geographically most widespread species. We discuss implications for ecological and evolutionary research in this taxon.     

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.