Olena Beskid

International study of factors affecting human chromosome translocations

Chromosome translocations in peripheral blood lymphocytes of normal, healthy humans increase with age, but the effects of gender, race, and cigarette smoking on background translocation yields have not been examined systematically. Further, the shape of the relationship between age and translocation frequency (TF) has not been definitively determined. We collected existing data from 16 laboratories in North America, Europe, and Asia on TFs measured in peripheral blood lymphocytes by fluorescence in situ hybridization whole chromosome painting among 1933 individuals. In Poisson regression models, age, ranging from newborns (cord blood) to 85 years, was strongly associated with TF and this relationship showed significant upward curvature at older ages versus a linear relationship (p<0.001). Ever smokers had significantly higher TFs than non-smokers (rate ratio (RR)=1.19, 95% confidence interval (CI), 1.09-1.30) and smoking modified the effect of age on TFs with a steeper age-related increase among ever smokers compared to non-smokers (p<0.001). TFs did not differ by gender. Interpreting an independent effect of race was difficult owing to laboratory variation. Our study is three times larger than any pooled effort to date, confirming a suspected curvilinear relationship of TF with age. The significant effect of cigarette smoking has not been observed with previous pooled studies of TF in humans. Our data provide stable estimates of background TF by age, gender, race, and smoking status and suggest an acceleration of chromosome damage above age 60 and among those with a history of smoking cigarettes.

Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24h with the following concentrations of tested chemicals: B[a]P: 1μM, 10μM, 25μM; EOMs: 1μg/ml, 10μg/ml, 25μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and NHEJ after treatment of human embryonic lung fibroblasts with B[a]P and complex mixtures containing PAHs.

Utilization of organic nitrogen by arbuscular mycorrhizal fungi—is there a specific role for protists and ammonia oxidizers?

Arbuscular mycorrhizal (AM) fungi can significantly contribute to plant nitrogen (N) uptake from complex organic sources, most likely in concert with activity of soil saprotrophs and other microbes releasing and transforming the N bound in organic forms. Here, we tested whether AM fungus (Rhizophagus irregularis) extraradical hyphal networks showed any preferences towards certain forms of organic N (chitin of fungal or crustacean origin, DNA, clover biomass, or albumin) administered in spatially discrete patches, and how the presence of AM fungal hyphae affected other microbes. By direct 15N labeling, we also quantified the flux of N to the plants (Andropogon gerardii) through the AM fungal hyphae from fungal chitin and from clover biomass. The AM fungal hyphae colonized patches supplemented with organic N sources significantly more than those receiving only mineral nutrients, organic carbon in form of cellulose, or nothing. Mycorrhizal plants grew 6.4-fold larger and accumulated, on average, 20.3-fold more 15N originating from the labeled organic sources than their nonmycorrhizal counterparts. Whereas the abundance of microbes (bacteria, fungi, or Acanthamoeba sp.) in the different patches was primarily driven by patch quality, we noted a consistent suppression of the microbial abundances by the presence of AM fungal hyphae. This suppression was particularly strong for ammonia oxidizing bacteria. Our results indicate that AM fungi successfully competed with the other microbes for free ammonium ions and suggest an important role for the notoriously understudied soil protists to play in recycling organic N from soil to plants via AM fungal hyphae.

Mycorrhizal symbiosis induces plant carbon reallocation differently in C 3 and C 4 Panicum grasses

AimsAlthough arbuscular mycorrhizal symbiosis is common in many plants with either C3 or C4 photosynthesis, it remains poorly understood whether photosynthesis type has any significant impact on carbon (C) fluxes in mycorrhizal plants. Thus, we compared mycorrhizal and non-mycorrhizal (NM) plants belonging to Panicum bisulcatum (C3) to its congeneric P. maximum (C4).MethodsPlants were or were not exposed to arbuscular mycorrhiza (AM) fungal inoculation and/or phosphorus (P) fertilization. Plants’ C budgets were assembled based on 13CO2 pulse-chase labelling and sequential harvesting.ResultsMycorrhizal plants allocated on average 3.9% more recently fixed C belowground than did their NM counterparts. At low P, mycorrhizal C3-Panicum plants allocated less C to aboveground respiration as compared to their respective NM controls. In contrast, mycorrhizal C4-Panicum increased the rates of photosynthesis and allocated more C to aboveground respiration than the respective NM controls. At high P, the differences were less prominent.ConclusionsWe demonstrated consistent differences in aboveground C allocation due to AM symbiosis formation in congeneric C3 and C4 grasses. Both grasses benefited from AM symbiosis in terms of improved P uptake (at least at low P). These results advocate a holistic (whole-plant) perspective in studying C fluxes in mycorrhizal plants.

Appropriate nonmycorrhizal controls in arbuscular mycorrhiza research: a microbiome perspective

Establishment of nonmycorrhizal controls is a “classic and recurrent theme” in mycorrhizal research. For decades, authors reported mycorrhizal plant growth/nutrition as compared to various nonmycorrhizal controls. In such studies, uncertainties remain about which nonmycorrhizal controls are most appropriate and, in particular, what effects the control inoculations have on substrate and root microbiomes. Here, different types of control and mycorrhizal inoculations were compared with respect to plant growth and nutrition, as well as the structure of root and substrate microbiomes, assessed by next-generation sequencing. We compared uninoculated (“absolute”) control to inoculation with blank pot culture lacking arbuscular mycorrhizal fungi, filtrate of that blank inoculum, and filtrate of complex pot-produced mycorrhizal inoculum. Those treatments were compared to a standard mycorrhizal treatment, where the previously sterilized substrate was inoculated with complex pot-produced inoculum containing Rhizophagus irregularis SYM5. Besides this, monoxenically produced inoculum of the same fungus was applied either alone or in combination with blank inoculum. The results indicate that the presence of mycorrhizal fungus always resulted in stimulation of Andropogon gerardii plant biomass as well as in elevated phosphorus content of the plants. The microbial (bacterial and fungal) communities developing in the differently inoculated treatments, however, differed substantially from each other and no control could be obtained comparable with the treatment inoculated with complex mycorrhizal inoculum. Soil microorganisms with significant biological competences that could potentially contribute to the effects of the various inoculants on the plants were detected in roots and in plant cultivation substrate in some of the treatments.

Little Cross-Feeding of the Mycorrhizal Networks Shared Between C3-Panicum bisulcatum and C4-Panicum maximum Under Different Temperature Regimes

Common mycorrhizal networks (CMNs) formed by arbuscular mycorrhizal fungi (AMF) interconnect plants of the same and/or different species, redistributing nutrients and draining carbon (C) from the different plant partners at different rates. Here, we conducted a plant co-existence (intercropping) experiment testing the role of AMF in resource sharing and exploitation by simplified plant communities composed of two congeneric grass species (Panicum spp.) with different photosynthetic metabolism types (C3 or C4). The grasses had spatially separated rooting zones, conjoined through a root-free (but AMF-accessible) zone added with 15N-labeled plant (clover) residues. The plants were grown under two different temperature regimes: high temperature (36/32°C day/night) or ambient temperature (25/21°C day/night) applied over 49 days after an initial period of 26 days at ambient temperature. We made use of the distinct C-isotopic composition of the two plant species sharing the same CMN (composed of a synthetic AMF community of five fungal genera) to estimate if the CMN was or was not fed preferentially under the specific environmental conditions by one or the other plant species. Using the C-isotopic composition of AMF-specific fatty acid (C16:1ω5) in roots and in the potting substrate harboring the extraradical AMF hyphae, we found that the C3-Panicum continued feeding the CMN at both temperatures with a significant and invariable share of C resources. This was surprising because the growth of the C3 plants was more susceptible to high temperature than that of the C4 plants and the C3-Panicum alone suppressed abundance of the AMF (particularly Funneliformis sp.) in its roots due to the elevated temperature. Moreover, elevated temperature induced a shift in competition for nitrogen between the two plant species in favor of the C4-Panicum, as demonstrated by significantly lower 15N yields of the C3-Panicum but higher 15N yields of the C4-Panicum at elevated as compared to ambient temperature. Although the development of CMN (particularly of the dominant Rhizophagus and Funneliformis spp.) was somewhat reduced under high temperature, plant P uptake benefits due to AMF inoculation remained well visible under both temperature regimes, though without imminent impact on plant biomass production that actually decreased due to inoculation with AMF.

Abstract 4648: Biomarkers of exposure and effecft short-term vs. chronic environmental exposure to carcinogenic polycyclic aromatic hydrocarbons

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background. Ostrava Region in the Northern Moravia (Silesia) is the most polluted region in the Czech Republic by particulate matter (PM10 and PM2.5) and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as benzo[a]pyrene (B[a]P). Sources of this pollution are industry (steel production, coke oven), traffic and local heating. In the most polluted district of Ostrava City Bartovice in the year 2009 was PM10 47.6±42.1 μg/m3, B[a]P was 9.2±4.2 ng/ m3. Objectives. The aim of the study is to gain new knowledge on the mechanisms of the effects of complex mixtures bound on dust particles (c-PAHs) in the ambient air on biomarkers of exposure and effect during the period of inversion in January-February 2010. Molecular epidemiology study. In the Ostrava City were selected 78 volunteers- working in the office, 31 city policemen from the near town Karvina, 12 city policemen from the near town Havirov, and 28 volunteers from Ostrava-Bartovice. As controls were city policemen from Prague (N=65). All volunteers were nonsmokers. The personal monitoring to c-PAHs was carried on in January-February 2010. The determined concentration of B[a]P was in the Ostrava region vs. Prague 14.8±13.3 vs. 2.80±1.87 ng/ m3. The impact of these concentration to biomarkers as DNA adducts, chromosomal aberrations, 8-oxodG, lipid peroxidation, genetic polymorphisms (metabolic and DNA repair genes) and gene expression profiles are followed. DNA adducts were analysed in lymphocytes by 32P-postlabeling assay, chromosomal aberrations by fluorescent in situ hybridization (FISH) by whole chromosome painting #1 and #4, and by the determination of micronuclei, oxidative damage as 8-oxodG and 15-F2t-IsoP. Obtained results indicate no differences between the level of biomarkers determined in the Ostrava region and Prague. But different results were observed in four subjects from Prague, who spent 3 weeks in Ostrava during this inversion (exposed), compared to controls from Prague. Analyzing 6000 cells/subject, the frequency of micronuclei in exposed group increased from 7.96±4.92 to 12.91±6.49 MN/1000 vs. 8.32±1.63 and 8.47±1.55 MN/1000 in controls (p<0.05). Similar tendency was observed using FISH and analysing 5000 cells/subject, genomic frequency of translocations (FG/100) in exposed group increased from 1.44±0.66 to 2.44±1.82 FG/100 vs. 1.51±0.61 and 1.45±0.0.52 FG/100. Conclusion: We may speculate, that high environmental exposure to B[a]P does not correspond to DNA damage according to dose effect relationship in a population affected by these concentrations for years, probably due to a possible adaptive response. Acknowledgement: Supported by the Czech Ministry of Environment (SP/1b3/8/08) and the Czech Ministry Education (2B08005). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4648. doi:10.1158/1538-7445.AM2011-4648